Safety verification for polysorbate 20, pharmaceutical excipient for intramuscular administration, in Sprague-Dawley rats and New Zealand White rabbits

Junhyung Kim; Seongsung Kwak; Mi-Sun Park; Chang-Hoon Rhee; Gi-Hyeok Yang; Jangmi Lee; Woo-Chan Son; Won-ho Kang

doi:10.1371/journal.pone.0256869

. 2021 Aug 27;16(8):e0256869. doi: 10.1371/journal.pone.0256869

Safety verification for polysorbate 20, pharmaceutical excipient for intramuscular administration, in Sprague-Dawley rats and New Zealand White rabbits

Junhyung Kim ^1,^‡, Seongsung Kwak ^1,^2,^‡, Mi-Sun Park ¹, Chang-Hoon Rhee ³, Gi-Hyeok Yang ¹, Jangmi Lee ², Woo-Chan Son ^4,^‡,^*, Won-ho Kang ^1,^‡,^*

Editor: Yasmina Abd‐Elhakim⁵

PMCID: PMC8396741 PMID: 34449810

Abstract

Human serum albumin (HSA) has been widely used as a pharmaceutical excipient in Botulinum toxin serotype A (BoNT/A) products that are indicated for use in therapeutics and cosmetics. However, HSA as a human-derived material has some concerns, such as the potential risk of transmission of infectious agents, an insufficient supply, and difficulty in maintaining a certain quality. For those reasons, newly developed BoNT/A products (CORETOX^®, Medytox, Inc., Republic of Korea) contained polysorbate 20, a non-human-derived excipient, to replace the HSA. However, most safety studies of polysorbate 20 have been conducted with non-invasive routes of administration, and thus there are a few studies on the safety of polysorbate 20 when administered intramuscularly. To secure the in vivo safety profile of polysorbate 20, a four-week repeated intramuscular dose toxicity study (0.02, 0.1, and 0.4 mg/kg, one injection every two weeks for a total of three injections) was conducted in 66 Sprague-Dawley (SD) rats. An intradermal irritation study was further conducted with 18 New Zealand White (NZW) rabbits. The toxicological evaluation of HSA (0.06 and 0.12 mg/kg) was also carried out as a comparative substance. Systemic and local toxicities were not observed in any of the SD rats or NZW rabbits based on clinical signs, body weight, hematology, clinical biochemistry, macroscopic findings on necropsy, histopathology of the injection site, and allergic reactions. The current study suggested that intramuscular administration of polysorbate 20 was considered to be safe at a level similar to that of HSA, which has an in vivo safety profile accumulated over the years. This provided the basis for the in vivo safety profile of polysorbate 20 administered intramuscularly and the scientific reliability of the use of polysorbate 20 as an alternative to HSA, which is used as an excipient for various pharmaceuticals in terms of its safety.

Introduction

Human serum albumin (HSA), the most abundant protein in human plasma, has the functions of regulating the colloid osmotic pressure, binding to and transporting various molecules, antioxidant action, anticoagulant action, regulating membrane permeability, and a direct neuroprotective action [1, 2]. Therefore, HSA has been used for therapeutic purposes in patients with hemorrhage, hypovolemia, and hypoalbuminemia since about the 19^th century [2]. In addition to therapeutic purposes, it is used as an excipient for various pharmaceuticals in biomedical aspects [1, 3].

Most botulinum toxin serotype A (BoNT/A) products that are indicated for use in therapeutics and cosmetics also use HSA as an excipient [4, 5]. In BoNT/A products, HSA plays a role in stabilizing the neurotoxic proteins during manufacturing, transportation, and administration and in preventing the aggregation of neurotoxic proteins [6]. However, there are several concerns with HSA, although it is produced under strict regulations and has a safety profiling accumulated over the years [2, 6]. Since the production of HSA mainly relies on human blood donation, the quality of HSA could differ depending on the population of the human blood donors (race or geographical distribution) [7]. Furthermore, the market demand for HSA has dramatically increased (over 500 tons), and it is one of the most widely used biopharmaceutical solutions today [7, 8]. This could lead to a worldwide shortage of HSA in the market [2]. Finally, the use of HSA has a theoretical risk of spreading blood-derived pathogens [6, 9]. Currently, the transmission of viral diseases, such as hepatitis B virus, human immunodeficiency virus, and West Nile virus are well controlled through strict management of HSA, but there are still concerns about the transmission of prion-derived variant Creutzfeldt-Jakob disease [10, 11].

For those reasons, Medytox, Inc. (Republic of Korea) has recently developed a complexing protein-free BoNT/A product (CORETOX^®) that uses polysorbate 20, a non-human-derived excipient, to replace the HSA [12, 13]. Polysorbates, a series of polyoxyethylenated sorbitan esters, are hydrophilic and nonionic surfactants that have been widely used as a food additive and stabilizer in pharmaceuticals [14, 15]. Among them, polysorbate 20, called polyoxyethylene-20-sorbitan monolaurate, has 20 repeating units of polyethylene glycol. It is mainly used as a direct/indirect additive in food and emulsifiers/surfactants in cosmetics that come into direct contact with the human skin, such as shampoo, bath soap, skin cleansing products, and hair spray [15]. It has also been used as an excipient in pharmaceuticals as a stabilizer, particularly in tablet and ophthalmic solutions. Hence, most of the safety studies on polysorbate 20 have been conducted on oral toxicity and skin irritation [14]. As a result, it was found that excessive dose of polysorbate20 could induce mild/temporary eye irritation and skin irritation [14–17]. In addition, when administered orally, it adversely affects the gastro-intestinal tract and, in severe cases, could cause death [14, 16]. However, there are a few studies on the safety of polysorbate 20 administered by other routes, especially intramuscular administration, which is the route of BoNT/A injection.

The objective of this study was to verify the in vivo safety properties of polysorbate 20 when administered intramuscularly. First, a four-week repeated intramuscular dose toxicity study in Sprague-Dawley (SD) rats (one injection every two weeks for a total of three injections) was carried out to evaluate its systemic toxicity. Second, an intradermal irritation study in New Zealand White (NZW) rabbits was carried out to evaluate local toxicity and allergic reactions. It was expected the current study could enable the evaluation of the safety of polysorbate 20 for intramuscular administration and would provide the basis for the in vivo safety profile of polysorbate 20 as a pharmaceutical excipient.

Materials and methods

Animals and facility

This study was reviewed and approved by the Institutional Animal Care and Use Committee of Medytox, Inc. (A-2019-009) and the Korea Testing & Research Institute (KTR, IAC2019-1500). In addition, the study design was established based on the ARRIVE guidelines [18].

In the four-week repeated intramuscular dose toxicity study performed at Medytox R&D Center (Republic of Korea), a total of 66 SD rats, consisting of 30 females (130.7 ~ 146.2g) and 36 males (163.5 ~ 187.2g), were used (Table 1). Healthy five-week-old SD rats were purchased from Orient Bio, Inc. (Republic of Korea). Upon receipt, all animals were macroscopically examined and acclimated for 7 days to the laboratory conditions (a specific pathogen-free animal room; temperature, 23 ± 3°C; humidity, 55 ± 15%; ventilation, 15 air changes per hour; light/dark cycle, 12 hours; light intensity, 150 ~ 300 Lux; polycarbonate cage; 2 ~ 3 animals per cage). Animals were divided into six groups with 11 animals (six male and five female SD rats) in each group. Group 1 (control group) were administered 0.9% saline every two weeks. Group 2, 3, and 4 were administered 0.02, 0.1, and 0.4 mg/kg of polysorbate 20 every two weeks, respectively. Group 5 and 6 were administered 0.06 and 0.12 mg/kg of HSA every two weeks, respectively.

Table 1. Study design of four-week repeated intramuscular dose toxicity study in Sprague-Dawley rats and intradermal irritation study in New Zealand white rabbits.

Study	Species	Group	Substance	Dose volume	Dose	Route and Frequency of administration	Number of animals (male/female)
Four-week repeated intramuscular dose toxicity study	Sprague-Dawley Rats	G1	Control (0.9% saline)	0.2 mL/kg	0 mg/kg	Intramuscular injection (Gastrocnemius muscle) Once per 2 weeks (Total 3 times)	11 (6/5)
		G2	Polysorbate 20		0.02 mg/kg		11 (6/5)
		G3	Polysorbate 20		0.1 mg/kg		11 (6/5)
		G4	Polysorbate 20		0.4 mg/kg		11 (6/5)
		G5	Human serum albumin		0.06 mg/kg		11 (6/5)
		G6	Human serum albumin		0.12 mg/kg		11 (6/5)
		Total					66 (36/30)
Intradermal irritation study	New Zealand White rabbits	G1	Control (0.9% saline)	0.4 mL/site (Total 5 sites)	0 mg/kg/site	Intradermal injection (Dorsal back skin) Once (Total 1 time)	3 (3/0)
		G2	Polysorbate 20		0.02 mg/kg/site		3 (3/0)
		G3	Polysorbate 20		0.1 mg/kg/site		3 (3/0)
		G4	Polysorbate 20		0.4 mg/kg/site		3 (3/0)
		G5	Human serum albumin		0.06 mg/kg/site		3 (3/0)
		G6	Human serum albumin		0.12 mg/kg/site		3 (3/0)
		Total					18 (18/0)

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In the intradermal irritation study performed at KTR (Republic of Korea), a total of 18 male NZW rabbits (2307.7 ~ 2947.8 g) were used (Table 1). Healthy three-month-old rabbits were purchased from DooYeol Biotech (Republic of Korea). Upon receipt, all animals were macroscopically examined and acclimated for 6 days to the laboratory conditions (specific pathogen-free animal room; temperature, 20.3 ~ 21.9°C; humidity, 54.5 ~ 64.9%; ventilation, 10~ 20 air changes per hour; light/dark cycle, 12 hours; light intensity, 150 ~ 300 Lux; stainless steel cage; 1 animal per cage). Animals were divided into six groups with 3 male NZW rabbits in each group. Group 1 (control group) were administered 0.9% saline once. Group 2, 3, and 4 were administered 0.02, 0.1, and 0.4 mg/kg/site of polysorbate 20 once, respectively. Group 5 and 6 were administered 0.06 and 0.12 mg/kg/site of HSA once, respectively.

Vehicle, positive control substance, and test substance

Saline (0.9% NaCl, DAI HAN PHARM. Co., Republic of Korea) was used as the vehicle in this study conducted in SD rats and NZW rabbits. HSA (Green Cross Corp., Republic of Korea) was selected as the comparative substance for the polysorbate 20 (Merck, Germany) which was the test substance of the current study. In the four-week repeated intramuscular dose toxicity study, the dose of HSA was calculated by multiplying the animal equivalent dose calculation factor (6.2) and the content of HSA in most BoNT/A products, 0.01 and 0.02 mg/kg (0.5 and 1.0 mg/vial, 50 kg human) [4, 19]. As a result, 0.06 and 0.12 mg/kg were selected as the dose of HSA. The dose of polysorbate 20 was selected based on the content of polysorbate 20 in CORETOX^® (1.0 mg/vial, 60 kg human) and the human dosage of CORETOX^® (0.2 ~ 3.6 vial) [12, 13]. The dose of polysorbate 20 was also calculated in the same manner and 0.02, 0.1, and 0.4 mg/kg were selected as the dose of polysorbate 20.

Doses of polysorbate 20 were calculated in the same way, and 0.02, 0.1 and 0.4 mg/kg were selected.

The same dose of HSA and polysorbate 20 used in the four-week repeated intramuscular dose toxicity study was applied in the intradermal irritation study in NZW rabbits. Detailed information is provided in Table 1.

In vivo administration

In the four-week repeated intramuscular dose toxicity study in SD rats, all substances (saline, HSA, polysorbate 20) were administered intramuscularly. The dosing formulation was prepared on the day of administration using saline (HSA: 0.3, 0.6 mg/mL; polysorbate 20: 0.1, 0.5, 2.0 mg/mL). The dose volume was selected as 0.2 mL/kg and the individual dose volume for each animal was calculated based on the body weight of the animal on the day of administration [20]. All substances were injected once every two weeks for four weeks (a total of 3 times; days 1, 15, 29) into one site of the gastrocnemius muscle of the right hindlimb. The dosing schedule (interval and frequency) was set based on the clinical application schedule (total 1 time, at least three month dosing interval) and the administration site (gastrocnemius muscle) was selected based on the site generally used to evaluate the effectiveness of BoNT/A in rodents [12, 21–23].

In the intradermal irritation study in NZW rabbits, all substances were administered via the intradermal route. The dosing formulation was prepared on the day of administration using saline (HSA: 0.15, 0.3 mg/mL; polysorbate 20: 0.05, 0.25, 1.0 mg/mL). The dose volume was selected as 0.4 mL/kg/site (total 2 mL/kg/head) and the individual dose volume for each animal was calculated based on the body weight of animal on the day of administration [20]. Before administration, the back skin of the rabbit was depilated of hair. All substances were then injected once into five sites (three left side and two right side) on the back skin (total 1 time; day 1) based on the guideline of the International Organization for Standardization (ISO) [24].

Clinical observation and body weights measurement

In the four-week repeated intramuscular dose toxicity study in SD rats, all animals were observed for clinical signs and general condition once daily throughout the study period (days 1 ~ 29). Their body weights were measured once a week (total five times), including the day of administration and at autopsy.

In the intradermal irritation study in NZW rabbits, clinical observation was conducted once daily throughout the study period (days 1 ~ 29). Furthermore, skin reactions at the administration site, including the degree of erythema, crust formation, and edema formation, were observed once daily based on the scoring system for skin reactions of the ISO [24]. Body weights were measured once a week (total five times), including on the day of administration and at autopsy.

Hematology, clinical biochemistry, and allergic reaction test

All animals (SD rats and NZW rabbits) were fasted overnight before the last day of the study. The SD rats were anesthetized with isoflurane and blood samples were collected from their abdominal vein. In the NZW rabbits, blood samples were collected from the marginal ear vein without anesthesia.

Approximately 1 mL of the blood sample of all animals was put into a tube containing EDTA. After that, the following parameters were analyzed in KTR using a hematological autoanalyzer (ADVIA 120, SIEMENS, Germany): white blood cell count (WBC), differential leucocyte count (neutrophils; lymphocytes; monocytes; eosinophils; basophils), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), RBC indices (mean corpuscular volume, MCV; mean corpuscular hemoglobin, MCH; mean corpuscular hemoglobin concentration, MCHC), reticulocyte, and platelets.

The blood samples (except those used in the hematological analysis) were centrifuged at 3,000 rpm for 10 minutes to obtain the serum. The following parameters were then examined in KTR using an automatic analyzer (TBA-120FR, TOSHIBA, Japan): total protein, albumin, albumin/globulin ratio, total bilirubin, total bile acid, alkaline phosphatase (ALP), aspartate aminotransferase (AST), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyltransferase (GGT), creatinine, blood urea nitrogen (BUN), total cholesterol, triglycerides (TG), glucose, calcium, inorganic phosphorus, creatine phosphokinase, cholinesterase, sodium, potassium, and chloride. Among these, total bile acid and cholinesterase were only analyzed for the NZW rabbits.

In addition, the levels of serum IgE and histamine of all animals were measured using an enzyme-linked immunosorbent assay (ELISA) kit.

Necropsy and histopathology

After blood sampling from the SD rats and NZW rabbits, autopsies were performed on all animals at day 29 according to the AVMA Guidelines for the Euthanasia of Animals [25]. All SD rats were euthanized by exsanguination from the posterior vena cava and abdominal aorta under isoflurane anesthesia. All NZW rabbits were euthanized by intravenous injection of T-61 solution (MSD animal health, Republic of Korea). At necropsy, their external appearance, internal organs, and injection sites were observed macroscopically. The injection site (one site of the gastrocnemius muscle of the right hindlimb for the SD rats and the five sites of the back skin for the NZW rabbits) were collected and stored in 10% neutral buffered formalin. Slides of all preserved tissues for histopathology were prepared through general tissue processing, including dehydration and paraffin embedding. All slides were then stained with hematoxylin and eosin and examined microscopically.

Statistical analysis

The Kruskal-Wallis test was performed to compare toxicological indicators, including body weights, hematology level, clinical biochemistry level, and serum IgE and histamine levels among the groups of SD rats and NZW rabbits. In the case of body weight, since there was a difference in the mean body weight among the groups before the first dose administration, the statistical analysis was performed using the ratio of body weight at the time of the measurement to the body weight before the first administration. The significance of intergroup difference between the control and administered groups was assessed using Dunn’s Rank Sum test. All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, CA) and a p-value of < 0.05 was accepted to indicate statistical significance. Data are represented as mean ± standard error of the mean (SEM).

Results

Mortality and clinical signs

In the four-week repeated intramuscular dose toxicity study, all SD rats remained well throughout the study and showed no clinical signs of disease, pain or distress. In the intradermal irritation study, no deaths or abnormal findings were observed in all NZW rabbits. Skin reactions, including erythema, eschar formation, and edema formation, were not observed regardless of the administered substance.

Body weights

For both the SD rats and NZW rabbits, their body weight was measured once a week for a total of five times. Individual body weights of all animals by time point are presented in the S1 and S2 Tables.

In the four-week repeated intramuscular dose toxicity study, a gradual body weight gain of the SD rats was observed regardless of the administered substance (Fig 1a). Compared to the body weight before the first administration, all male SD rats had a body weight of 138.4% at day 8, 173.6% at day 15, 200.4% at day 22, and 225.0% at day 29, while all female SD rats had a body weight of 132.2% at day 8, 153.1% at day 15, 173.1% at day 22, and 187.3% at day 29. There was no significant increase or decrease of the body weight of the SD rats administered polysorbate 20 and HSA compared to the control group at any of the time points.

Fig 1 — Body weight changes measured once a week in a) SD rats and b) NZW rabbits, for a total of five times during the study period. In SD rats, all substances were injected once every two weeks for four weeks (total three times; days 1, 15, 29) into one site of the gastrocnemius muscle of the right hindlimb. Neither male nor female rat groups showed significant differences in body weight on days 8, 15, 22, and 29 (Kruskal-Wallis test). In NZW rabbits, all substances were injected once into five sites of the back skin (total one time; day 1). There was no significant difference in body weight on days 8, 15, 22, and 29 in all groups (Kruskal-Wallis test).

In the intradermal irritation study, a gradual body weight gain of the NZW rabbits was also observed regardless of the administered substance (Fig 1b). Compared to their body weight before administration, all NZW rabbits had a weight of 105.0% at day 8, 111.0% at day 15, 116.7% at day 22, and 120.8% at day 29. There was also no significant difference in body weight of the NZW rabbits administered polysorbate 20 and HSA compared to the control group at any of the time points.

Hematology, clinical biochemistry, and serum IgE and histamine levels

Hematological tests and clinical biochemistry tests were performed on all SD rats and NZW rabbits. Individual results are presented in the S3 and S4 Tables. In both male and female SD rats, there were no polysorbate 20 or HSA related changes in the hematology parameters. In clinical biochemistry, compared to the control group, the albumin/globulin ratio significantly decreased in male group 6 (p < 0.05), the level of glucose significantly decreased in male group 3, 5, and 6 (p < 0.05, respectively), and the level of sodium significantly increased in male group 4 (p < 0.01) and 6 (p < 0.05) (Table 2). In females, the level of total bilirubin significantly decreased in group 2 and 4 (p < 0.05, respectively) and the level of chloride significantly increased in group 2 (p < 0.05) and 3 (p < 0.01), compared to the control group (Table 3). Furthermore, in the allergic reaction test (ELISA analysis of serum IgE and histamine), significantly higher serum histamine levels were found in the female group 5 and 6 compared to the control group (p < 0.05).

Table 2. Hematology, clinical biochemistry, and serum IgE and histamine levels in male Sprague-Dawley (SD) rats.

Category		Male
Category		G1	G2	G3	G4	G5	G6
Hematological findings	RBC (10 ⁶ cells/μL)	7.37±0.13	7.38±0.14	7.31±0.11	7.48±0.14	7.50±0.10	7.37±0.14
	HGB (g/dL)	14.52±0.20	14.57±0.22	14.57±0.31	14.58±0.23	14.7±0.12	14.37±0.24
	HCT (%)	46.30±0.40	46.15±0.68	45.87±0.85	46.22±0.57	46.42±0.36	45.67±0.89
	MCV (fL)	62.95±0.81	62.57±0.55	62.73±0.56	61.82±0.57	61.95±1.00	62.02±0.09
	MCH (pg)	19.72±0.27	19.73±0.16	19.90±0.17	19.50±0.31	19.57±0.27	19.50±0.13
	MCHC (g/dL)	31.33±0.19	31.60±0.07	31.72±0.24	31.55±0.23	31.62±0.12	31.48±0.19
	RETIC (10 ⁹ cells/μL)	218.7±8.4	213.2±9.4	225.7±11.4	238.7±4.2	198.7±11.3	222.8±10.0
	PLT (10 ³ cells/μL)	1156±63	1168±55	1161±66	1210±39	1228±28	1170±47
	WBC (10 ³ cells/μL)	10.81±1.14	10.90±0.81	9.68±0.95	8.56±0.44	7.15±0.76	8.14±0.87
Clinical biochemical findings	TP (g/dL)	5.90±0.06	6.07±0.08	5.97±0.11	6.00±0.09	6.10±0.09	6.13±0.10
	ALB (g/dL)	3.85±0.04	3.88±0.03	3.83±0.06	3.85±0.03	3.97±0.04	3.88±0.06
	A/G ratio	1.88±0.04	1.78±0.03	1.78±0.04	1.80±0.04	1.87±0.04	1.73±0.02^*
	T-BIL (mg/dL)	0.04±0.00	0.05±0.00	0.04±0.00	0.03±0.00	0.03±0.00	0.05±0.00
	ALP (U/L)	596.3±33.6	711.0±57.2	532.3±38.5	577.2±30.6	560.2±35.6	647.5±47.3
	AST (U/L)	90.33±6.35	119.2±30.2	92.67±4.34	81.00±4.46	106.5±4.8	101.5±9.7
	ALT (U/L)	28.33±3.46	45.50±16.37	27.17±2.85	22.83±1.56	29.00±2.66	26.17±2.44
	CREA (mg/dL)	0.44±0.01	0.45±0.02	0.46±0.02	0.44±0.02	0.40±0.01	0.42±0.01
	BUN (mg/dL)	11.78±0.60	12.60±1.00	11.93±0.54	10.42±0.74	11.17±0.48	10.90±0.41
	T-CHO (mg/dL)	71.00±3.39	78.50±4.65	76.33±4.26	72.33±5.33	65.83±5.68	82.00±3.01
	TG (mg/dL)	80.33±7.06	99.67±13.84	112.5±15.3	96.83±13.26	81.33±9.54	93.67±17.89
	GLU (mg/dL)	127.7±2.9	111.0±4.2	103.5±3.7^*	117.0±7.9	99.33±3.33^*	99.50±6.82^*
	CA (mg/dL)	10.15±0.11	10.23±0.05	10.13±0.10	10.32±0.06	10.00±0.10	10.23±0.14
	IP (mg/dL)	8.90±0.18	8.68±0.17	8.63±0.15	8.78±0.13	9.02±0.09	8.90±0.11
	GGT (IU/L)	0.39±0.16	0.34±0.18	0.69±0.26	0.34±0.16	0.19±0.09	0.48±0.26
	CK (U/L)	529.7±45.1	529.7±29.3	578.5±60.3	452.5±39.0	745.5±50.4	652.3±79.4
	Na (mmol/L)	143.0±0.3	144.4±0.5	143.6±0.3	145.2±0.4^**	144.3±0.4	144.9±0.4^*
	K (mmol/L)	4.68±0.08	4.68±0.06	4.76±0.08	4.87±0.12	4.95±0.05	4.89±0.10
	Cl (mmol/L)	101.4±0.3	102.8±0.5	101.4±0.8	103.1±0.5	101.5±0.6	102.3±0.6
ELISA analysis	IgE (ng/mL)	0.233±0.014	0.270±0.001	0.246±0.015	0.181±0.008	0.200±0.016	0.188±0.004
ELISA analysis	Histamine (ng/mL)	0.480±0.040	0.383±0.038	0.402±0.034	0.423±0.026	0.519±0.052	0.390±0.040

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Data are presented as mean ± SEM.

All data were analyzed using Kruskal-Wallis test and the significance of intergroup difference between the control and administered groups was assessed using Dunn’s Rank Sum test.

*vs. Control group: p < 0.05,

** vs. Control group: p < 0.01

Table 3. Hematology, clinical biochemistry, and serum IgE and histamine levels in female Sprague-Dawley (SD) rats.

Category		Female
Category		G1	G2	G3	G4	G5	G6
Hematological findings	RBC (10 ⁶ cells/μL)	7.14±0.20	6.72±0.17	7.06±0.08	7.30±0.09	7.47±0.22	7.30±0.13
	HGB (g/dL)	14.08±0.30	13.78±0.31	13.96±0.20	14.58±0.12	14.74±0.28	14.52±0.28
	HCT (%)	42.54±0.88	42.08±1.01	42.06±0.46	44.42±0.16	44.60±0.88	44.40±0.76
	MCV (fL)	59.70±0.89	62.70±1.13	59.60±0.91	60.92±0.69	59.84±0.92	60.84±0.60
	MCH (pg)	19.76±0.22	20.46±0.06	19.78±0.36	20.00±0.26	19.76±0.29	19.88±0.19
	MCHC (g/dL)	33.12±0.27	32.70±0.50	33.20±0.15	32.84±0.17	33.00±0.28	32.70±0.15
	RETIC (10 ⁹ cells/μL)	156.9±10.2	170.9±16.1	151.9±14.7	169.0±5.2	143.1±13.7	159.8±7.6
	PLT (10 ³ cells/μL)	995.6±167.9	858.2±201.0	1208±111	1164±78	1097±56	1217±63
	WBC (10 ³ cells/μL)	3.48±0.43	4.24±0.97	5.62±1.00	5.88±1.13	4.71±0.35	4.38±0.37
Clinical biochemical findings	TP (g/dL)	6.10±0.05	6.06±0.14	6.10±0.23	6.04±0.04	6.02±0.09	6.20±0.13
	ALB (g/dL)	4.14±0.06	4.02±0.12	3.94±0.19	3.90±0.06	3.90±0.03	4.04±0.09
	A/G ratio	2.10±0.05	1.98±0.08	1.84±0.11	1.84±0.06	1.86±0.04	1.88±0.04
	T-BIL (mg/dL)	0.07±0.00	0.03±0.00^*	0.05±0.01	0.03±0.01^*	0.04±0.01	0.04±0.01
	ALP (U/L)	354.8±16.1	483.2±45.1	448.2±37.2	433.0±49.5	442.4±44.8	474.6±48.6
	AST (U/L)	83.00±2.51	96.60±6.17	92.60±13.1	92.00±2.00	82.00±4.32	84.20±7.04
	ALT (U/L)	26.20±0.80	27.00±2.55	31.20±5.00	25.40±2.38	33.20±2.06	29.60±1.96
	CREA (mg/dL)	0.43±0.02	0.46±0.03	0.43±0.01	0.41±0.02	0.39±0.01	0.43±0.02
	BUN (mg/dL)	19.88±2.57	15.58±1.01	15.94±0.88	13.46±1.22	14.62±0.86	16.86±1.08
	T-CHO (mg/dL)	86.80±9.28	85.80±3.81	87.00±4.55	86.60±6.59	86.40±3.82	97.80±9.41
	TG (mg/dL)	76.20±19.14	47.60±7.47	48.60±3.66	47.60±16.46	52.40±6.54	63.80±7.65
	GLU (mg/dL)	114.0±4.4	124.0±6.5	104.0±2.7	103.6±5.2	113.2±4.6	120.4±6.6
	CA (mg/dL)	10.04±0.10	9.96±0.13	10.16±0.20	10.08±0.14	10.28±0.04	10.44±0.10
	IP (mg/dL)	7.36±0.25	7.30±0.23	7.48±0.23	7.50±0.22	7.76±0.17	8.00±0.13
	GGT (IU/L)	0.34±0.09	0.17±0.07	0.21±0.13	0.32±0.14	0.20±0.13	0.43±0.12
	CK (U/L)	606.4±15.2	683.2±67.8	533.6±118.0	491.4±28.3	431.6±45.0	508.6±114.3
	Na (mmol/L)	140.8±0.3	141.6±0.5	141.7±0.7	141.9±0.3	141.1±0.3	141.2±0.3
	K (mmol/L)	4.44±0.07	4.29±0.14	4.50±0.16	4.44±0.09	4.54±0.12	4.43±0.22
	Cl (mmol/L)	100.2±0.3	102.5±0.4^*	103.0±0.6^**	101.6±0.9	102.3±0.4	101.5±0.3
ELISA analysis	IgE (ng/mL)	0.222±0.023	0.211±0.008	0.247±0.035	0.211±0.025	0.187±0.022	0.183±0.016
ELISA analysis	Histamine (ng/mL)	0.253±0.030	0.311±0.043	0.385±0.027	0.257±0.034	0.570±0.128^*	0.510±0.063^*

Open in a new tab

Data are presented as mean ± SEM.

All data were analyzed using Kruskal-Wallis test and the significance of intergroup difference between the control and administered groups was assessed using Dunn’s Rank Sum test.

*vs. Control group: p < 0.05,

** vs. Control group: p < 0.01

In NZW rabbits, there were no changes in the hematology and clinical biochemistry parameters in the polysorbate 20 or HSA administered animals. There was also no significant change in the level of serum IgE and histamine in NZW rabbits administered polysorbate 20 and HSA compared to the control group.

Necropsy and histopathology

During the necropsy on all SD rats, no polysorbate 20 or HSA related macroscopic findings were observed such as changes in external appearance, internal organs, and administration sites. In histopathological examination of the administration site, mild macrophage infiltration and neovascularization were observed with a similar degree and frequency in all administered groups, including the control group. In addition, inflammatory cells infiltration, fat infiltration, giant cells, and necrosis were observed only in some individuals regardless of the administered substance and dose (Fig 2).

Fig 2 — Hematoxylin and eosin stained histopathological section (original magnification 100x) of the gastrocnemius muscle of the SD rats: a) Control group, male; b) Control group, female; c) Polysorbate 20–0.02 mg/kg, male; d) Polysorbate 20–0.02 mg/kg, female; e) Polysorbate 20–0.1 mg/kg, male; f) Polysorbate 20–0.1 mg/kg, female; g) Polysorbate 20–0.4 mg/kg, male; h) Polysorbate 20–0.4 mg/kg, female; i) Human serum albumin-0.06 mg/kg, male; j) Human serum albumin-0.06 mg/kg, female; k) Human serum albumin-0.12 mg/kg, male; l) Human serum albumin-0.12 mg/kg, female. Mild macrophage infiltration, neovascularization, inflammatory cells infiltration, and fat infiltration were observed with a similar degree and frequency in all administered groups. An arrow pointing up (↑) indicated macrophage infiltration and an arrow pointing down (↓) indicated neovascularization.

In the case of the NZW rabbits, macroscopic findings at necropsy and histopathological findings at the sites of administration were not observed regardless of the administered substance (Fig 3).

Fig 3 — Hematoxylin and eosin stained histopathological section (original magnification 50x) of the dorsal back skin of the NZW rabbits: a) Control group; b) Polysorbate 20–0.02 mg/kg; c) Polysorbate 20–0.1 mg/kg; d) Polysorbate 20–0.4 mg/kg; e) Human serum albumin (HSA)-0.06 mg/kg; f) HSA-0.12 mg/kg, male. No histopathological findings were found at the site of administration in all administered groups.

Discussion

Polysorbate 20 was approved by the regulatory authorities as an additive in foods, cosmetics, eye drops, and tablets in various countries [14, 15]. Therefore, most clinical and nonclinical safety studies of polysorbate 20 have been conducted using non-invasive routes of administration (e.g., oral, skin, and ophthalmic administration) [16, 17]. In particular, as a food additive, polysorbate 20 has various in vivo safety data, including in vitro genotoxicity and DNA reactivity based on structural alerts [26]. Recently, polysorbate 20, which was one of the excipients of CORETOX^® approved by the Ministry of Food and Drug Safety (MFDS, Republic of Korea), has been used in humans via an intramuscular route of administration [12]. However, there are few studies on the safety of polysorbate 20 when administered intramuscularly. In a study conducted in four monkeys, it was reported that intramuscular injection of 275 mg/kg of polysorbate20 for 20 days did not cause toxicity in the liver and kidney [27]. This study did not evaluate clinical signs, body weight, or hematology and clinical biochemistry, which are essential criteria for evaluating toxicity [28]. Therefore, the current study was conducted to verify the safety of intramuscular administration of polysorbate 20 through four-week repeated intramuscular dose toxicity in SD rats and an intradermal irritation study in NZW rabbits. This study is the first to fully evaluate the in vivo safety of polysorbate 20 when administered intramuscularly based on current guidelines of non-clinical safety research.

To evaluate the systemic toxicity of intramuscular injection of polysorbate 20, a four-week repeated dose toxicity study in SD rats was conducted. According to the MFDS, the clinical dosage of CORETOX^®, which contains 1 mg of polysorbate 20 in one vial, ranged from 0.2 vial (treatment of forehead wrinkles) to 3.6 vials (stiff muscles in the upper limbs after a stroke) [12]. Thus, the dose (0.02, 0.1, and 0.4 mg/kg) of polysorbate 20 in the current study was established based on the therapeutic indication of CORETOX^® in humans and the animal equivalent dose calculation factor [19]. In addition, 0.9% saline was set as the vehicle control substance, while 0.06 and 0.12 mg/kg doses of HSA, which has an in vivo safety profile accumulated over the years for intramuscular administration, was set as a comparative substance [2, 6].

Generally, mortality, body weight, and clinical signs have the most significance in repeated dose toxicity studies [29, 30]. Furthermore, changes in the levels of hematology and clinical biochemistry in laboratory animals could enable more accurate predictions of human toxicity [31, 32]. In the current study, adverse effects, including mortality, clinical signs, and macroscopic findings on necropsy, were not found at any dose level of HSA and polysorbate 20 administered during the study period in all SD rats. In addition, significant changes in the body weight and the level of hematology were not observed in the polysorbate 20 and HSA administered groups compared to the control group. In the clinical biochemistry test and allergy reaction test, significant decrease in albumin/globulin ratio (p < 0.05), glucose (p < 0.05), and total bilirubin (p < 0.05) and significant increase in sodium (p < 0.05 or p < 0.01), chloride (p < 0.05 or p < 0.01), and serum histamine (p < 0.05) were observed in the polysorbate 20 or HSA administered group compared to the control group. However, these changes had no toxicological significance as they were not dose-responsive and the levels were within the normal range for the same age of SD rats [33, 34]. This indicated that intramuscular administration of 0.02, 0.1, and 0.4 mg/kg dose of polysorbate 20 did not cause systemic toxicity in SD rats.

Histopathological examination of the administration site was conducted to observe the local toxicity of the polysorbate 20 in all SD rats. At the administration site, macrophage infiltration, neovascularization, giant cell, fibrosis, and necrosis were observed to a similar degree and frequency in all administered groups regardless of the administered substance. However, these findings were observed in most of the administered groups without a dose response, including the control group. Furthermore, inflammatory lesions could occur in the body of SD rats as a defense mechanism against intramuscular injection of any foreign substances [35]. Therefore, these findings were presumed to be due to the intramuscular injection rather than any effect of polysorbate 20. In other words, there were no toxicological changes at the site of administration after intramuscular administration of polysorbate 20 based on histopathology. Furthermore, serum IgE and histamine levels were measured to determine whether allergic reactions occurred after the administration of polysorbate 20 in all SD rats. In addition to clinical signs, increased levels of serum IgE and histamine are representative indicators of an allergic reaction [36–38]. In the current study, allergic reactions were not observed in response to the administration of polysorbate 20, which indicated that polysorbate 20 is not an allergen in SD rats.

However, despite its very low frequency, side effects in the allergy category have been reported in humans using shampoos (8.4% polysorbate 20, 2 cases among 5.88 million uses), cuticle softeners (2% polysorbate 20, 24 cases among 131 million use), and paste masks (2% polysorbate 20, 11 cases among 120.7 million uses) containing polysorbate 20 [14, 15]. Therefore, an additional intradermal irritation study was conducted in NZW rabbits to confirm that polysorbate 20 was not an allergen in vivo. Traditionally, the intradermal test is the gold standard method for diagnosing allergic reactions [39, 40]. If an immune response, such as erythema, crust formation, and edema formation, occurs at the administration site after intradermal administration of a substance, it could be concluded that the substance was an allergen to the administered subjects [24]. In the current study, immune responses of the administration site were not observed at all polysorbate 20 treated NZW rabbits after intradermal administration during the study period, which is the same as the results in the control group and the HSA administered group. Furthermore, in the polysorbate 20 administered group, toxicological changes based on the histopathological examination of the site of administration and increased levels of serum IgE and histamine were not observed. There were also no toxicologically significant changes, such as changes in the body weight or the hematology and clinical biochemistry parameters in all rabbits administered polysorbate 20 and all toxicological values in NZW rabbits were within the normal range for the same age [41, 42]. These results of the intradermal irritation study indicated that polysorbate 20 is not an allergen in NZW rabbits, which was consistent with the results of the four-week repeated dose toxicity study in SD rats.

Conclusions

The current study, a four-week repeated intramuscular dose toxicity study in SD rats and an intradermal irritation study in NZW rabbits, provides the basis for the in vivo safety profile of intramuscular administration of polysorbate 20, an excipient of newly developed BoNT/A products (CORETOX^®). Polysorbate 20, which did not induce systemic and local toxicities in this study, was considered to be a safe substance for intramuscular administration, just like HSA for which a safety profile has been obtained over the years. In addition, since polysorbate 20 is a synthetic product rather than an animal-derived product, it has the advantages of a consistent quality of the product, ease of manufacture and supply, and a low risk of pathogen transmission compared to HSA. Therefore, the findings of this study provide the scientific reliability of the use of polysorbate 20 as an alternative to HSA, which is used as an excipient for most BoNT/A products, particularly in terms of its safety. Furthermore, this study suggest the possibility that polysorbate 20, which is mainly used as an additive in foods, cosmetics, eye drops, and tablets, could be used as a safe excipient for various pharmaceuticals administered intramuscularly.

Supporting information

S1 Table. Individual body weights of male (n: 36) and female (n: 30) Sprague-Dawley (SD) rats in the current study.

(XLSX)

Click here for additional data file.^{(15KB, xlsx)}

S2 Table. Individual body weights of male New Zealand White (NZW) rabbits (n: 18) in the current study.

(XLSX)

Click here for additional data file.^{(11.3KB, xlsx)}

S3 Table. Individual levels of hematology, clinical biochemistry, and serum IgE and histamine in Sprague-Dawley (SD) rats in the current study.

(XLSX)

Click here for additional data file.^{(35.2KB, xlsx)}

S4 Table. Individual level of hematology, clinical biochemistry, and serum IgE and histamine in New Zealand White (NZW) rabbits in the current study.

(XLSX)

Click here for additional data file.^{(19.5KB, xlsx)}

Acknowledgments

The authors thanks Do Yeon Lee and Min-Seo Choi, colleagues at the Medytox, Inc., for their helpful discussion and careful reading of the manuscript.

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

This study was supported by the Technology Innovation Program, establishment of risk management platform with aim of reduce attrition of new drugs and its service [grant number: 10067737, 2016], funded by the Ministry of Trade, Industry & Energy in Republic of Korea (WCS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors [JK, SK, MSP, CHR, GHY, WhK] are employed by a commercial company, Medytox Inc., which only provided support in the form of salaries for authors, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.

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PLoS One. doi: 10.1371/journal.pone.0256869.r001

Decision Letter 0

Arun K Yadav

13 Apr 2021

PONE-D-21-03019

Safety verification for polysorbate 20, pharmaceutical excipient of newly developed Botulinum toxin serotype A products, in Sprague-Dawley rats and New Zealand White rabbits

PLOS ONE

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Comments from Staff Editor:

Please address the following points as part of your revisions and include comments in your response to reviewers.

1) Why did you measure IgE levels and not for example HSA/PS specific IgG?

2) What was the rationale for using the gastrocnemius muscle for injection rather than the thigh muscles?

3) We recommend rephrasing Lines 202-203 to removed the toxicologists' bias of looking for death in a study ("all animals survived..") to "..all animals remained well throughout the study and showed no clinical signs of disease, pain or distress.". Please also replace uses of the word 'symptoms' (what a patient reports) instead of 'signs' (what is observable).

4) We note that the manuscript focuses mainly on the implications of this study for the development of cosmetics, and therefore request that you explicitly assert and emphasize the medical implications of this study in order to show the full range of applications of your findings.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments are mentioned in the attached pdf file. Although a nice work, statistics needs to be reviewed. A little bit of language errors. Histological images needs to be marked with arrows pointing to the damage or change seen.

Reviewer #2: The paper mentioned the use of polysorbate-20 as a replacement of the HSA in the BoNT/A formulation.

Major and minor correction required for the paper:

1. The study mentioned the use of Polysorbate-20 as an excipient for the BoNT products. But no where in the paper any results mention about presence of Botulinum neurotoxin along with polysorbate-20. According to study results polysorbate-20 is nontoxic excipient, and similar as HSA for the any pharmaceutical products administer through transdermal or intramuscular route. Not just limited for the BoNT. Additionally, Polysorbate-20 is already approved exicipient from FDA, so significance of this study is limited.

2. The title as well the introduction should be rewritten without emphasizing the Botulinum toxin. The current data seems the controls used for the formulation study. However, authors can include the possible use of polysorbated-20 as an excipient for Botulinum toxin products in the discussion but should not be only therapeutic product.

3. To use the polysorbated-20 as an excipient in the BoNT based formulation required in depth efficacy, safety, pharmacokinetics and pharmacodynamic study of the Botulinum toxin in the presence of polysorbate-20. The data should be compared with the existing formulation like Botox or dysport etc. The study having BoNT formulation with Polysorbate-20 has already published and mentioned in the paper as a reference # 18 and 19. So the significance of this particular control study reduces.

4. Data table 2 and 3 title are same. Please specify if table 2 belong to polysorbate-20 or for HSA.

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2021 Aug 27;16(8):e0256869. doi: 10.1371/journal.pone.0256869.r002

Author response to Decision Letter 0

3 Jun 2021

[We have upload a separate file labeled 'Response to Reviewers'.]

Dear editor and reviewers,

We would like to thank the editor and the reviewers for helpful and constructive comments and critiques. We have done our best to revise the manuscript in order to satisfactorily address each of the points raised. We believe that this revised manuscript has greatly benefited from these changes and hope that it will be considered satisfactory for publication in PLOS ONE.

Response to Editor’s comment

- We are grateful to the editor for the comments and suggestions, which have helped us to improve our manuscript. We have revised the manuscript based on the editor’s comments and have provided our point-by-point responses to each comment below.

1. Why did you measure IgE levels and not for example HSA/PS specific IgG?

- Thank you for your comments. Generally, allergic reactions are divided into four types (Type I, II, III, and IV), of which type I and IV are the most common types of drug-related allergy (#1). Therefore, we conducted in vivo test, a standard method, to determine whether polysorbate 20 induces type I or IV allergies. Then, we confirmed that there was no allergic reaction, including abnormalities at the site of administration, due to polysorbate 20 in both studies (four-week repeated intramuscular dose toxicity study in SD rats and intradermal irritation study in NZW rabbits). After in vivo test, since only IgE-mediated anaphylaxis cases (type I allergy) were reported due to polysorbates (#2), we further measured the levels of serum IgE and histamine, which were type I allergy-related factors. The level of IgE and histamine also did not increase after administration of polysorbate 20 in both studies.

-#1: Böhm R, Proksch E, Schwarz T, Cascorbi I. Drug hypersensitivity: diagnosis, genetics, and prevention. Dtsch Arztebl Int. 2018;115: 501.

-#2: Maggio E. Polysorbates, biotherapeutics, and anaphylaxis: a review. Bioprocess Int. 2017.

2. What was the rationale for using the gastrocnemius muscle for injection rather than the thigh muscles?

- We thank you for pointing this out. In the case of BOTOX®, the first FDA-approved Botulinum toxin serotype A (BoNT/A) product, the route of administration was set to gastrocnemius muscle rather than thigh muscle in the toxicity study in rodents (Biologic license application number: 103000). In addition, in the compound muscle action potential (CMAP) and digit abduction scoring (DAS) techniques, which are generally used to evaluate the effectiveness of BoNT/A products, the route of administration was also set to gastrocnemius muscle rather than thigh muscle in the toxicity study in rodents (#1, 2, 3). Therefore, in the current study, the administration route was set to the gastrocnemius muscle for accurate comparison with the previously reported results. We have added the rationale for setting the route of administration to the revised manuscript.

-#1: Kim S-H, Kim S-B, Yang G-H, Rhee C-H. Mouse compound muscle action potential assay: An alternative method to conduct the LD50 botulinum toxin type A potency test. Toxicon. 2012;60: 341–347.

-#2: Aoki KR. A comparison of the safety margins of botulinum neurotoxin serotypes A, B, and F in mice. Toxicon. 2001;39: 1815–1820.

-#3: Oh H-M, Park JH, Song DH, Chung ME. Efficacy and safety of a new botulinum toxin type A free of complexing proteins. Toxins (Basel). 2016;8: 4.

3. We recommend rephrasing Lines 202-203 to removed the toxicologists' bias of looking for death in a study ("all animals survived..") to "..all animals remained well throughout the study and showed no clinical signs of disease, pain or distress.". Please also replace uses of the word 'symptoms' (what a patient reports) instead of 'signs' (what is observable).

- Thank you for your recommendation. As you suggest, we have revised the phrase “all SD rats survived throughout the study period” in the revised manuscript. In addition, we have modified the word ‘symptoms’ to ‘signs’ throughout the revised manuscript.

4. We note that the manuscript focuses mainly on the implications of this study for the development of cosmetics, and therefore request that you explicitly assert and emphasize the medical implications of this study in order to show the full range of applications of your findings.

- Thank you for your recommendation. As you suggest, we have modified the “Introduction” and “Conclusion” part accordingly in the revised manuscript. We have emphasized the significance of this study and mentioned the scope of application of our findings in the future.

Response to Reviewers' comments (Reviewer #1)

Comments are mentioned in the attached pdf file. Although a nice work, statistics needs to be reviewed. A little bit of language errors. Histological images needs to be marked with arrows pointing to the damage or change seen.

- Thank you for your encouraging feedback. Based on the comments in PDF file, we have revised the manuscript, including language errors and histological images. We have provided our point-by-point responses to each comment below.

1. Line 18: Modify "were" to "have". In addition, only one author can be the corresponding author.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

- According to the PLOS ONE's submission guidelines, the following is stated in relation to the corresponding author: “Only one corresponding author can be designated in the submission system, but this does not restrict the number of corresponding authors that may be listed on the article in the event of publication. Whoever is designated as a corresponding author on the title page of the manuscript file will be listed as such upon publication. Include an email address for each corresponding author listed on the title page of the manuscript." Therefore, we kept two corresponding authors in the current study.

2. Line 25-27, 51-52, 80-81: Modify "few" to "a few".

-Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

3. Line 90 “Animals and Facility”: Mention the grouping of animals in this section. For example, Animals were divided into 5 groups with 11 animals in each group. Group 1 were administered 0.01 ml of substance x for y days and so on. Kindly do this for both the substances.

- Thank you for your comments. As you suggest, we have revised the text accordingly in the revised manuscript.

4. Line 95, 101: Provide weight range of animals

- Thank you for your comments. As you suggest, we have revised the text accordingly in the revised manuscript.

5. Line 101: Females are considered to be more sensitive. It would have been better if female animals were included. It will help in determining if there are any sex related differences.

- Thank you for pointing this out. In the ISO guideline, the following is stated in relation to the intradermal irritation study (#1): “Healthy young adult albino rabbits of either sex from a single strain, weighing not less than 2 kg, shall be used. A minimum of three animals shall initially be used to evaluate the test material. Inject intracutaneously 0,2 ml of the extract obtained with polar or non-polar solvent at five sites on one side of each rabbit.” Since the test substance was administered to five sites on the back skin, we selected a male as a test system, which was expected to have less interference between the administered substances because it is larger than that of female. In the current study, since there was no reaction by the administration of polysorbate 20 in the male SD rats, it is expected that there will be no side effects by the administration of polysorbate 20 in female SD rats.

-#1: Iso B, STANDARD B. Biological evaluation of medical devices. Part. 2009;1: 10993.

6. Were the rats and rabbits placed in the same animal room or different ones? Since the temperatures are different, is it that the mentioned temperatures are required for their survival?

- Thank you for your comments. The four-week repeated intramuscular dose toxicity study in SD rats were conducted in Medytox R&D Center (Republic of Korea) and all SD rats were purchased from Orient Bio, Inc. (Republic of Korea). The intradermal irritation study in NZW rabbits were performed at KTR (Republic of Korea), and all NZW rabbits were purchased from DooYeol Biotech (Republic of Korea). SD rats and NZW rabbits used in these two studies were placed in different facilities. In addition, the overall environment of the facilities for SD rats and NZW rabbits was optimally set based on the guidelines (#1 and #2)

-#1: ARRP. Guideline 20: Guidelines for the Housing of Rats in Scientific Institutions. Animal Research Review Panel. 2007.

-#2: ARRP. Guideline 18: Guidelines for the Housing of Rabbits in Scientific Institutions. Animal Research Review Panel. 2003

7. Line 178-179: Modify “based on” to “according to”.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

8. Line 182-183: Delete “using the naked eye”.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

9. Line 187-188: Modify “Then, all slides were” to “All slides were then” and “hematoxylin & eosin” to “hematoxylin and eosin”. In addition, delete “at KTR”. Avoid mentioning everytime that it was done at KTR. One time mention will suffice.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

10. Line 227-230 “In SD rats, all substances were injected once every two weeks for four weeks (total three times; days 1, 15, 29) into one site of the gastrocnemius muscle of the right hindlimb.”: What was the basis for selection of these days for dosing. Why not daily as in acute toxicity studies?

- Thank you for your comments. The objective of this study was to verify the in vivo safety properties of polysorbate 20, an excipient of CORETOX® (newly developed BoNT/A product), when administered intramuscularly. Therefore, the dosing schedule (interval and frequency) was set based on the clinical application schedule of BoNT/A products (#1). The clinical application interval of BOTOX® (first FDA-approved BoNT/A product), which is similar to that of CORETOX®, is once every 3 months (Biologic license application number: 103000). In addition, according to the FDA approval document of BOTOX®, a 4-week repeated dose intramuscular toxicity study in rats was performed. Therefore, we have selected the dosing schedule of polysorbate 20 (once every two weeks for four weeks, total three times; days 1, 15, 29) based on clinical application dosing schedule, information of FDA-approved BoNT/A product, and ICH guideline. Furthermore, we thought that this four-week repeated intramuscular dose toxicity study could sufficiently replace the acute toxicity study. Because the dose of polysorbate 20 in the current study (0.02, 0.1, and 0.4 mg/kg) is more than six times the clinical application dose (0.003 ~ 0.06 mg/kg) (#2 and #3) and polysorbate 20 (with CORETOX®) is administered every 3 months (or single dose) in human, as mentioned above. Since the commented part is the figure legend section, we have modified the text accordingly in the method section.

-#1: Guideline ICHHT. Guidance on nonclinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals M3 (R2). International conference on harmonisation of technical requirements for registration of pharmaceuticals for human use. 2009.

-#2: Lee J, Chun MH, Ko YJ, Lee S-U, Kim DY, Paik N-J, et al. Efficacy and safety of MT10107 (Coretox®) in post-stroke upper limb spasticity treatment: A randomized, double-blind, active drug-controlled, multi-center, phase III clinical trial. Arch Phys Med Rehabil. 2020.

-#3: Kwak S, Kang W, Rhee C-H, Yang G-H, Cruz DJM. Comparative Pharmacodynamics Study of 3 Different Botulinum Toxin Type A Preparations in Mice. Dermatologic Surg. 2020;46: e132–e138.

11. Line 231-232 “In NZW rabbits, all substances were injected once into five sites of the back skin (total one time; day 1).”: What is the basis of dosing selection?

- Thank you for your comments. As mentioned above (Q5), the following is stated in relation to the intradermal irritation study in the ISO guideline (#1): “Inject intracutaneously (intradermally) 0,2 ml of the extract obtained with polar or non-polar solvent at five sites on one side of each rabbit.” In addition, polysorbate 20 (with CORETOX®) is administered every 3 months (or single dose) in human, as mentioned above (Q10). Therefore, we have selected the dosing method of polysorbate 20 based on the ISO guideline and clinical application dosing schedule. Since the commented part is the figure legend section, we have modified the text accordingly in the method section.

12. Line 235-236, 332, Table 2 : Modify “hematology, clinical chemistry, serum IgE, and serum histamine” to “hematology, clinical chemistry and serum IgE and histamine”. Also, instead of chemistry, I feel the appropriate word used could be clinical biochemistry.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

13. Line 239-240: Doses have already been mentioned in methods section. Repetition unnecessary. If any changes were observed, then mention the doses on which changes were observed only.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

14. Line 241, 244: Significanlty higher or lower, please mention. In addition, modify “group administered” to “group that were administered”.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

15. Line 242, 245, 284, 294, 342, 363: Delete “Vehicle”. Mention in the beginning of the manuscript that control group was administered the vehicle only. And then just mention control in all the places in the manuscript.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

16. Line 248: If the doses are already mentioned in the methods section, there is no need to mention them again and again. It can be simply mentioned that no changes were seen in all the administered doses. If there is a change, them mention the specific dose at which the change was seen.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

17. Line 253 and 256: Why arent the data represented as mean=/- standard error of mean? Furthermore, ANOVA or Kruskal-Wallis test are usually followed by a post hoc test such as Dunnet's test, Bonferoni's test or Tukey's test. However, none of the tests have been done to make comparasions. Authors kindly perform a test which best suits your data represented to make comparasions between test and control values and one test and another test values. Remove the shadings, significant values can be represented by a superscript *

- Thank you for pointing this out. The Kruskal-Wallis test performed to compare toxicological indicators in the current study is a ranking-based nonparametric statistical test. Therefore, we presented the median and range in Tables 2 and 3 to show the data without statistical bias. However, since the raw data of the information presented in Tables 2 and 3 are all presented in S3 table, we have modified Tables 2 and 3 (mean ± standard error of the mean, no shadings) accordingly in the revised manuscript. In addition, the Dunn's Rank Sum test was performed as a post hoc test of Kruskal-Wallis test for the significance of intergroup difference between the control and administered groups. We have modified the text accordingly in the method section.

18. Line 257: Modify "in" to "of".

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

19. Line 258: Modify "polysorbate 20 or HSA related macroscopic findings were not observed ~" to " no polyabsorbate 20 or....... macroscopic findings were observed such as changes in external appearance......".

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

20. Line 259-276: Changes observed should be represented as the mean or average changes observed in the whole group, not individual animal changes. Do not mention individual animal changes, rather describe the mean value obtained from the observed group. Don’t mention individual rat results, mention what was most commonly observed. Follow this for all the below results too. Please mention only the most common effects seen over all. Please refer some toxicity papers and observe how data are represented.

- Thank you your recommendations. In the current study, there was no significant histopathological change according to the administered substance in SD rats. Therefore, we have deleted individual changes and only represented the change of the whole groups for the concise histopathology results. We have modified the text accordingly in the revised manuscript.

21. Line 267, 364: Modify “administration group” to “administered group”.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

22. Line 332-334: Mention p value. And Modify “a” to “the”.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

23. Figure 2: Histological images needs to be marked with arrows pointing to the damage or change seen.

- Thank you for pointing this out. In Figure 2, macrophage infiltration and neovascularization were seen in all administered group (Fig 2a ~ l). In Figure 3, No histopathological findings were seen in all administered groups (Fig 3a ~ f). Therefore, we have modified the Figure 2 and Figure legend 2 accordingly in the revised manuscript.

Response to Reviewers' comments (Reviewer #2)

The paper mentioned the use of polysorbate-20 as a replacement of the HSA in the BoNT/A formulation.

- Thank you for your overall comments, which have substantially improved the manuscript. We have provided our point-by-point responses to each comment below.

- Thank you for your comments. Polysorbate 20 is an inactive ingredient for drug products that have already been approved by the FDA for auricular, intramuscular, intravenous, nasal, ophthalmic, oral, subcutaneous, topical, and vaginal administration (Drug Approvals and Databases). However, since it is mainly used as an excipient in tablet and ophthalmic solutions, most of the safety studies on polysorbate 20 have been conducted on oral toxicity and skin irritation (#1 and 2). There are few studies on the safety of polysorbate 20 when administered intramuscularly. Therefore, the current study was conducted to obtain in vivo safety data for intramuscular administration of polysorbate 20, which has little data currently accumulated. Findings of the current study suggest that polysorbate 20 could be used as a safe excipient for various pharmaceuticals administered intramuscularly. In addition, BoNT/A product (CORETOX®) that uses polysorbate 20 as an excipient have already been approved by the Ministry of Food and Drug Safety (Republic of Korea) and are on the market. In the approval process, in vivo safety and efficacy studies were already conducted in the presence of Botulinum neurotoxin along with polysorbate 20 and the relevant results were submitted to regulatory authorities. Therefore, in the current study, the safety of Botulinum neurotoxin along with polysorbate 20 was not investigated.

-#1: Moore J. Final report on the safety assessment of polysorbates 20, 21, 40, 60, 61, 65, 80, 81, and 85. J Am Coll Toxicol. 1984;3: 1–82.

-#2: Lanigan RS. Final report on the safety assessment of PEG-20 sorbitan cocoate; PEG-40 sorbitan diisostearate; PEG-2,-5, and-20 sorbitan isostearate; PEG-40 and-75 sorbitan lanolate; PEG-10,-40,-44,-75, and-80 sorbitan laurate; PEG-3, and-6 sorbitan oleate; PEG-80 sorb. Int J Toxicol. 2000;19: 43–89.

- Thank you for your recommendation. As you suggest, we have revised the title to “Safety verification for polysorbate 20, pharmaceutical excipient for intramuscular administration, in Sprague-Dawley rats and New Zealand White rabbits”. Throughout the ‘Introduction’ part, we have rewritten the manuscript to emphasize polysorbate 20 and HSA rather than Botulinum toxin. In addition, in ‘Introduction’ and ’Conclusions’ part, we have revised the manuscript to emphasize that the findings of the current study could be applied to a various therapeutic product, not limited to BoNT/A product.

- Thank you for your comments. As mentioned in the answer to Q2, in vivo safety, efficacy, pharmacodynamic studies were already conducted in the presence of Botulinum neurotoxin along with polysorbate 20 and the relevant results were submitted to regulatory authorities. Considering the characteristics of Botulinum toxin (local diffusion at the injection site, followed by rapid systemic metabolism and excretion), pharmacokinetic studies were not conducted. In those studies, the efficacy and toxicity of BOTOX® (uses HSA as an excipient) and CORETOX® (uses polysorbate 20 as an excipient) were also compared. In addition, since polysorbate 20 is an inactive ingredient, it is considered that efficacy, pharmacokinetics, and pharmacodynamic studies other than safety study are unnecessary when used alone. Therefore, the current study was conducted to obtain in vivo safety data of polysorbate 20. Although polysorbate 20 have already been approved by the FDA for the pharmaceutical excipient, the current study is considered worthwhile given the lack of safety data on intramuscular administration of polysorbate 20 (#1 and #2). It is expected that findings of the current study provides the basis for the in vivo safety profile of polysorbate 20 administered intramuscularly and the scientific reliability of the use of polysorbate 20 as an excipient for various pharmaceuticals in terms of its safety.

-#1: Moore J. Final report on the safety assessment of polysorbates 20, 21, 40, 60, 61, 65, 80, 81, and 85. J Am Coll Toxicol. 1984;3: 1–82.

4. Data table 2 and 3 title are same. Please specify if table 2 belong to polysorbate-20 or for HSA.

- Thank you for your comments. Table 2 shows the level of hematology, clinical biochemistry, and serum IgE and histamine in male SD rats, while Table 3 shows the same values as table 2 in female SD rats.

Attachment

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PLoS One. doi: 10.1371/journal.pone.0256869.r003

Decision Letter 1

Yasmina Abd‐Elhakim

9 Aug 2021

PONE-D-21-03019R1

Safety verification for polysorbate 20, pharmaceutical excipient for intramuscular administration, in Sprague-Dawley rats and New Zealand White rabbits

PLOS ONE

Dear Dr. Kang,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: All queries have been addressed. An interesting work. Some minor language errors. References require a little bit of checking. The comments are mentioned in the attached pdf file.

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

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PLoS One. 2021 Aug 27;16(8):e0256869. doi: 10.1371/journal.pone.0256869.r004

Author response to Decision Letter 1

16 Aug 2021

[We have upload a separate file labeled 'Response to Reviewers'.]

Dear editor and reviewers,

We appreciate the editor and the reviewers to review our manuscript. Our manuscript has been remarkably improved due to their valuable comments. Our responses to the comments are provided below.

Reviewers' comments

[Reviewer #1]

- Thank you for your feedback. We have provided our point-by-point responses to each comment below.

Q1. Abstract does not contain keywords?

- We have already submitted keywords within the submission system (Botulinum toxin type A products; Pharmaceutical excipient; Polysorbate 20; Human serum albumin). However, we have added keywords in the revised manuscript.

- Original version: N/A

- Revised version: [Line 52-53] Keywords: Botulinum toxin type A products, Pharmaceutical excipient, Polysorbate 20, Human serum albumin

Q2. Line 57: 19 superscript th

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

- Original version: [Line 57] Therefore, HSA has been used for therapeutic purposes in patients with hemorrhage, hypovolemia, and hypoalbuminemia since about the 19th century [2].

- Revised version: [Line 60] Therefore, HSA has been used for therapeutic purposes in patients with hemorrhage, hypovolemia, and hypoalbuminemia since about the 19th century [2].

Q3. Line 126-127 (Table 1): Number of animals (male/female)

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

- Original version: [Table 1] No. animals (Male/Female)

- Revised version: [Table 1] Number of animals (male/female)

Q4. Line 131: Which

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

- Original version: [Line 131] HSA (Green Cross Corp., Republic of Korea) was selected as the comparative substance for the polysorbate 20 (Merck, Germany) that was the test substance of the current study.

- Revised version: [Line 135] HSA (Green Cross Corp., Republic of Korea) was selected as the comparative substance for the polysorbate 20 (Merck, Germany) which was the test substance of the current study.

Q5. Line 137-139: Delete ‘Then’ and ‘manner as when determining the dose of HSA’. Join this sentence (As a result, 0.02, 0.1, and 0.4 mg/kg were selected as the dose of polysorbate 20.) with the earlier sentence.

- Thank you for comments. We have modified the text accordingly in the revised manuscript.

- Original version: [Line 137-139] Then, the dose of polysorbate 20 was also calculated in the same manner as when determining the dose of HSA. As a result, 0.02, 0.1, and 0.4 mg/kg were selected as the dose of polysorbate 20.

- Revised version: [Line 141-142] The dose of polysorbate 20 was also calculated in the same manner and 0.02, 0.1, and 0.4 mg/kg were selected as the dose of polysorbate 20.

Q6. Line 189, 206: Delete ‘Then’. Please do not start a sentence with then. Kindly correct in other places also.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

- Original version: [Line 41] Then, an intradermal irritation study was further conducted with 18 New Zealand White (NZW) rabbits.

[Line 111] Then, animals were divided into six groups with 11 animals (six male and five female SD rats) in each group.

[Line 122] Then, animals were divided into six groups with 3 male NZW rabbits in each group.

[Line 160] Then, all substances were injected once into five sites (three left side and two right side) on the back skin (total 1 time; day 1) based on the guideline of the International Organization for Standardization (ISO) [24].

[Line 178] Then, the SD rats were anesthetized with isoflurane and blood samples were collected from their abdominal vein.

[Line 189] Then, the following parameters were examined in KTR using an automatic analyzer (TBA-120FR, TOSHIBA, Japan):

[Line 206] Then, the injection site (one site of the gastrocnemius muscle of the right hindlimb for the SD rats and the five sites of the back skin for the NZW rabbits) were collected and stored in 10% neutral buffered formalin.

- Revised version: [Line 41] An intradermal irritation study was further conducted with 18 New Zealand White (NZW) rabbits.

[Line 115] Animals were divided into six groups with 11 animals (six male and five female SD rats) in each group.

[Line 125] Animals were divided into six groups with 3 male NZW rabbits in each group.

[Line 165] All substances were then injected once into five sites (three left side and two right side) on the back skin (total 1 time; day 1) based on the guideline of the International Organization for Standardization (ISO) [24].

[Line 183] The SD rats were anesthetized with isoflurane and blood samples were collected from their abdominal vein.

[Line 194] The following parameters were then examined in KTR using an automatic analyzer (TBA-120FR, TOSHIBA, Japan):

[Line 211] The injection site (one site of the gastrocnemius muscle of the right hindlimb for the SD rats and the five sites of the back skin for the NZW rabbits) were collected and stored in 10% neutral buffered formalin.

Q7. Line 213: Please mention that data are represented as mean ± standard error of mean (SEM).

- Thank you for comments. We have modified the text accordingly in the revised manuscript.

- Original version: NA

- Revised version: [Line 227-228] Data are represented as mean ± standard error of the mean (SEM).

Q8. Line 232: Delete ‘Changes in’.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

- Original version: [Line 232] Changes in body weights

- Revised version: [Line 238] Body weights

Q9. Line 235 and 264: In a sentence wherever the word table comes please make the ‘t’ small. While mentioning as a caption or a table then the ‘t’ can be capitalised. Kindly make these corrections in other locations as well.

- Thank you for comments. According to the PLOS ONE's submission guidelines, the following is stated in relation to tables:

- [Table citations] Tables should be cited as “Table 1”, “Table 2”, etc.

- [Examples] Vestibulum adipiscing urna ut lectus gravida, et bland Table 1.

- Therefore, we have kept the existing table citation form.

Q10. Line 237: Is it Fig. 1a or Fig 1a? Please kindly refer the journal guidelines once.

- Thank you for comments. According to the PLOS ONE's submission guidelines, the following is stated in relation to the figures:

- [Figure citations] Cite figures as “Fig 1”, “Fig 2”, etc.

- [Examples] Vestibulum adipiscing urna ut lectus gravida, vitae (Fig 1) interdum. Nam sit amet nulla lacus a, Figs 1 and 2 ultrices tellus.

- Therefore, we have kept the existing figure citation form.

Q11. Line 239, 241: “respectively” is inappropriate here, since you have already mentioned the weight immediately against the day. If days were mentioned consecutively followed by weights consecutively, then respectively may be used.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

- Original version: [Line 237-241] Compared to the body weight before the first administration, all male SD rats had a body weight of 138.4% at day 8, 173.6% at day 15, 200.4% at day 22, and 225.0% at day 29, respectively, while all female SD rats had a body weight of 132.2% at day 8, 153.1% at day 15, 173.1% at day 22, and 187.3% at day 29, respectively.

- Revised version: [Line 243-246] Compared to the body weight before the first administration, all male SD rats had a body weight of 138.4% at day 8, 173.6% at day 15, 200.4% at day 22, and 225.0% at day 29, while all female SD rats had a body weight of 132.2% at day 8, 153.1% at day 15, 173.1% at day 22, and 187.3% at day 29.

Q12. Line 261-262: Delete “Findings of”. Delete “the level of” (hematology is a study not level). Change “serum IgE and histamine” to “serum IgE and histamine levels”.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

- Original version: [Line 261-262] Findings of the level of hematology, clinical biochemistry, and serum IgE and histamine

- Revised version: [Line 267-268] Hematology, clinical biochemistry, and serum IgE and histamine levels

Q13. Line 266-272: Delete “was”.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

- Original version: [Line 266-272] In clinical biochemistry, compared to the control group, the albumin/globulin ratio was significantly decreased in male group 6 (p < 0.05), the level of glucose was significantly decreased in male group 3, 5, and 6 (p < 0.05, respectively), and the level of sodium was significantly increased in male group 4 (p < 0.01) and 6 (p < 0.05) (Table 2).

In females, the level of total bilirubin was significantly decreased in group 2 and 4 (p < 0.05, respectively) and the level of chloride was significantly increased in group 2 (p < 0.05) and 3 (p < 0.01), compared to the control group (Table 3).

- Revised version: [Line 271-277] In clinical biochemistry, compared to the control group, the albumin/globulin ratio significantly decreased in male group 6 (p < 0.05), the level of glucose significantly decreased in male group 3, 5, and 6 (p < 0.05, respectively), and the level of sodium significantly increased in male group 4 (p < 0.01) and 6 (p < 0.05) (Table 2).

In females, the level of total bilirubin significantly decreased in group 2 and 4 (p < 0.05, respectively) and the level of chloride significantly increased in group 2 (p < 0.05) and 3 (p < 0.01), compared to the control group (Table 3).

Q14. Line 277: Change “increase or decrease” to “change”.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

- Original version: [Line 276-278] There was also no significant increase or decrease in the level of serum IgE and histamine in NZW rabbits administered polysorbate 20 and HSA compared to the control group.

- Revised version: [Line 282-284] There was also no significant change in the level of serum IgE and histamine in NZW rabbits administered polysorbate 20 and HSA compared to the control group.

Q15. Line 279 and 284: Delete “The level of”, and “in the current study”. Change “IgE and histamine” to “IgE and histamine levels”.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

- Original version: [Line 279] Table 2. The level of hematology, clinical biochemistry, and serum IgE and histamine in male Sprague-Dawley (SD) rats in the current study.

[Line 284-285] Table 3. The level of hematology, clinical biochemistry, and serum IgE and histamine in female Sprague-Dawley (SD) rats in the current study.

- Revised version: [Line 285] Table 2. Hematology, clinical biochemistry, and serum IgE and histamine levels in male Sprague-Dawley (SD) rats.

[Line 290] Table 3. Hematology, clinical biochemistry, and serum IgE and histamine levels in female Sprague-Dawley (SD) rats.

Q16. Line 280-282 and 286-288: Change “Data were presented as mean ± standard error of the mean” to “Data ae presented as mean ± SEM”. Delete “All statistical analyses were performed using GraphPad Prism 282 8.0 (GraphPad Software, Inc., San Diego, CA)”

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

- Original version: [Line 280-282] Data were presented as mean ± standard error of the mean. All data were analyzed using Kruskal-Wallis test and the significance of intergroup difference between the control and administered groups was assessed using Dunn's Rank Sum test. All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, CA).

[Line 286-288] Data were presented as mean ± standard error of the mean. All data were analyzed using Kruskal-Wallis test and the significance of intergroup difference between the control and administered groups was assessed using Dunn's Rank Sum test. All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, CA).

- Revised version: [Line 286-288] Data are presented as mean ± SEM. All data were analyzed using Kruskal-Wallis test and the significance of intergroup difference between the control and administered groups was assessed using Dunn's Rank Sum test. [deleted]

[Line 291-293] Data are presented as mean ± SEM. All data were analyzed using Kruskal-Wallis test and the significance of intergroup difference between the control and administered groups was assessed using Dunn's Rank Sum test. [deleted]

Q17. Line 290: Delete “Findings of”.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

- Original version: [Line 290] Findings of necropsy and histopathology

- Revised version: [Line 295] Necropsy and histopathology

Q18. Line 371: Change “those” to “these”.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

- Original version: [Line 371-372] Therefore, those findings were presumed to be due to the intramuscular injection rather than any effect of polysorbate 20.

- Revised version: [Line 376] Therefore, these findings were presumed to be due to the intramuscular injection rather than any effect of polysorbate 20.

Q19. Line 381: Just 2% will be better instead of 2.0%.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

- Original version: [Line 379-382] However, despite its very low frequency, side effects in the allergy category have been reported in humans using shampoos (8.4% polysorbate 20, 2 cases among 5.88 million uses), cuticle softeners (2.0% polysorbate 20, 24 cases among 131 million use), and paste masks (2.0% polysorbate 20, 11 cases among 120.7 million uses) containing polysorbate 20 [14,15].

- Revised version: [Line 384-387] However, despite its very low frequency, side effects in the allergy category have been reported in humans using shampoos (8.4% polysorbate 20, 2 cases among 5.88 million uses), cuticle softeners (2% polysorbate 20, 24 cases among 131 million use), and paste masks (2% polysorbate 20, 11 cases among 120.7 million uses) containing polysorbate 20 [14,15].

Q20. Line 393-396: Change “hematology and clinical biochemistry” to “hematology and clinical biochemistry parameters”. Delete “of the NZW rabbits”.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

- Original version: [Line 393-396] There were also no toxicologically significant changes, such as changes in the body weight or the hematology and clinical biochemistry in all rabbits administered polysorbate 20 and all toxicological values in NZW rabbits were within the normal range for the same age of the NZW rabbits [41,42].

- Revised version: [Line 398-401] There were also no toxicologically significant changes, such as changes in the body weight or the hematology and clinical biochemistry parameters in all rabbits administered polysorbate 20 and all toxicological values in NZW rabbits were within the normal range for the same age [41,42].

Q21. Line 397: Change “was” to “is”.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

- Original version: [Line 396-398] These results of the intradermal irritation study indicated that polysorbate 20 was not an allergen in NZW rabbits, which was consistent with the results of the four-week repeated dose toxicity study in SD rats.

- Revised version: [Line 401-403] These results of the intradermal irritation study indicated that polysorbate 20 is not an allergen in NZW rabbits, which was consistent with the results of the four-week repeated dose toxicity study in SD rats.

Q22. Line 415: Authors maybe can wish to thank the institutions the works was carried out and the people who assisted in the work, if they wish to. Not applicable is not the appropriate term.

- Thank you for your comments. We have added thanks to colleagues who helped with the works.

- Original version: [Line 415] Not applicable

- Revised version: [Line 420-421] The authors thanks Do Yeon Lee and Min-Seo Choi, colleagues at the Medytox, Inc., for their helpful discussion and careful reading of the manuscript.

Q23. Line 466: Please check the journal abbreviation.

- Thank you for pointing this out. We have modified the text accordingly in the revised manuscript.

- Original version: [Line 464-466] Diehl K, Hull R, Morton D, Pfister R, Rabemampianina Y, Smith D, et al. A good practice guide to the administration of substances and removal of blood, including routes and volumes. J Appl Toxicol An Int J. 2001;21: 15–23.

- Revised version: [Line 470-472] Diehl K, Hull R, Morton D, Pfister R, Rabemampianina Y, Smith D, et al. A good practice guide to the administration of substances and removal of blood, including routes and volumes. J Appl Toxicol. 2001;21: 15–23.

Q24. Line 490 and 519: Please check the journal abbreviation (use standard abbreviations and check LTWA/ISSN website).

- Thank you for your comments. According to the PLOS ONE's submission guidelines, the following is stated in relation to the reference format:

- “PLOS uses the reference style outlined by the International Committee of Medical Journal Editors (ICMJE). Journal name abbreviations should be those found in the National Center for Biotechnology Information (NCBI) databases.”

- We have checked journal name abbreviations (NCBI database) and kept the existing reference citation form.

[Reviewer #2]

- Thank you for the encouraging feedback, including previous reviews.

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PLoS One. doi: 10.1371/journal.pone.0256869.r005

Decision Letter 2

Yasmina Abd‐Elhakim

18 Aug 2021

Safety verification for polysorbate 20, pharmaceutical excipient for intramuscular administration, in Sprague-Dawley rats and New Zealand White rabbits

PONE-D-21-03019R2

Dear Dr. Kang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Yasmina Abd‐Elhakim

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0256869.r006

Acceptance letter

Yasmina Abd‐Elhakim

20 Aug 2021

PONE-D-21-03019R2

Safety verification for polysorbate 20, pharmaceutical excipient for intramuscular administration, in Sprague-Dawley rats and New Zealand White rabbits

Dear Dr. Kang:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

Dr. Yasmina Abd‐Elhakim

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Individual body weights of male (n: 36) and female (n: 30) Sprague-Dawley (SD) rats in the current study.

(XLSX)

Click here for additional data file.^{(15KB, xlsx)}

S2 Table. Individual body weights of male New Zealand White (NZW) rabbits (n: 18) in the current study.

(XLSX)

Click here for additional data file.^{(11.3KB, xlsx)}

S3 Table. Individual levels of hematology, clinical biochemistry, and serum IgE and histamine in Sprague-Dawley (SD) rats in the current study.

(XLSX)

Click here for additional data file.^{(35.2KB, xlsx)}

S4 Table. Individual level of hematology, clinical biochemistry, and serum IgE and histamine in New Zealand White (NZW) rabbits in the current study.

(XLSX)

Click here for additional data file.^{(19.5KB, xlsx)}

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Submitted filename: Review PLONE.pdf

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Submitted filename: Response to Reviewers.docx

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Attachment

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Data Availability Statement

All relevant data are within the manuscript and its Supporting information files.

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PERMALINK

Safety verification for polysorbate 20, pharmaceutical excipient for intramuscular administration, in Sprague-Dawley rats and New Zealand White rabbits

Junhyung Kim

Seongsung Kwak

Mi-Sun Park

Chang-Hoon Rhee

Gi-Hyeok Yang

Jangmi Lee

Woo-Chan Son

Won-ho Kang

Roles

Abstract

Introduction

Materials and methods

Animals and facility

Table 1. Study design of four-week repeated intramuscular dose toxicity study in Sprague-Dawley rats and intradermal irritation study in New Zealand white rabbits.

Vehicle, positive control substance, and test substance

In vivo administration

Clinical observation and body weights measurement

Hematology, clinical biochemistry, and allergic reaction test

Necropsy and histopathology

Statistical analysis

Results

Mortality and clinical signs

Body weights

Fig 1. Body weight changes in a four-week repeated intramuscular dose toxicity study in Sprague-Dawley (SD) rats and an intradermal irritation study in New Zealand White (NZW) rabbits.

Hematology, clinical biochemistry, and serum IgE and histamine levels

Table 2. Hematology, clinical biochemistry, and serum IgE and histamine levels in male Sprague-Dawley (SD) rats.

Table 3. Hematology, clinical biochemistry, and serum IgE and histamine levels in female Sprague-Dawley (SD) rats.

Necropsy and histopathology

Fig 2. Histopathological section of the administration site in the four-week repeated intramuscular dose toxicity study in Sprague-Dawley (SD) rats.

Fig 3. Histopathological section of the administration site in the intradermal irritation study in New Zealand White (NZW) rabbits.

Discussion

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Arun K Yadav

Roles

Author response to Decision Letter 0

Decision Letter 1

Yasmina Abd‐Elhakim

Roles

Author response to Decision Letter 1

Decision Letter 2

Yasmina Abd‐Elhakim

Roles

Acceptance letter

Yasmina Abd‐Elhakim

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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