Abstract
1-(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione (MDM hydantoin) is a commonly used antiseptic preservative in cosmetics. However, limited toxicity information data are available for this chemical. The aim of this study was to obtain toxicity data for MDM hydantoin through single- and repeated-dose toxicity studies in Sprague–Dawley (SD) rats. In the single-dose toxicity study, MDM hydantoin was administered once orally to SD rats at four doses (5, 50, 300, and 2000 mg/kg/day). There was no significant difference in mortality, clinical signs, and body weight change for 14 days among the animals treated with the different doses in this study. Hence, the approximate lethal dose of MDM hydantoin was considered higher than 2000 mg/kg/day. Based on the results of the dose-range finding study, a 28-day repeated-dose oral toxicity study was conducted. MDM hydantoin was administered orally to SD rats at doses of 125, 250, 500, and 1000 mg/kg/day throughout an experimental period of 28 days. In the repeated-dose oral toxicity study, the adverse effects caused by MDM hydantoin were not detected in terms of body weight, clinical signs, food and water intake, hematology, organ weights, gross pathology, and histopathology. Therefore, the no-observed-adverse-effect level of MDM hydantoin was considered to be greater than 1000 mg/kg/day.
Keywords: MDM hydantoin, Repeated-dose 28-day oral toxicity study, Adverse effect, NOAEL
Introduction
1-(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione (MDM hydantoin) is a formaldehyde releaser (FAR) used as a preservative at concentrations up to 0.2% in cosmetic products [1]. Its high reactivity makes it suitable for use as a biocide or a preservative agent (e.g., in cosmetics) [2].
The International Agency for Research on Cancer (IARC) classifies formaldehyde as carcinogenic to humans, based on evidence in humans and animals studies [3]. Many of the FAR compounds used in cosmetics are added as preservatives including DMDM-hydantoin; quaternium-15; imidazolidinyl urea; diazolidinyl urea; and benzalkonium chloride [4, 5]. Because preservatives are widely added to prevent bacterial and fungal contamination.
Numerous studies have reported the various toxicity of preservative through in vitro analyses, experiments with animal. Cahill J et al. studied Q15 renders major damage to the skin such as allergic contact dermatitis [6]. Benzalkonium chloride is used as an active ingredient in many consumer and household products. Administration of topical drugs containing benzalkonium chloride can lead to much higher prevalence of disorders [7].
Moreover, although several studies on the toxicity of well-known preservatives [4–7], including the oral toxicity study, the dermal toxicity study and eye irritant study, have been reported, the potential toxicity of the other preservative compounds has not been investigated. A number of previous studies have focused on the toxicity of 1,3-Bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione (DMDM hydantoin) when used for various purposes such as in cosmetics, as a preservative, and other industrial uses, with regard to allergic contact dermatitis [4, 8, 9]. The composition of DMDM hydantoin and MDM hydantoin as determined by gas chromatography is 94–98% DMDM hydantoin (two molecules of formaldehyde) and 2.5–3.0% MDM hydantoin (one molecule formaldehyde; monomethylol dimethyl hydantoin), respectively. However, there are no scientific reports on MDM hydantoin, and its safety profile remains to be established. In particular, information regarding the oral toxicity of MDM hydantoin when administered singly and in repeated doses is insufficient.
Rats have been found to be sensitive to the toxic effects of a variety of drugs and chemicals. Rats are also considered a standard animal model for repeated-dose toxicology studies as per regulatory requirements. Hence, in this study, we aimed to evaluate the single- and repeated-dose toxicity and safety of MDM hydantoin in Sprague‐Dawley (SD) rats. To this end, a 7-day dose-range finding (DRF) study was performed primarily to determine the dosage range for a subsequent 28-day study. In the 28-day repeated-dose oral administration study in SD rats, we aimed to elucidate the toxic effects of MDM hydantoin and determine its no-observed-adverse-effect level (NOAEL) so as to provide toxicological data for assessing the safety of MDM hydantoin.
The present paper is part of documentation supporting a clinical trial, including toxicity studies. We believe that by producing toxicity data and providing toxicity information through this study, we have laid the ground for a scientific database for regulating public safety management.
Materials and methods
Test substance
MDM hydantoin (1-hydroxymethyl-5,5-dimethyl hydantoin; C6H10N2O3, molecular weight 158.16 gmol, CAS: 116–25-6, purity 98%) was obtained from Leapchem (Hangzhou, China) and used as received unless otherwise noted. MDM hydantoin for oral administration or treatment was formulated in sterile distilled water.
Experimental animals and husbandry
Specific pathogen‐free Sprague‐Dawley rats (Crl:CD (SD)) were used for general toxicity studies. The animals were acclimated for 7 days after arrival at the laboratory animal facility of the National Institute of Food and Drug Safety Evaluation of the Ministry of Food and Drug Safety (Osong, Korea). The studies were approved by the Institutional Animal Care and Use Committee (IACUC) (2018, Approval NO. MFDS-18–127). The animals were housed at a temperature of 22 ± 3 °C and relative humidity of 50% ± 20%. The animal rooms were maintained under a 12‐h light‐dark cycle, and 10‐20 air changes per hour. The animals were allowed access to feed (LabDiet 5002; PMI Nutrition, Richmond, VA, USA) and water (autoclaved water) ad libitum.
Each animal was dosed by oral gavage. For all animals, the dose was administered daily (7 days/week) for a period of 28 days. The dose mixtures were maintained on a magnetic stir plate during dose administration. All doses were administered volumetrically at 10 mL/kg. The control group received the vehicle only at the same dose volume as the test animals. The first day of administration was considered Day 1 of the study. Dosing was carried out at approximately the same time each day (± 2 h). Residual dose preparations were properly discarded following daily administration and sampling.
Single-dose oral toxicity study
Six‐week‐old male and female SD rats (n = 5 per sex and group) were orally treated with MDM hydantoin at doses of 0, 5, 50, 300, and 2,000 mg/kg. The study was performed according to “Toxicity Test Standards for Drugs” from the Korean Ministry of Food and Drug Safety [10]. Animals were housed in stainless steel cages. No more than three animals were housed per cage during the quarantine and acclimation periods, and the animals were individually housed during the dosing and observation periods. The animals were constantly monitored for clinical signs and mortality for the first 30 min, and then every hour until 6 h after the oral treatment and daily for 14 days subsequently. During the 15‐day experimental period, the body weight of all rats was recorded, and gross findings were observed at necropsy.
28-day repeated-dose oral toxicity study
We performed 7-day repeated-dose oral toxicity studies as DRF studies for the 28-day repeated-dose oral study. Briefly, the DRF studies were conducted at doses of 0, 100, 200, 500, 1000, and 2000 mg/kg. Individual dosing volumes were calculated based on 10 mL/kg body weight. During the 7‐day experimental period, the body weight of all rats was recorded, and gross findings were observed at necropsy. There were significant differences in body weight between the vehicle and 2000 mg/kg MDM hydantoin-treated male groups (Fig. 1). No other clinical signs of toxicity or mortality related to MDM hydantoin administration were observed in the rats during the 7-day experimental period at any given dose. The 28-day repeated oral toxicity study was conducted based on the DRF study results.
Fig. 1.
Effect of MDM hydantoin oral administration on body weight in male and female rats. a Single-dose oral toxicity. b 7-day dose oral toxicity. c 28-day dose oral toxicity. *Significantly different from the vehicle group (p < 0.05). Data are expressed as the mean ± SD values
The 28-day repeated-dose oral toxicity study was performed according to “Toxicity Test Standards for Drugs” from the Korean Ministry of Food and Drug Safety [10]. In this study, 6‐week-old male and female SD rats (n = 5–8 per sex and group) were orally administered with MDM hydantoin at doses of 0, 125, 250, 500, and 1000 mg/kg/day for 28 days. Five to eight animals of each sex were added to the vehicle group and the high‐dose (1000 mg/kg/day) group to evaluate the recovery potential. The body weight ranges at the initiation of dosing were 173.95–212.19 g for males and 115.82–142.31 g for females.
Clinical signs and body weights
The animals were checked once a day to observe any clinical sign and mortality, and the type, date of occurrence, and severity of signs were recorded individually. The body weights of all rats were recorded before the initiation of dosing (day 1) and once a week during the experimental period. Before necropsy, all rats were fasted overnight, and the body weight of all rats was recorded at necropsy. Food and water intake was checked on the same days as the body weight measurements were recorded.
Hematology and clinical chemistry
At necropsy, the animals were anesthetized by CO2 inhalation. Blood samples were collected from the abdominal aorta for hematological and serum biochemical testing. Approximately 1 mL of blood was placed in a CBC bottle (Vacutainer 3 mL; BD) with anticoagulant EDTA‐2 K. The following hematology parameters were measured using a Coulter counter (ADVIA 2120; Siemens): white blood cell (WBC), red blood cell (RBC), platelet (PLT), neutrophil (NEUT), lymphocyte (LYM), monocyte (MONO), eosinophil (EOS), basophil (BASO), and reticulocyte (Retic) counts; hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).
Serum biochemical parameters were measured using a serum biochemistry analyzer (Konelab 20; Thermo Scientific). Approximately 2 mL of the blood sample was added into a 5 mL Vacutainer tube (SST™ II Advance; BD) containing a clot activator. The blood was coagulated by maintaining it at room temperature (22 ± 3 °C) for 15‐20 min and then centrifuged for 10 min (3000 rpm, Large capacity Table-top centrifuge, UNION 32R; Hanil) to collect the serum sample. The parameters examined were alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase(ALP), gamma-glutamyl transferase (GGT), blood urea nitrogen (BUN), creatinine (CREA), total protein (TP), albumin (ALB), cholesterol (T-CHOL), glucose (GLU), triglyceride (TG), total bilirubin (T-BIL), direct bilirubin (D-BIL), lactate dehydrogenase (LDH), creatine kinase (CK), uric acid (UA), calcium (CA), phosphorus (IP), high-density lipoprotein (HDL), and low-density lipoprotein (LDL).
Necropsy, organ weight, and histopathology
After the blood sampling, the animals were killed by exsanguination from the abdominal aorta. Gross findings including the body surface, subcutis, head, and all organs in the abdominal and thoracic cavities were recorded. Thereafter, the following organs were dissected: liver, spleen, heart, kidney (both), adrenal gland, lung, brain, pituitary gland, thymus, urinary bladder, stomach, intestine, testis, ovary, epididymis, uterus, prostate, seminal vesicle, trachea, esophagus, thyroid gland, salivary gland, skin, femur, and Harderian gland. Then, the following organs were weighed using an electronic balance (Sartorius AG): liver, spleen, heart, kidney (both), adrenal gland, lung, brain, pituitary gland, thymus, testis, prostate, seminal vesicle, thyroid gland, and salivary gland. Organ weights (%) relative to the terminal body weights were also calculated. Tissue slides were prepared from fixed tissues with gross findings of all animals. Sections (5-μm thick) were stained with hematoxylin and eosin and a histopathological examination was performed.
Statistical analysis
All data were analyzed by one-way ANOVA followed by post hoc Dunnett’s multiple comparison test using GraphPad prism. A value of p < 0.05 was considered significant, and data are expressed as mean ± standard of mean (SD) values.
Results
Single-dose oral toxicity
In the single-dose oral toxicity study, single oral administration of MDM hydantoin at doses of 0, 5, 50, 300, and 2000 mg/kg body weight to rats did not result in any mortality. None of the treated rats showed any visible sign of toxicity or behavioral change, and the animals were found to be normal throughout the 14-day study period, similar to the rats in the vehicle group. The mean body weights of the treated and vehicle groups are shown in Fig. 1. There was a gradual increase in the mean body weights of the treated and vehicle groups during the study period. There was no statistical difference in the mean body weights between the treated and vehicle groups from the onset up to the end of the study period (p > 0.05). At the end of the study period, the percentage body weight gain at doses of 5, 50, 300, and 2000 mg/kg body weight in the treated group was similar (p > 0.05) to that in the vehicle group.
Clinical signs and body weights
No clinical signs of toxicity or mortality related to the MDM hydantoin administration at any dose were observed in the rats during the 28-day study. There were no significant changes in body weight of rats of both sexes between the vehicle control and MDM hydantoin-treated groups (Fig. 1). There were no mortalities on single-, 7-day or 28-day repeated oral dose toxicity test. Furthermore, there were no significant changes in food and water intake of rats of both sexes between the vehicle and MDM hydantoin-treated groups (Fig. 2).
Fig. 2.
Effect of MDM hydantoin oral administration on weekly food and water consumption in male and female rats for 28 days. a Weekly food consumption. b Weekly water consumption. Data are expressed as the mean ± SD values
Hematology and clinical chemistry
Hematology and clinical biochemistry parameters determined at the end of the 28-day period among rats treated with MDM hydantoin doses of 125, 250, 500, and 1000 mg/kg body weight compared to those among the rats in the vehicle group rats are presented in Tables 1, 2, and 3. The hematology parameters of both sexes were similar to those of the vehicle group, with the exception of NEUT and MONO counts in the male rats in the MDM hydantoin-treated groups (p < 0.05). The NEUT count was significantly higher in the MDM hydantoin-treated male group (1000 mg/kg) than in the male rats in the vehicle group (p < 0.05). MONO count significantly decreased in all the treated male groups (p < 0.05).
Table 2.
Clinical biochemistry parameters of MDM hydantoin-treated male rats
| Parameter | Groups (mg/kg/day) | ||||
|---|---|---|---|---|---|
| 0 | 125 | 250 | 500 | 1000 | |
| ALT (U/L) | 49.60 ± 7.50 | 45.71 ± 5.35 | 48.50 ± 5.47 | 47.63 ± 4.03 | 55.43 ± 12.18 |
| AST (U/L) | 147.00 ± 13.51 | 131.14 ± 23.80 | 143.00 ± 14.71 | 121.88 ± 18.53 | 127.71 ± 26.06 |
| ALP (U/L) | 175.00 ± 29.21 | 156.71 ± 12.30 | 179.00 ± 20.82 | 164.25 ± 20.94 | 145.29 ± 30.28 |
| GGT (U/L) | 0.80 ± 0.84 | 1.67 ± 0.52 | 1.20 ± 0.45 | 1.13 ± 0.99 | 1.00 ± 1.15 |
| BUN (mg/dL) | 22.06 ± 2.24 | 24.41 ± 4.13 | 22.45 ± 1.89 | 23.51 ± 1.86 | 22.06 ± 3.04 |
| CREA (mg/dL) | 0.62 ± 0.04 | 0.63 ± 0.05 | 0.65 ± 0.05 | 0.64 ± 0.05 | 0.64 ± 0.05 |
| TP (g/L) | 6.40 ± 0.55 | 6.14 ± 1.07 | 7.00 ± 0.00 | 6.75 ± 0.46 | 6.14 ± 0.38 |
| ALB (g/dL) | 3.52 ± 0.04 | 3.46 ± 0.44 | 3.60 ± 0.06 | 3.66 ± 0.07 | 3.46 ± 0.18 |
| T-CHO (mg/dL) | 99.26 ± 30.38 | 83.66 ± 9.94 | 103.72 ± 18.14 | 105.71 ± 11.43 | 96.39 ± 13.79 |
| GLU (mg/dL) | 161.00 ± 33.86 | 175.43 ± 43.53 | 165.35 ± 27.41 | 165.23 ± 69.12 | 138.21 ± 22.27 |
| TG (mmol/L) | 66.61 ± 11.93 | 61.43 ± 8.62 | 54.62 ± 11.47 | 53.14 ± 14.78 | 63.36 ± 15.09 |
| T-BIL (mg/dL) | 0.20 ± 0.00 | 0.19 ± 0.04 | 0.20 ± 0.00 | 0.19 ± 0.04 | 0.20 ± 0.00 |
| D-BIL (mg/dL) | 0.10 ± 0.00 | 0.10 ± 0.00 | 0.10 ± 0.00 | 0.10 ± 0.00 | 0.10 ± 0.00 |
| LDH (U/L) | 2248.80 ± 301.47 | 2603.57 ± 808.31 | 1627.67 ± 452.22 | 2041.63 ± 682.78 | 1869.86 ± 313.64 |
| CK (U/L) | 611.87 ± 110.18 | 587.29 ± 137.18 | 638.25 ± 248.85 | 421.16 ± 129.67 | 448.57 ± 107.56 |
| UA (mg/dL) | 1.64 ± 0.43 | 1.66 ± 0.28 | 1.67 ± 0.28 | 1.73 ± 0.22 | 1.83 ± 0.45 |
| CA (mg/dL) | 11.08 ± 0.57 | 11.03 ± 1.05 | 11.34 ± 0.23 | 11.57 ± 0.32 | 10.82 ± 0.64 |
| IP (mg/dL) | 10.37 ± 0.47 | 10.02 ± 1.12 | 10.60 ± 0.36 | 10.07 ± 0.53 | 9.53 ± 1.11 |
| HDL (mg/dL) | 76.91 ± 20.11 | 69.39 ± 8.77 | 85.95 ± 15.75 | 87.15 ± 9.52 | 74.61 ± 6.87 |
| LDL (mg/dL) | 23.07 ± 10.86 | 16.58 ± 5.16 | 26.27 ± 5.88 | 24.67 ± 4.10 | 25.24 ± 8.24 |
Data are expressed as mean ± SD values (n = 5–8/group)
ALT alanine aminotransferase, AST aspartate aminotransferase, ALP alkaline phosphatase, GGT gamma-glutamyl transferase, BUN blood urea nitrogen, CREA creatinine, TP total protein, ALB albumin, T-CHOL cholesterol, GLU glucose, TG triglyceride, T-BIL total bilirubin, D-BIL direct bilirubin, LDH lactate dehydrogenase, CK creatine kinase, UA uric acid, CA calcium, IP phosphorus, HDL high-density lipoprotein, LDL low-density lipoprotein
*Significantly different from the vehicle group (p < 0.05). Statistics: One-way analysis of variance (ANOVA) followed by the Dunnett's test
Table 3.
Clinical biochemistry parameters of MDM hydantoin-treated female rats
| Parameter | Groups (mg/kg/day) | ||||
|---|---|---|---|---|---|
| 0 | 125 | 250 | 500 | 1000 | |
| ALT (U/l) | 40.60 ± 7.89 | 42.00 ± 4.28 | 45.43 ± 5.06 | 39.63 ± 5.04 | 48.88 ± 8.49 |
| AST (U/l) | 149.40 ± 15.13 | 151.43 ± 19.07 | 143.00 ± 11.78 | 135.00 ± 21.01 | 158.13 ± 28.16 |
| ALP (U/l) | 113.00 ± 15.81 | 122.43 ± 15.82 | 123.57 ± 17.39 | 119.63 ± 22.14 | 131.75 ± 21.87 |
| GGT (U/l) | 3.60 ± 1.34 | 3.14 ± 1.35 | 4.00 ± 0.58 | 3.00 ± 1.07 | 4.25 ± 1.04 |
| BUN (mg/dl) | 18.48 ± 3.38 | 20.63 ± 1.46 | 22.64 ± 2.71 | 21.50 ± 2.87 | 25.21 ± 4.65 |
| CREA (mg/dl) | 0.62 ± 0.04 | 0.69 ± 0.04 | 0.71 ± 0.04 | 0.66 ± 0.05 | 0.76 ± 0.05 |
| TP (g/l) | 5.80 ± 0.45 | 6.00 ± 0.00 | 6.14 ± 0.38 | 6.75 ± 0.46* | 6.63 ± 0.52* |
| ALB (g/d) | 3.36 ± 0.11 | 3.46 ± 0.05 | 3.66 ± 0.21* | 3.74 ± 0.13* | 3.73 ± 0.16* |
| T-CHO (mg/dl) | 104.74 ± 13.32 | 103.84 ± 14.21 | 121.50 ± 16.21 | 115.05 ± 16.09 | 121.05 ± 18.88 |
| GLU (mg/dl) | 107.66 ± 14.26 | 103.17 ± 7.43 | 104.39 ± 17.06 | 133.16 ± 16.88* | 135.86 ± 20.35* |
| TG (mmol/l) | 48.66 ± 20.13 | 57.74 ± 40.56 | 71.07 ± 53.50 | 55.96 ± 18.12 | 65.27 ± 27.93 |
| T-BIL (mg/dl) | 0.18 ± 0.04 | 0.17 ± 0.05 | 0.17 ± 0.05 | 0.19 ± 0.04 | 0.19 ± 0.04 |
| D-BIL (mg/dl) | 0.16 ± 0.13 | 0.11 ± 0.04 | 0.10 ± 0.00 | 0.10 ± 0.00 | 0.10 ± 0.00 |
| LDH (U/l) | 1056.80 ± 673.80 | 2383.71 ± 727.19* | 1716.57 ± 866.28 | 2207.88 ± 483.21* | 1922.25 ± 307.61 |
| CK (U/l) | 753.76 ± 139.83 | 482.55 ± 74.26* | 517.98 ± 104.90 | 585.97 ± 211.31 | 587.41 ± 204.15 |
| UA (mg/dl) | 1.82 ± 0.16 | 1.66 ± 0.30 | 1.79 ± 0.33 | 1.86 ± 0.47 | 1.91 ± 0.26 |
| CA (mg/dl) | 9.67 ± 0.17 | 9.88 ± 0.26 | 10.40 ± 0.24 | 11.04 ± 0.91* | 12.13 ± 1.08* |
| IP (mg/dl) | 9.59 ± 0.68 | 9.41 ± 0.89 | 10.15 ± 0.94 | 9.02 ± 1.49 | 10.56 ± 1.09 |
| HDL (mg/dl) | 90.12 ± 12.14 | 87.63 ± 10.45 | 98.65 ± 11.82 | 95.57 ± 12.09 | 99.15 ± 14.32 |
| LDL (mg/dl) | 20.37 ± 1.34 | 18.54 ± 4.70 | 24.56 ± 6.10 | 21.17 ± 4.32 | 21.97 ± 5.95 |
Data are expressed as mean ± SD values (n = 5–8/group)
ALT alanine aminotransferase, AST aspartate aminotransferase, ALP alkaline phosphatase, GGT gamma-glutamyl transferase, BUN blood urea nitrogen, CREA creatinine, TP total protein, ALB albumin, T-CHOL cholesterol, GLU glucose, TG triglyceride, T-BIL total bilirubin, D-BIL direct bilirubin, LDH lactate dehydrogenase, CK creatine kinase, UA uric acid, CA calcium, IP phosphorus, HDL high-density lipoprotein, LDL low-density lipoprotein
*Significantly different from the vehicle group (p < 0.05). Statistics: One-way analysis of variance (ANOVA) followed by the Dunnett's test
No significant changes were noted in clinical biochemistry parameters. However, significant differences in TP, ALB, LDH, CK, and CA levels were found among the treated female groups, as shown in Table 3. Because the changes were minimal and there was no evidence of a dose–response relationship, the changes in these parameters were considered random biological variations.
Necropsy, organ weight, and histopathology
The relative organ weights of the dissected organs of the treated and vehicle groups, which were recorded during necropsy, are shown in Tables 4 and 5. According to results, there were no significant differences between the vehicle and MDM hydantoin-treated groups, except in the 1000 mg/kg MDM hydantoin-treated male group (increase in adrenal gland weight), 50 mg/kg MDM hydantoin-treated male group (increase in testis weight), and 500 and 1000 mg/kg MDM hydantoin-treated female groups (increase in adrenal gland weight). Based on the histopathology results, there were no abnormalities attributable to the administration of MDM hydantoin (Fig. 3).
Table 4.
Toxicity of MDM hydantoin on percent relative organ weight in male rats
| Organ (g/100 g body weight) | Groups (mg/kg/day) | ||||
|---|---|---|---|---|---|
| 0 | 125 | 250 | 500 | 1000 | |
| Liver | 2.76 ± 0.22 | 2.71 ± 0.13 | 2.68 ± 0.12 | 2.78 ± 0.15 | 2.85 ± 0.13 |
| Kidney-R | 0.36 ± 0.03 | 0.36 ± 0.02 | 0.36 ± 0.02 | 0.37 ± 0.01 | 0.37 ± 0.02 |
| Kidney-L | 0.35 ± 0.02 | 0.36 ± 0.02 | 0.35 ± 0.02 | 0.38 ± 0.03 | 0.36 ± 0.01 |
| Adrenal gland-R (mg) | 7.18 ± 1.08 | 7.20 ± 0.64 | 7.20 ± 1.07 | 7.92 ± 0.71 | 8.78 ± 1.49* |
| Adrenal gland-L (mg) | 7.54 ± 0.77 | 7.44 ± 0.88 | 7.91 ± 0.61 | 8.30 ± 0.91 | 9.18 ± 1.44* |
| Heart | 0.36 ± 0.02 | 0.36 ± 0.01 | 0.35 ± 0.01 | 0.36 ± 0.03 | 0.36 ± 0.03 |
| Lung | 0.47 ± 0.03 | 0.47 ± 0.02 | 0.45 ± 0.02 | 0.46 ± 0.03 | 0.46 ± 0.03 |
| Brain | 0.53 ± 0.01 | 0.54 ± 0.02 | 0.55 ± 0.04 | 0.55 ± 0.03 | 0.55 ± 0.04 |
| Pituitary gland | 2.94 ± 0.43 | 3.16 ± 0.18 | 3.55 ± 0.54 | 3.34 ± 0.58 | 3.41 ± 0.36 |
| Spleen | 0.22 ± 0.02 | 0.22 ± 0.03 | 0.23 ± 0.03 | 0.23 ± 0.02 | 0.22 ± 0.01 |
| Thymus | 0.13 ± 0.01 | 0.15 ± 0.02 | 0.15 ± 0.01 | 0.13 ± 0.02 | 0.12 ± 0.01 |
| Testis-R | 0.56 ± 0.03 | 0.56 ± 0.02 | 0.61 ± 0.04* | 0.58 ± 0.02 | 0.59 ± 0.03 |
| Testis-L | 0.58 ± 0.03 | 0.56 ± 0.02 | 0.61 ± 0.04 | 0.59 ± 0.03 | 0.59 ± 0.03 |
| Prostate | 0.10 ± 0.02 | 0.11 ± 0.03 | 0.13 ± 0.02 | 0.11 ± 0.02 | 0.11 ± 0.02 |
| Seminal vesicle | 0.46 ± 0.02 | 0.40 ± 0.07 | 0.50 ± 0.07 | 0.41 ± 0.12 | 0.46 ± 0.05 |
| Salivary gland | 0.18 ± 0.02 | 0.19 ± 0.02 | 0.19 ± 0.02 | 0.19 ± 0.01 | 0.18 ± 0.01 |
| Thyroid gland-R (mg) | 2.84 ± 0.70 | 2.98 ± 0.48 | 3.44 ± 0.62 | 3.55 ± 0.73 | 3.66 ± 0.51 |
| Thyroid gland-L (mg) | 3.21 ± 0.56 | 3.54 ± 0.79 | 3.49 ± 0.58 | 3.10 ± 0.61 | 3.63 ± 1.20 |
Data are expressed as mean ± SD values (n = 5–8/group)
R right, L left
*Significantly different from the vehicle group (p < 0.05). Statistics: One-way analysis of variance (ANOVA) followed by the Dunnett's test
Table 5.
Toxicity of MDM hydantoin on percent relative organ weight in female rats
| Organ (g/100 g body weight) | Groups (mg/kg/day) | ||||
|---|---|---|---|---|---|
| 0 | 125 | 250 | 500 | 1000 | |
| Liver | 2.76 ± 0.15 | 2.73 ± 0.14 | 2.80 ± 0.09 | 2.75 ± 0.12 | 2.92 ± 0.23 |
| Kidney-R | 0.35 ± 0.01 | 0.36 ± 0.02 | 0.37 ± 0.01 | 0.38 ± 0.01 | 0.37 ± 0.02 |
| Kidney-L | 0.36 ± 0.01 | 0.36 ± 0.02 | 0.37 ± 0.03 | 0.37 ± 0.02 | 0.36 ± 0.02 |
| Adrenal gland-R (mg) | 14.81 ± 1.22 | 17.26 ± 3.02 | 16.65 ± 1.17 | 18.62 ± 2.27* | 18.10 ± 1.53* |
| Adrenal gland-L (mg) | 15.97 ± 1.74 | 17.92 ± 3.32 | 17.66 ± 2.11 | 18.50 ± 2.37 | 18.54 ± 1.63 |
| Heart | 0.39 ± 0.02 | 0.40 ± 0.03 | 0.41 ± 0.03 | 0.42 ± 0.03 | 0.41 ± 0.03 |
| Lung | 0.58 ± 0.03 | 0.58 ± 0.03 | 0.70 ± 0.29 | 0.61 ± 0.05 | 0.63 ± 0.04 |
| Brain | 0.85 ± 0.04 | 0.83 ± 0.07 | 0.83 ± 0.05 | 0.84 ± 0.03 | 0.84 ± 0.06 |
| Pituitary gland | 5.99 ± 1.06 | 6.21 ± 0.58 | 5.42 ± 0.60 | 6.39 ± 0.46 | 6.04 ± 0.70 |
| Spleen | 0.28 ± 0.02 | 0.26 ± 0.02 | 0.28 ± 0.02 | 0.28 ± 0.01 | 0.29 ± 0.02 |
| Thymus | 0.18 ± 0.02 | 0.18 ± 0.03 | 0.18 ± 0.03 | 0.17 ± 0.01 | 0.18 ± 0.06 |
| Ovary-R | 22.28 ± 4.03 | 23.16 ± 3.20 | 20.47 ± 3.62 | 22.54 ± 5.79 | 26.68 ± 7.35 |
| Ovary-L | 24.03 ± 3.70 | 22.51 ± 3.45 | 22.06 ± 2.95 | 21.57 ± 3.63 | 24.42 ± 3.19 |
| Uterus | 0.30 ± 0.18 | 0.19 ± 0.03 | 0.19 ± 0.02 | 0.24 ± 0.06 | 0.28 ± 0.16 |
| Salivary gland | 0.19 ± 0.04 | 0.23 ± 0.03 | 0.22 ± 0.03 | 0.22 ± 0.03 | 0.23 ± 0.02 |
| Thyroid gland-R (mg) | 4.47 ± 1.14 | 5.10 ± 1.29 | 4.10 ± 0.91 | 5.03 ± 1.85 | 4.68 ± 1.56 |
| Thyroid gland-L (mg) | 4.34 ± 1.01 | 4.70 ± 0.99 | 4.15 ± 1.10 | 5.25 ± 1.79 | 5.04 ± 1.69 |
Data are expressed as mean ± SD values (n = 5–8/group)
R right, L left
*Significantly different from the vehicle group (p < 0.05). Statistics: One-way analysis of variance (ANOVA) followed by the Dunnett's test
Fig.3.
Histopathological changes of rats in the 28-day oral administration toxicity test. There were no significant histophathological lesion both control and high dose groups. H&E stain, × 100 (scale bar: 200um) (a male, b female)
Discussion
MDM hydantoin is a formaldehyde-releasing agent or a formaldehyde releaser (FAR) and is often used as a preservative in cosmetics, in lacquers, and as an antimicrobial agent in metalworking fluids and paints [8, 11, 12]. FARs are chemicals that release formaldehyde as a result of decomposition and/or chemicals synthesized from formaldehyde that may still contain residues of free formaldehyde [12, 13]. Previous studies have shown that positive patch tests for FARs are often accompanied by concomitant reactions to formaldehyde [11] and commonly used preservatives such as parabens, DMDM hydantoin, imidazolidinyl urea, diazolidinyl urea, and 2-bromo-2-nitropropane-1,3diol [13, 14]. Numerous studies have reported the allergic reactions of chemicals in cosmetics (e.g., quaternium-15 [15], diazolidinyl urea [16], and DMDM hydantoin [9]). However, there is little published information available on the effect of MDM hydantoin in humans. Thus, in this study, we evaluated the toxicity associated with MDM hydantoin exposure in rats.
We performed single- and repeated-dose oral toxicity studies of MDM hydantoin. Repeated-dose oral toxicity studies are used to predict and determine safe doses in humans in accordance with NOAELs and are standard tests in the field of toxicology [17, 18]. No mortality was observed in the single-dose oral toxicity study. Furthermore, no behavioral alterations were recorded during the first 30 min or at checkpoints 6 h after the administration of MDM hydantoin. Hence, the LD50 in rats was considered to be higher than 2000 mg/kg body weight. In the 28-day repeated-dose oral toxicity study, no clinical sign or mortality due to toxicity was observed in the vehicle and MDM hydantoin-treated groups. Furthermore, MDM hydantoin administration up to 1000 mg/kg/day induced no significant change in body weight or food consumption. A significant increase in NEUT count and decrease in MONO count (Table 1) were observed in 1000 mg/kg of MDM hydantoin-treated male rats. There were significant differences in clinical biochemistry parameters in 1000 mg/kg/day MDM hydantoin-treated female rats, which appeared to be due to random biological variations. Although, there was a dose-dependent increase in glucose, the data value is within the reference range of the reference data. Therefore, it is referred to as random biological variations [19]. The results of GLU and CA were changed in a dose dependent manner. Even though there was a dose-dependent increase in female of GLU, CA, AST, BUN, CREA etc., which are indicators related with hypercalcemia, bone or renal failure, are not statistically significant. It appears to be a temporary effect due to increased capacity of the test substance. It is considered that there is no toxicological effect by the test substance. With regard to changes in organ weight, there were no significant differences in organ weights at any given dose of MDM hydantoin, except for in the weights of the adrenal gland and testis. However, these changes seemed to be normal variations because dose-dependent outcomes were not observed. They were not considered adverse as they were within the normal range, showed a lack of a dose–response relationship, or were not accompanied by any significant changes in the relevant parameters. In 28-day oral toxicity study, our results suggest that the NOAEL of MDM hydantoin is 1000 mg/kg/day in both sex.
Table 1.
Hematological parameter of MDM hydantoin in male and female rats
| Groups (mg/kg/day) | |||||
|---|---|---|---|---|---|
| Parameter | 0 | 125 | 250 | 500 | 1000 |
| Male | |||||
| WBCs (× 103 cells/μL) | 9.46 ± 1.52 | 8.39 ± 1.20 | 10.07 ± 1.10 | 8.49 ± 1.37 | 8.63 ± 2.48 |
| RBCs (× 106 cells/μL) | 8.27 ± 0.28 | 8.45 ± 0.33 | 8.30 ± 0.22 | 8.41 ± 0.40 | 8.22 ± 0.29 |
| HGB (g/dL) | 16.14 ± 0.92 | 16.14 ± 0.52 | 16.27 ± 0.49 | 16.48 ± 0.54 | 16.00 ± 0.51 |
| HCT (%) | 48.18 ± 2.30 | 48.40 ± 1.54 | 48.09 ± 1.52 | 49.15 ± 1.91 | 47.74 ± 1.55 |
| MCV (fL) | 58.28 ± 1.09 | 57.34 ± 0.99 | 57.97 ± 0.93 | 58.50 ± 0.94 | 58.14 ± 1.90 |
| MCH (pg) | 19.50 ± 0.47 | 19.13 ± 0.45 | 19.61 ± 0.23 | 19.58 ± 0.46 | 19.47 ± 0.54 |
| MCHC (g/dL) | 33.44 ± 0.54 | 33.37 ± 0.30 | 33.83 ± 0.35 | 33.46 ± 0.42 | 33.51 ± 0.45 |
| PLTs (× 103 cells/μL) | 1031.60 ± 77.35 | 1000.71 ± 159.96 | 1039.14 ± 32.88 | 1028.38 ± 47.10 | 1023.43 ± 157.99 |
| NEUTs (% of WBCs) | 8.06 ± 4.83 | 12.83 ± 2.79 | 13.74 ± 5.16 | 13.06 ± 3.12 | 20.39 ± 2.64* |
| LYMs (% of WBC) | 80.20 ± 4.79 | 83.43 ± 1.88 | 82.41 ± 5.59 | 83.56 ± 3.04 | 75.11 ± 3.05 |
| MONOs (% of WBCs) | 7.10 ± 5.87 | 2.44 ± 0.73* | 2.17 ± 0.60* | 1.80 ± 0.45* | 2.10 ± 0.38* |
| EOSs (% of WBCs) | 0.80 ± 0.19 | 1.11 ± 0.62 | 0.66 ± 0.09 | 0.70 ± 0.16 | 0.55 ± 0.22 |
| BASO (% of WBCs) | 0.30 ± 0.06 | 0.24 ± 0.07 | 0.29 ± 0.06 | 0.24 ± 0.05 | 0.21 ± 0.08 |
| Retic (%) | 2.76 ± 0.25 | 2.27 ± 0.17 | 2.49 ± 0.46 | 2.31 ± 0.29 | 2.45 ± 0.36 |
| Female | |||||
| WBCs (× 103 cells/μL) | 6.49 ± 1.14 | 6.63 ± 1.69 | 6.68 ± 0.77 | 7.42 ± 2.57 | 6.38 ± 1.57 |
| RBCs (× 106 cells/μL) | 7.98 ± 0.36 | 7.85 ± 0.39 | 7.97 ± 0.25 | 8.02 ± 0.27 | 7.81 ± 0.36 |
| HGB (g/dL) | 15.58 ± 0.56 | 15.53 ± 0.87 | 15.49 ± 0.61 | 15.70 ± 0.49 | 15.19 ± 0.44 |
| HCT (%) | 46.36 ± 1.66 | 45.73 ± 2.95 | 46.00 ± 1.85 | 45.76 ± 1.69 | 45.16 ± 1.48 |
| MCV (fL) | 58.10 ± 1.48 | 58.16 ± 1.68 | 57.73 ± 2.75 | 57.06 ± 0.86 | 57.89 ± 1.81 |
| MCH (pg) | 19.52 ± 0.47 | 19.77 ± 0.32 | 19.41 ± 0.49 | 19.56 ± 0.39 | 19.45 ± 0.51 |
| MCHC (g/dL) | 33.64 ± 1.00 | 34.03 ± 0.73 | 33.67 ± 1.23 | 34.28 ± 0.69 | 33.63 ± 0.73 |
| PLTs (× 103 cells/μL) | 1068.20 ± 64.70 | 1057.71 ± 50.16 | 1108.29 ± 125.98 | 1094.75 ± 71.29 | 1110.86 ± 92.75 |
| NEUTs (% of WBCs) | 12.76 ± 2.87 | 10.27 ± 3.47 | 8.91 ± 1.98 | 12.08 ± 3.40 | 13.83 ± 5.73 |
| LYMs (% of WBCs) | 83.26 ± 3.07 | 85.84 ± 3.59 | 87.33 ± 2.42 | 83.41 ± 3.66 | 81.64 ± 5.72 |
| MONOs (% of WBCs) | 2.04 ± 0.53 | 2.00 ± 0.62 | 1.96 ± 0.55 | 2.35 ± 0.71 | 2.65 ± 0.75 |
| EOSs (% of WBCs) | 0.98 ± 0.33 | 0.81 ± 0.12 | 0.66 ± 0.33 | 0.86 ± 0.59 | 0.84 ± 0.52 |
| BASOs (% of WBCs) | 0.18 ± 0.04 | 0.20 ± 0.08 | 0.23 ± 0.07 | 0.24 ± 0.11 | 0.21 ± 0.13 |
| Retic (%) | 2.27 ± 0.24 | 2.23 ± 0.27 | 2.28 ± 0.39 | 2.28 ± 0.61 | 2.36 ± 0.58 |
Data are expressed as mean ± SD values (n = 5–8/group)
WBC white blood cell, RBC red blood cell, HGB hemoglobin, HCT hematocrit, MCV mean corpuscular volume, MCH mean corpuscular hemoglobin, MCHC mean corpuscular hemoglobin concentration, PLT platelets, NEUT neutrophil, LYM lymphocyte, MONO monocyte, EOS eosinophil, BASO basophil, Retic reticulocyte
*Significantly different from the vehicle group (p < 0.05). Statistics: One-way analysis of variance (ANOVA) followed by the Dunnett's test
Acknowledgements
This work was supported by a grant from the Ministry of Food and Drug Safety (grant number 18181MFDS362 [2019]).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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