Toxicological evaluation of a recombinant inulosucrase (Convero®) produced by precision fermentation: In vitro genotoxicity studies and 28-day oral toxicity study in rats

Abstract

Convero® is an inulosucrase powder preparation produced via precision fermentation using a genetically modified strain of Komagataella phaffii as the production organism. It is intended for use in food applications due to its ability to catalyse the conversion of sucrose into inulin-type fructooligosaccharides. To support the safety of Convero®_, a bacterial reverse mutation assay, an in vitro micronucleus assay in mammalian cells, and a 28-day repeated-dose oral toxicity study were performed. All experimental procedures were performed in compliance with Good Laboratory Practice (GLP) and adhered to the Organisation for Economic Co-operation and Development (OECD) test guidelines for chemicals. No evidence of mutagenicity, aneugenicity or clastogenicity was detected. No evidence of oral toxicity was observed and no treatment-related adverse effects were observed in the 28-day repeated-dose oral toxicity study in rats at doses up to 2000 mg TOS/kg bw/day, the highest dose tested. Isolated minor, non-adverse changes in clinical pathology, urinalysis, and organ weights were considered incidental and within physiological variability. Overall, the combined in vitro and in vivo data indicate no toxicologically relevant effects of Convero® under the conditions tested.

Graphical Abstract

Highlights

• •Convero® is proposed to be used as food ingredient.
• •Safety evaluation followed GLP and OECD guidelines.
• •In vitro studies indicated no concerns regarding genotoxicity.
• •No adverse effects observed in rats given 2000 mg/kg bw/day for 28 days

.

1. Introduction

Inulosucrases (EC 2.4.1.9) are enzymes belonging to the family of fructosyltransferases that catalyse the transfer of fructosyl units from sucrose to suitable acceptor molecules, resulting in the formation of inulin-type fructans and related oligosaccharides [1]. Due to their catalytic properties, these enzymes are used in food processing for the controlled modification of carbohydrate structures and the production of specific ingredients with defined technological functionalities [2], [3].

The production of food ingredients through precision fermentation has expanded considerably in recent years [4]. This approach uses selected and well-characterised microorganisms to produce target compounds under controlled conditions, enabling consistent quality and scalable manufacturing. Precision fermentation is increasingly applied for the production of enzymes, proteins, and other food ingredients, including those that may not be readily obtained from conventional sources. As a result, it has become an important technological platform within the food sector [5], [6]. Convero® contains a recombinant inulosucrase with a modified amino acid sequence and is produced using a genetically engineered strain of Komagataella phaffii, a microorganism widely used in the industrial production of fermentation-derived food ingredients [8], [7]. The enzyme preparation is intended for use as an ingredient in sugar-containing food products.

The introduction of ingredients obtained through such processes requires a comprehensive safety evaluation to support their use in food. Regulatory frameworks in multiple jurisdictions may require toxicological data when the available information is not sufficient to establish safety [11], [9], [10]. These evaluations are typically conducted in accordance with internationally recognised guidelines, such as those developed by the Organisation for Economic Co-operation and Development (OECD) and follow a tiered approach [12]. Within this framework, genotoxicity and repeated-dose toxicity studies represent key and complementary components of the safety assessment. As independent endpoints, these studies address different aspects of toxicological risk and are commonly required to support the safety evaluation of novel food ingredients.

To date, toxicological data specifically addressing inulosucrase enzymes are limited. Safety data are available for related fructosyltransferases and their carbohydrate products [13], [14], [15]. Despite the increasing number of precision fermentation–derived food ingredients entering the market, there remains a lack of clarity regarding the extent of toxicological data required to support their safety assessment, particularly for recombinant enzymes with modified amino acid sequences. While regulatory authorities apply a weight-of-evidence approach, the decision to perform in vivo toxicological studies is often made on a case-by-case basis and is not always transparent. In this context, well-documented case studies combining standard genotoxicity assays with repeated-dose toxicity studies can provide valuable insight into how such data contribute to the overall safety assessment of precision fermentation–derived enzymes.

This work aimed to assess the preclinical safety profile of Convero®, under the experimental conditions employed, to support safety and regulatory risk assessment. Its genotoxic potential was investigated using a bacterial reverse mutation assay together with an in vitro micronucleus test. In addition, a 28-day repeated oral toxicity study in rats was performed to evaluate possible adverse effects. Such studies are typically required by regulatory authorities to support the safety assessment of novel substances intended for use in human consumption. Additionally, this work aims to contribute to the broader understanding of how standard toxicological datasets support the safety evaluation of precision fermentation–derived food enzymes within current regulatory frameworks.

2. Materials and methods

2.1. Study design and regulatory compliance

All studies were performed in compliance with Good Laboratory Practice (GLP). The bacterial reverse mutation (Ames) test was conducted in line with OECD Test Guideline 471 [16], employing the ‘treat and wash’ method because of the proteinaceous nature of the test item [17]. The in vitro micronucleus assay using human peripheral blood lymphocytes followed OECD Test Guideline 487 [18]. In addition, the 28-day repeated-dose oral toxicity study was carried out according to OECD Test Guideline 407 [19]. All toxicological studies were undertaken by Anthem Biosciences Ltd.

2.2. Test item characterisation

Convero® contains a recombinant inulosucrase whose amino acid sequence is derived from a Lactobacillus. The production organism is a genetically modified strain of Komagataella phaffii. The final enzyme differs from its native counterpart, containing several amino acid substitutions (to increase its stability during digestion). The manufacturing process is conducted under controlled conditions representative of industrial-scale production. A Hazard Analysis and Critical Control Points (HACCP) plan is implemented throughout the process to ensure food safety, including the identification and management of potential biological, chemical, and physical hazards.

The inulosucrase preparation had a protein purity of > 85%, with the remaining fraction comprising ash, moisture, fermentation-derived carbohydrates (Table 1), and residual host cell proteins from Komagataella phaffii. The test item consisted of the spray-dried enzyme preparation, without any additional carrier, and is representative of Convero®. Comprehensive product characterisation, including assessments of viable production organisms, residual recombinant DNA, host cell proteins, microbiological quality, and batch-to-batch consistency, was performed separately as part of the product quality package and is therefore not described in detail in this toxicological study.

Table 1.

Characterisation of the test item (Convero®).

Parameter	Result
Protein (Nx6.25)	74.1%
Inulosucrase content	63.6%
Total fat content	< 0.3%
Carbohydrates	18.6%
Moisture content	5.7%
Ash content	1.6%
Total organic solids (TOS)	92.7%
Water activity	0.268

2.3. Bacterial reverse mutation test

Bacterial strains used for the bacterial reverse mutation test were all purchased from Molecular Toxicology Inc. (NC, USA). Chemical compounds were all purchased from Sigma-Aldrich, Tokyo Chemical Industry, Thermo Fisher, CDH, SRL Pvt Ltd, Spectrochem Pvt Ltd or HiMedia Laboratories. The S9 fraction was produced in-house by Anthem Biosciences Ltd.

The study was performed according to the OECD 471 guideline, with modifications proposed by EFSA [17], on four histidine deficient mutant tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, and TA100) and one tryptophan deficient tester strain of Escherichia coli (WP2 uvrA (pKM101)). Convero® was tested in two independent experiments, in the absence and in the presence of metabolic activation (10% v/v rat liver S9 mix in all the experiments). The test system included a basal control, vehicle controls, test item, and positive controls to ensure the validity and reliability of the assay. The basal control (bacteria plus medium only, no vehicle) was included to measure the spontaneous revertant frequency. Two vehicle controls were used to account for the solvents present in the experiment: sterile ultrapure water, corresponding to the test item vehicle, and dimethyl sulfoxide (DMSO), corresponding to the positive controls’ vehicle. The test item was dissolved in water, forming a homogeneous suspension, and the positive controls were dissolved in DMSO in which they were readily soluble. The inclusion of both vehicle controls ensures that any observed effects are attributable to the test item or positive control, and not to the solvents themselves. All tests were performed by ‘treat and wash’ methodology. The initial toxicity mutation test was conducted with 8 different concentration levels, from 2 to 5000 µg TOS/plate. In the main mutation test, the concentration used were 20.07, 50.00, 150.00, 500.00, 1500.00 and 5000.00 µg TOS/plate.

Dose formulation analysis was performed using reverse phase high performance liquid chromatography (RP-HPLC). The analysis of Inulosucrase protein concentrations confirmed that the determined concentrations of Convero® dose formulation (top layer, middle layer and bottom layer) of Low test concentration (LTC) - 0.20 mg TOS/mL, Mid test concentration (MTC) - 5.00 mg TOS/mL and High test concentration (HTC)- 50.00 mg TOS/mL) were within acceptable range (% mean recovery 100 ± 30%; %RSD ≤20%).

Positive controls used in absence of metabolic activation were acridine mutagen ICR 191 for strain TA1537, sodium azide for TA1535, 4-nitroquinoline N-oxide for TA100, 2-nitrofluorene for TA98 and 4-nitroquinoline N-oxide for E. coli WP2 uvrA (pKM101). In presence of metabolic activation, the positive control was always 2-aminoanthracene. Plates were maintained in triplicates for each test concentration. The numbers of revertant colonies were recorded after 48–72 h incubation period at 37 ± 1°C.

2.4. In vitro mammalian cell micronucleus test

Chemical compounds were all purchased from Sigma-Aldrich, Tokyo Chemical Industry, Thermo Fisher or HiMedia Laboratories. The rat liver S9 fraction was produced in-house by Anthem Biosciences Limited. The study was performed according to OECD 487 guideline, by using cultured human peripheral blood lymphocytes. For the main test, blood was drawn from a healthy male volunteer (29-year-old, non-smoking, non-alcoholic, and free from recent exposure to radiation, drugs and chemicals). The whole blood withdrawal was performed by a trained phlebotomist. Human peripheral blood lymphocytes were cultured using RPMI−1640 medium supplemented with 10% (v/v) fetal bovine serum, 100 U of penicillin G and 100 µg/mL of streptomycin. The culture conditions were maintained at 37 ± 1 ºC with 5 ± 1% CO₂ using a CO₂ incubator. 4 mL of commercially procured Phytohemagglutinin-M solution was added to 196 mL of RPMI−1640 complete medium to prepare a mitogen medium with 2% v/v of PHA-M in the culture medium. This mitogen medium was prepared fresh on the day of the experiment.

Before conducting the main study, Convero® was evaluated for cytotoxicity in the absence and presence of metabolic activation (1% v/v S9 mix). The cytotoxicity test was performed at concentrations from 156.3 μg TOS/mL to 5000 μg TOS/mL. Based on the cytotoxicity test results, Convero® was evaluated for its potential to induce micronuclei at concentration levels of 1250, 2500 and 5000 μg TOS/mL, in the absence and presence of metabolic activation in phase I (4 h exposure) and the absence of metabolic activation in phase II (24 h exposure).

Ultrapure water was used as vehicle control, whereas vinblastine sulphate, cyclophosphamide monohydrate and mitomycin C were used as positive control, in the absence and presence of metabolic activation for 4 h, and in the absence of metabolic activation for 24 h, respectively.

2.5. Repeated dose oral toxicity study

The oral toxicity of Convero® was evaluated in a repeated dose 28-day oral toxicity study in Sprague Dawley rats following oral gavage, conducted in accordance with OECD 407 guidelines. The study was conducted in compliance with the Committee for Control and Supervision of Experiments on Animals (CCSEA) guidelines for laboratory animal facilities (Compendium of CPCSEA, 2018) and the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). The experimental protocol (Form-B) was reviewed and approved by the Institutional Animal Ethics Committee (IAEC Protocol No. ABD/IAEC/PR/394–25–26; approval date: 15 May 2025) prior to study commencement.

The animal study comprised 80 healthy Sprague Dawley rats (CD(SD)IGS), 40 males and 40 females aged 6–7 weeks at study initiation, and the body weight variation among the rats was within ± 20% of the mean body weight for each sex. Rats were housed in groups of 2 rats/cage/sex during the study period. Animals were housed with autoclaved corncob bedding and maintained under standard laboratory conditions. A gamma irradiated rat maintenance diet manufactured by Altromin Spezialfutter GmbH & Co. KG was provided ad libitum throughout acclimatization and the study period, except during scheduled fasting intervals. Autoclaved reverse osmosis UV-treated water was available ad libitum throughout the study. Analytical certificates confirmed the absence of known contaminants in the feed, water, and bedding that could have interfered with the study objectives.

Animals were randomly allocated (acclimatized for a period of 5–6 days prior to randomization) to four groups of 10 rats/sex/group: Group 1 (vehicle control), Group 2 (low dose), Group 3 (mid dose), and Group 4 (high dose). Test material was administered once daily by oral gavage at dose levels of 0, 500, 1000, and 2000 mg total organic solids (TOS)/kg body weight (bw)/day for 28 consecutive days. No recovery group was considered. The highest dose of 2000 mg TOS/kg bw/day was selected to provide a sufficiently high exposure for hazard identification while remaining technically feasible for repeated oral administration, as no toxicity was anticipated based on the available information on the test item.

A constant dose volume of 5 mL/kg bw/day was used throughout the study. Dose formulations were prepared as suspensions in ultrapure Type 1 water. The homogeneity of the formulations were analytically verified by determining the concentration of Inulosucrase protein content using RP-HPLC prior to the start of dosing and during the final week of treatment. Formulation stability was demonstrated for up to 5 days under refrigerated conditions and for 24 h at room temperature. Animals were observed twice daily for morbidity and mortality and once daily for clinical signs throughout the treatment period. Body weight and feed consumption were recorded weekly. Ophthalmological examinations were performed before treatment initiation in all animals and repeated during the final treatment week in the control and high-dose groups. A functional observational battery was conducted in the control and high-dose groups during the last week of treatment. On Day 29, blood and urine samples were collected for haematology, clinical chemistry, coagulation, and urinalysis assessments. At scheduled termination, all animals underwent gross necropsy. Specified organs were collected, weighed, and preserved in 10% neutral buffered formalin, Davidson’s fluid (eyes) and/or modified Davidson’s fluid (Testes) for subsequent histopathological examination.

2.6. Data interpretation and statistical analysis

In the bacterial reverse mutation test, results were interpreted based on biological relevance criteria: a test item was considered mutagenic if it produced a reproducible, dose-related and biologically significant increase in revertant colonies in at least one strain, with fold-increase thresholds of ≥ 3-fold for S. typhimurium TA1535 and TA1537, and ≥ 2-fold for TA98, TA100 and E. coli WP2 uvrA (pKM101), compared with vehicle control. A lack of such increases across independent experiments was considered indicative of a negative response, while inconsistent or marginal effects were classified as equivocal.

Statistical analysis in the in vitro micronucleus test was performed on the results by Chi-square test for pair-wise comparison between each treated and vehicle control group using GraphPad Prism Software (version 5.03). The significance level was chosen at P < 0.05 for all statistical analysis in comparison to the respective vehicle control.

Statistical analyses in the repeated dose oral toxicity study were performed using GraphPad Prism Software (version 5.03). Data sets, including body weight, body weight gain, feed consumption, organ weights, clinical pathology parameters, urinalysis, and functional observational endpoints, were screened for outliers and analysed separately for males and females. Normality and homogeneity of variance were assessed using the Shapiro–Wilk and Bartlett tests, respectively. Parametric data were analysed by one-way ANOVA followed by Dunnett’s post hoc test comparing each treated group (G2–G4) with the vehicle control group (G1). Where assumptions for parametric analysis were not met, log transformation was applied; if criteria remained unmet, non-parametric analysis was conducted using the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Statistical significance was set at p < 0.05.

3. Results

3.1. Bacterial reverse mutation test

The test item Convero® was initially evaluated for solubility in sterile ultrapure water. A stock concentration of 50.0 mg TOS/mL was prepared, vortexed, and sonicated for 10 min to ensure homogeneity. The resulting suspension was homogeneous. Further dilutions at 15.0 and 5.0 mg TOS/mL also formed homogeneous suspensions, confirming adequate solubility across the tested range. No precipitation was observed at 50.0, 15.0, or 5.0 mg TOS/mL in top agar in the absence of S9 metabolic activation. Based on these results, test concentrations of 50.00, 15.00, 5.00, 1.50, 0.50, 0.15, 0.05, and 0.02 mg TOS/mL (equivalent to 5000.00, 1500.00, 500.00, 150.00, 50.00, 15.00, 5.00, and 2.00 μg TOS/plate) were selected for the preliminary study.

In the preliminary cytotoxicity assay, Convero® was evaluated in Salmonella typhimurium TA100 and Escherichia coli WP2 uvrA (pKM101) at concentrations ranging from 50.00 to 0.02 mg TOS/mL, in the presence and absence of S9 metabolic activation using the treat and wash method. No cytotoxic effects were observed at any tested concentration, as indicated by the absence of growth inhibition or relevant reductions in revertant colony counts according to the study criteria. Therefore, the tested concentration range was considered appropriate for the main mutagenicity assay.

The main bacterial reverse mutation assay was conducted at concentrations of 50.00, 15.00, 5.00, 1.50, 0.50, and 0.20 mg TOS/mL (corresponding to 5000.00–20.07 μg TOS/plate) using the treat and wash method, with and without S9 metabolic activation (Table 2). Overall, no biologically relevant increase in revertant colony numbers was observed in any tester strain, either in the presence or absence of metabolic activation. Slight increases in the mutagenicity factor (up to 1.3 in isolated cases) were observed at the highest tested concentrations; however, these responses did not meet the criteria for biological relevance, as they did not reach a 2-fold increase and were not accompanied by a dose–response relationship. Convero® did not induce any biologically or statistically significant increase in revertant colony numbers in any of the tested strains (S. typhimurium TA98, TA100, TA1535, TA1537, and E. coli WP2 uvrA (pKM101)) under any condition.

Table 2.

Reverse mutation assay results in absence and presence of S9 (results reported as “average ± 1 standard deviation (mutagenicity factor)”).

		Convero® Dose (µg/plate)						Controls
Strain	Metabolic activation	20.07	50	150	500	1500	5000	Basal control (Spontaneous)	Vehicle (water)	Vehicle (DMSO)	Positive control
WP2 uvrA (pKM101)	-S9	68.3 ± 1.7 (0.9)	68.7 ± 4.1 (1.0)	68.7 ± 7.4 (1.0)	78.3 ± 4.8 (1.1)	87.3 ± 6.5 (1.2)	96.7 ± 4.8 (1.3)	60.7 ± 1.2 (NA)	72.0 ± 0.8 (NA)	55.7 ± 3.1 (NA)	617.3 ± 27.8 (11.1) (4-NON)
	+S9	118.3 ± 6.6 (1.0)	120.3 ± 6.3 (1.0)	134.7 ± 6.2 (1.1)	136.0 ± 5.7(1.1)	133.3 ± 9.0 (1.1)	141.7 ± 7.1 (1.1)	110.3 ± 9.0 (NA)	123.3 ± 9.1(NA)	106.3 ± 9.7(NA)	717.3 ± 22.9 (6.7) (2-AA)
TA98	-S9	29.3 ± 3.9 (1.0)	27.0 ± 4.1 (0.9)	28.0 ± 4.5 (0.9)	33.7 ± 4.0 (1.1)	34.7 ± 4.0 (1.1)	31.0 ± 4.3 (1.0)	26.3 ± 3.1 (NA)	30.3 ± 2.9 (NA)	29.3 ± 3.1 (NA)	229.3 ± 3.9 (7.8) (2-NF)
	+S9	33.0 ± 1.6 (1.1)	33.0 ± 2.9 (1.1)	30.0 ± 2.9 (1.0)	30.3 ± 3.8 (1.0)	33.3 ± 2.5 (1.1)	34.7 ± 2.1 (1.2)	31.0 ± 3.3 (NA)	29.3 ± 1.7 (NA)	26.7 ± 1.7 (NA)	588.0 ± 13.1 (22.1) (2-AA)
TA100	-S9	176.3 ± 9.0 (1.1)	174.7 ± 9.8 (1.1)	164.7 ± 9.8 (1.0)	175.7 ± 6.2 (1.1)	193.7 ± 0.9 (1.2)	214.0 ± 1.6 (1.3)	186.3 ± 9.7 (NA)	165.7 ± 13.2 (NA)	169.3 ± 8.2 (NA)	862.7 ± 10.0 (5.1) (4-NON)
	+S9	177.3 ± 7.6 (1.1)	172.3 ± 10.7 (1.0)	167.0 ± 7.3 (1.0)	179.7 ± 3.4 (1.1)	169.3 ± 10.5 (1.0)	178.0 ± 3.3 (1.1)	178.0 ± 8.5 (NA)	167.7 ± 3.7 (NA)	147.0 ± 7.0 (NA)	494.7 ± 10.0 (3.4) (2-AA)
TA1535	-S9	13.3 ± 0.9 (1.0)	13.3 ± 1.9 (1.0)	12.0 ± 1.6 (0.9)	13.3 ± 2.9 (1.0)	12.0 ± 0.8 (0.9)	12.7 ± 1.7 (1.0)	9.0 ± 0.8 (NA)	13.0 ± 0.8 (NA)	10.3 ± 1.7 (NA)	219.0 ± 6.7 (21.2) (SA)
	+S9	14.3 ± 0.9 (1.0)	14.7 ± 3.4 (1.0)	12.0 ± 2.2 (0.9)	14.0 ± 0.8 (1.0)	12.3 ± 2.1 (0.9)	15.7 ± 1.7 (1.1)	11.0 ± 1.4 (NA)	14.0 ± 2.2 (NA)	12.3 ± 1.2 (NA)	100.7 ± 6.2 (8.2) (2-AA)
TA1537	-S9	10.0 ± 2.2 (0.9)	10.3 ± 1.2 (0.9)	11.3 ± 0.9 (1.0)	11.3 ± 2.1 (1.0)	10.0 ± 1.6 (0.9)	10.0 ± 2.4 (0.9)	8.3 ± 1.2 (NA)	11.0 ± 0.8 (NA)	8.7 ± 0.9 (NA)	432.0 ± 16.1 (49.8) (ICR 191)
	+S9	11.3 ± 0.5 (0.9)	11.0 ± 1.4 (0.9)	13.0 ± 2.2 (1.1)	12.0 ± 2.9 (1.0)	13.7 ± 1.2 (1.1)	12.3 ± 1.2 (1.0)	13.3 ± 1.2 (NA)	12.3 ± 1.7 (NA)	10.7 ± 1.2 (NA)	129.3 ± 6.2 (12.1) (2-AA)

Positive controls:2-NF: 2- nitrofluorene; 2-AA: 2-aminoanthracene; 4-NON: 4-Nitroquinoline N-Oxide; SA: Sodium azide; NA: not applicable.

S9: rat liver S9 metabolic activation system; DMSO: dimethyl sulfoxide.

All control values, including vehicle and positive controls, were within the established historical control ranges. Acceptance criteria for the assay were fulfilled based on the expected response of the strain-specific positive controls, which produced the required fold increase and fell within the historical data distribution (mean ± 2 standard deviations), confirming the validity of the assay and the performance of the metabolic activation system. Positive controls induced clear and expected increases in revertant colonies, demonstrating the sensitivity of the assay and the metabolic competence of the S9 system. Sterility controls showed no microbial contamination (0 CFU/plate).

3.2. In vitro mammalian cell micronucleus test

A preliminary cytotoxicity assessment was conducted in mitogen-stimulated human peripheral blood lymphocytes following exposure to Convero® at concentrations ranging from 156.3 to 5000 µg TOS/mL under three treatment conditions: 4 h without metabolic activation (S9), 4 h with S9, and 24 h without S9. Cytotoxicity was evaluated using the cytokinesis-block proliferation index (CBPI), and the corresponding percentage cytotoxicity was calculated from CBPI values. Under the 4 h treatment in the absence of S9, Convero® induced limited cytotoxicity across the tested range, with values from 3.18% to 15.92%. The highest concentration tested (5000 µg TOS/mL) produced 15.92% cytotoxicity. In the presence of S9 during the 4 h treatment, cytotoxicity ranged from 1.56% to 12.50%, again indicating only modest effects on cell proliferation at the highest concentration. Following the 24 h treatment in the absence of S9, greater cytotoxicity was observed, reaching 29.11% at 5000 µg TOS/mL and 12.03% at 2500 µg TOS/mL, whereas concentrations of 1250 µg TOS/mL and below showed minimal or no cytotoxicity. No precipitation of the test item was observed at any concentration, including the highest tested concentration of 5000 µg TOS/mL, under any treatment condition. As no substantial cytotoxicity was achieved and no solubility limitations were identified, the three highest analysable concentrations (5000, 2500, and 1250 µg TOS/mL) were selected for micronucleus evaluation in all treatment conditions.

In the main micronucleus assay, Convero® was evaluated under three treatment conditions: 4 h without metabolic activation, 4 h with metabolic activation, and 24 h without metabolic activation (Table 3). 2000 binucleated cells were scored per concentration. Across all treatment conditions, only low to moderate cytotoxicity was observed, with the highest effect at 5000 µg TOS/mL, reaching 16.22% after 4 h without S9, 12.81% after 4 h with S9, and 28.75% after 24 h without S9. No precipitation was observed at any tested concentration.

Table 3.

In vitro micronucleus assay results in absence and presence of S9.

Treatment condition	Treatment	Concentration	Mean CBPI (n = 2)	Cytotoxicity (%)	Binucleated cells with Micronuclei (n = 2)	%MNBN
4 h, -S9	Vehicle control - Ultrapure water (10% v/v)	—	1.635	0	3	0.15
	Positive control - Vinblastine sulfate (µg/mL)	0.05	1.429	32.44	31	1.55*
	Convero® (µg TOS/mL)	5000	1.532	16.22	3	0.15
		2500	1.608	4.25	3	0.15
		1250	1.606	4.57	2	0.1
4 h, +S9	Vehicle control - Ultrapure water (10% v/v)	—	1.64	0	2	0.1
	Positive control - Cyclophosphamide monohydrate (µg/mL)	10	1.465	27.34	31	1.55*
	Convero® (µg TOS/mL)	5000	1.558	12.81	4	0.2
		2500	1.587	8.28	3	0.15
		1250	1.616	3.75	2	0.1
24 h, -S9	Vehicle control - Ultrapure water (10% v/v)	—	1.633	0	4	0.2
	Positive control - Mitomycin C (µg/mL)	0.1	1.43	32.07	33	1.65*
	Convero® (µg TOS/mL)	5000	1.451	28.75	4	0.2
		2500	1.548	13.43	2	0.1
		1250	1.616	2.69	1	0.05

CBPI: Cytokinesis Block Proliferation Index; BN: Binucleated; MNBN: Binucleated cell with micronuclei; TOS: Total Organic Solids; S9: rat liver S9 metabolic activation system;.

* Statistically significant compared to the vehicle control at p < 0.05 (Chi-square Test).

Micronucleus frequencies in Convero®-treated cultures remained low and comparable to concurrent vehicle controls in all treatment conditions. In the absence of S9 (4 h), %MNBN values ranged from 0.10% to 0.15% versus 0.15% in the vehicle control. In the presence of S9 (4 h), values ranged from 0.10% to 0.20% versus 0.10% in the vehicle control. Following 24 h treatment without S9, values ranged from 0.05% to 0.20% versus 0.20% in the vehicle control. No statistically significant increases were observed at any concentration (Chi-square test, p > 0.05), and no concentration-related trend was evident. All values were within the laboratory historical negative control ranges. The positive controls demonstrated clear assay sensitivity and validity. Vinblastine sulphate (0.05 µg/mL), cyclophosphamide monohydrate (10 µg/mL), and mitomycin C (0.1 µg/mL) produced statistically significant increases in micronucleus frequencies of 1.55%, 1.55%, and 1.65%, respectively (p < 0.05).

3.3. Repeated dose oral toxicity study

Dose formulation analysis confirmed the validity and homogeneity of the test item formulations throughout the study period. The analytical concentration of formulations prepared prior to Day 1 administration and at Week 4 ranged between 88.71% and 101.50% of nominal concentrations, remaining within the predefined acceptance criteria of 100 ± 30%. Formulations prepared for administration at different concentrations were considered homogenous. No test item was detected in the vehicle control samples, confirming the absence of cross-contamination.

No mortality or morbidity was observed in any group throughout the treatment period. All animals remained clinically normal in the control and test item-treated groups during the study. No treatment-related clinical signs were observed. Feed consumption remained comparable between control and treated groups in both sexes throughout the study period, with no treatment-related effects observed. No ophthalmological abnormalities were observed at any dose level. Functional observation battery parameters, including neurobehavioral and neuromuscular endpoints, were comparable between control and high-dose groups (2000 mg TOS/kg bw/day), indicating no neurotoxic potential. No treatment-related effects on body weight or body weight gain were observed in either sex. A statistically significant decrease in body weight gain was observed in males at 500 mg TOS/kg bw/day during Week 1 (−10.51% change to control). This isolated finding was not dose-dependent, not sustained, and terminal body weights were comparable to controls; therefore, it was considered toxicologically irrelevant (Table 4).

Table 4.

Summary of group mean body weight gain in males and females during the duration of the study (Mean ± SD).

Sex	Study days	1–8	8–15	15–22	22–28	1–28 (%)
	Group (Dose mg TOS/kg bw/day)
Male	G1 (0)	73.22 ± 5.98	59.36 ± 10.06	44.11 ± 9.17	29.88 ± 9.43	80.68 ± 4.77
	G2 (500)	65.53 ± 7.44 *	58.95 ± 9.38	42.18 ± 6.19	33.44 ± 7.80	78.04 ± 6.72
	G3 (1000)	73.61 ± 4.28	59.67 ± 4.67	41.06 ± 5.09	35.24 ± 10.55	82.08 ± 3.41
	G4 (2000)	71.17 ± 6.58	61.12 ± 9.84	44.05 ± 5.92	30.65 ± 5.51	81.19 ± 8.05
Female	G1 (0)	27.98 ± 5.42	19.06 ± 6.17	22.89 ± 9.00	5.36 ± 2.95	38.20 ± 4.29
	G2 (500)	32.06 ± 9.81	24.41 ± 6.09	20.10 ± 9.05	7.81 ± 5.02	43.00 ± 6.19
	G3 (1000)	27.11 ± 7.14	23.92 ± 4.65	21.19 ± 8.19	7.84 ± 4.58	39.15 ± 4.28
	G4 (2000)	29.14 ± 8.57	18.85 ± 6.18	22.27 ± 7.20	7.98 ± 3.47	40.04 ± 5.85

Data are presented as mean ± SD. N = 10 for all groups and parameters throughout the study. Weekly body weight change is in grams and terminal (day 1–28) changes in %.

*: Statistically significant (p < 0.05). Statistical comparisons were performed separately for each sex using one-way ANOVA followed by Dunnett's multiple comparison test. P < 0.05 versus the concurrent vehicle control group (Group 1).

TOS: Total Organic Solids.

No biologically relevant, test item-related effects were observed in the evaluated haematology or coagulation parameters in either sex at terminal sacrifice (Table 5). Statistically significant differences were isolated, of low magnitude, and lacked consistency across dose levels, sexes, or related endpoints. In males, statistically significant decreases in hematocrit at 500 mg TOS/kg bw/day (−3.26% compared to control group) and in mean corpuscular volume at 2000 mg TOS/kg bw/day (−3.42% compared to control group), as well as a decrease in monocyte counts at 500 mg TOS/kg bw/day (−29.85% compared to control group) were noted. However, these changes were small in magnitude, lacked a consistent dose–response relationship, and were not accompanied by concordant alterations in haemoglobin, erythrocyte counts, or other red cell indices, while still being within the ranges reported in the historical data of the laboratory.

Table 5.

Haematological parameters in male and female rats following 28-day oral administration of test item (Mean ± SD).

Sex	Male				Female
Group (Dose mg TOS/kg bw/day)	G1 (0)	G2 (500)	G3 (1000)	G4 (2000)	G1 (0)	G2 (500)	G3 (1000)	G4 (2000)
Parameter (Unit)
WBC (103cells/ µL)	8.52 ± 1.02	7.67 ± 1.13	9.50 ± 1.81	9.20 ± 2.56	8.01 ± 1.52	7.03 ± 2.23	6.47 ± 1.06	6.95 ± 1.76
RBC (106cells/ µL)	7.90 ± 0.36	7.61 ± 0.33	8.07 ± 0.38	7.92 ± 0.17	8.02 ± 0.36	7.61 ± 0.37 ↓*	7.58 ± 0.22 ↓*	7.88 ± 0.34
HGB (g/dL)	16.34 ± 0.69	15.80 ± 0.42	16.41 ± 0.56	15.93 ± 0.57	15.89 ± 0.52	15.68 ± 0.69	15.55 ± 0.53	15.79 ± 0.55
HCT (%)	50.06 ± 1.98	48.25 ± 1.54 ↓*	49.86 ± 1.59	48.50 ± 1.43	47.04 ± 0.96	45.81 ± 2.04	45.50 ± 1.32	46.20 ± 1.24
MCV (fL)	63.37 ± 1.26	63.43 ± 1.60	61.84 ± 2.07	61.20 ± 1.18 ↓*	58.75 ± 1.70	60.19 ± 1.41	60.06 ± 1.48	58.66 ± 1.50
MCH (pg)	20.68 ± 0.40	20.79 ± 0.80	20.34 ± 0.78	20.12 ± 0.64	19.86 ± 0.62	20.59 ± 0.50 ↑*	20.56 ± 0.70 ↑*	20.05 ± 0.56
MCHC (g/dL)	32.64 ± 0.26	32.78 ± 0.50	32.91 ± 0.62	32.87 ± 0.60	33.79 ± 0.67	34.23 ± 0.38	34.20 ± 0.45	34.19 ± 0.54
Neu (103cells/ µL)	1.80 ± 0.62	1.47 ± 0.30	2.17 ± 0.75	1.71 ± 0.55	1.19 ± 0.44	1.15 ± 0.61	1.00 ± 0.39	1.06 ± 0.51
Lymph (103cells/ µL)	6.16 ± 0.86	5.78 ± 1.10	6.80 ± 1.33	6.93 ± 2.33	6.43 ± 1.27	5.54 ± 1.71	5.13 ± 1.13	5.54 ± 1.24
Mono (103cells/ µL)	0.34 ± 0.10	0.24 ± 0.05 ↓*	0.31 ± 0.05	0.32 ± 0.11	0.22 ± 0.07	0.18 ± 0.07	0.19 ± 0.02	0.19 ± 0.09
Eos (103cells/ µL)	0.10 ± 0.02	0.09 ± 0.03	0.12 ± 0.04	0.12 ± 0.08	0.10 ± 0.04	0.09 ± 0.04	0.08 ± 0.02	0.09 ± 0.04
Baso (103cells/ µL)	0.02 ± 0.01	0.01 ± 0.00	0.01 ± 0.01	0.01 ± 0.01	0.01 ± 0.00	0.01 ± 0.00	0.01 ± 0.01	0.01 ± 0.01
Platelet (103cells/ µL)	1056.20 ± 100.10	967.90 ± 124.68	991.60 ± 108.51	972.00 ± 120.24	1116.70 ± 134.14	928.0 ± 156.08 ↓*	1040.30 ± 132.93	984.30 ± 126.60
Retic (109cells/ L)	298.68 ± 31.58	288.75 ± 29.78	283.42 ± 36.83	279.61 ± 24.67	237.49 ± 26.86	254.79 ± 35.98	262.04 ± 26.45	250.53 ± 36.09
PT (Sec)	22.72 ± 0.84	22.60 ± 1.08	22.50 ± 1.32	22.26 ± 1.03	22.66 ± 1.03	22.12 ± 0.81	22.00 ± 1.11	22.03 ± 1.10
APTT (Sec)	16.46 ± 0.66	16.73 ± 0.75	17.08 ± 0.77	17.19 ± 0.72	16.23 ± 0.76	16.85 ± 0.59	16.80 ± 0.60	17.13 ± 0.73 ↑*

Data are presented as mean ± SD. N = 10 for all groups and parameters throughout the study. SD = standard deviation; * / ↓ / ↑ = statistically significant difference from control (p < 0.05, one-way ANOVA followed by Dunnett’s post hoc test).

WBC, White Blood Cell count; RBC, Red Blood Cell count; HGB, Hemoglobin; HCT, Hematocrit; MCV, Mean Corpuscular Volume; MCH, Mean Corpuscular Hemoglobin; MCHC, Mean Corpuscular Hemoglobin Concentration; Neu, Neutrophils; Lymph, Lymphocytes; Mono, Monocytes; Eos, Eosinophils; Baso, Basophils; Platelet, Platelet count; Retic, Reticulocytes; PT, Prothrombin Time; APTT, Activated Partial Thromboplastin Time; TOS: Total Organic Solids.

In females, statistically significant decreases in erythrocyte counts at 500 and 1000 mg TOS/kg bw/day were observed together (−5.05 and −5.46% compared to control group, respectively) with isolated increases in mean corpuscular haemoglobin at the same dose levels (+3.68% and +3.52% compared to control group), and a slight increase in activated partial thromboplastin time at 2000 mg TOS/kg bw/day (+5.55% compared to control group). Additionally, a decrease in platelet count was observed at 500 mg TOS/kg bw/day (−16.90% compared to control group); however, this was not dose-dependent and remained within physiological variability (historical data) and comparable to the control group. It has been reported that some clinical pathology parameters can occasionally be altered by stress, such as red blood cell and platelet counts [20]. In fact, monocyte counts and platelet numbers are among the most variable leukocyte and thrombocyte parameters in laboratory rodents and are not considered robust standalone indicators of toxicity in the absence of corroborative changes in related immune or coagulation endpoints.

No consistent pattern of change was observed across leukocyte differentials, coagulation endpoints, and no corroborative findings were detected across sexes. Importantly, all values remained within expected physiological ranges for the species and were not associated with any changes in clinical condition, organ weights, or histopathological findings. Therefore, the observed statistically significant differences were considered incidental, not treatment-related, and not toxicologically relevant.

No biologically relevant, test item-related effects were observed in serum clinical chemistry parameters in either sex following 28 days of administration (Table 6). In males, statistically significant increases in phosphorus concentrations were observed at 1000 and 2000 mg TOS/kg bw/day (+6.28% and +5.73% compared to control group), whereas potassium concentrations were significantly decreased at 500 and 2000 mg TOS/kg bw/day (−9.25% and –11.82% compared to control). In addition, a slight decrease in calcium was noted at 2000 mg TOS/kg bw/day (−4.58% compared to control). However, these changes were of low magnitude, remained within expected physiological ranges, and were not associated with corroborative alterations in renal function markers (blood urea nitrogen, creatinine), hepatic enzymes (ALT, AST, ALP), or related electrolyte disturbances.

Table 6.

Clinical chemistry parameters in male and female rats following 28-day oral administration of test item (Mean ± SD).

Sex	Male				Female
Group (Dose mg TOS/kg bw/day)	G1 (0)	G2 (500)	G3 (1000)	G4 (2000)	G1 (0)	G2 (500)	G3 (1000)	G4 (2000)
Parameter (Unit)
TBI(mg/dL)	0.11 ± 0.07	0.12 ± 0.04	0.13 ± 0.07	0.10 ± 0.07	0.20 ± 0.07	0.13 ± 0.05	0.09 ± 0.06 ↓*	0.10 ± 0.00 ↓*
TP(g/L)	6.67 ± 0.44	6.57 ± 0.25	6.70 ± 0.24	6.61 ± 0.15	6.71 ± 0.22	6.79 ± 0.36	6.94 ± 0.25	6.81 ± 0.28
GLOB(g/dL)	5.36 ± 0.44	5.22 ± 0.23	5.39 ± 0.22	5.26 ± 0.11	5.29 ± 0.15	5.42 ± 0.28	5.52 ± 0.26	5.45 ± 0.22
ALP (U/L)	188.10 ± 43.17	200.40 ± 24.22	176.90 ± 29.86	206.00 ± 25.15	114.20 ± 14.79	113.60 ± 27.26	123.60 ± 20.52	98.70 ± 20.59
TGL(mg/dL)	49.10 ± 21.25	44.60 ± 14.33	36.20 ± 6.80	35.50 ± 11.35	25.30 ± 4.62	25.60 ± 4.17	29.70 ± 5.70	23.60 ± 2.55
CHOL(mg/dL)	61.60 ± 14.30	55.30 ± 8.12	53.90 ± 15.77	50.10 ± 8.43	62.20 ± 7.22	59.40 ± 11.38	63.80 ± 14.15	64.10 ± 12.88
PHOS(mg/dL)	7.16 ± 0.36	7.15 ± 0.37	7.61 ± 0.41 ↑*	7.57 ± 0.29 ↑*	6.33 ± 0.55	6.79 ± 0.51	6.47 ± 0.21	6.96 ± 0.34 ↑*
AST (U/L)	98.60 ± 9.69	102.30 ± 18.39	92.80 ± 16.75	110.70 ± 37.98	96.40 ± 8.03	102.40 ± 13.17	96.30 ± 11.50	91.40 ± 11.74
ALB (g/L)	1.31 ± 0.06	1.35 ± 0.10	1.31 ± 0.10	1.35 ± 0.07	1.42 ± 0.10	1.37 ± 0.16	1.42 ± 0.10	1.36 ± 0.11
ALT (U/L)	41.40 ± 5.93	48.40 ± 8.50	43.90 ± 11.05	53.70 ± 26.51	41.60 ± 5.50	39.60 ± 6.04	39.40 ± 5.44	40.60 ± 9.05
BUN(mg/dL)	10.10 ± 2.42	10.10 ± 1.29	10.50 ± 1.65	10.90 ± 1.52	12.80 ± 1.81	13.90 ± 1.45	13.60 ± 3.34	13.40 ± 3.03
GLUC(mg/dL)	112.20 ± 15.08	106.70 ± 9.15	111.10 ± 11.71	110.10 ± 6.61	102.20 ± 7.73	105.90 ± 12.51	107.30 ± 9.44	100.60 ± 9.71
Ca(mg/dL)	10.27 ± 0.30	10.26 ± 0.22	10.06 ± 0.26	9.80 ± 0.18 ↓*	10.18 ± 0.16	10.09 ± 0.32	9.77 ± 0.30 ↓*	10.03 ± 0.18
CREA(mg/dL)	0.29 ± 0.07	0.31 ± 0.06	0.29 ± 0.07	0.35 ± 0.09	0.37 ± 0.07	0.41 ± 0.07	0.37 ± 0.10	0.33 ± 0.08
Na+(mmol/ L)	144.22 ± 1.08	144.44 ± 0.94	144.60 ± 1.07	144.89 ± 0.72	142.99 ± 1.66	142.82 ± 1.24	142.97 ± 1.07	142.92 ± 1.11
K+(mmol/ L)	4.16 ± 0.30	3.78 ± 0.24 ↓*	3.88 ± 0.37	3.67 ± 0.28 ↓*	4.07 ± 0.26	4.04 ± 0.38	4.17 ± 0.44	3.84 ± 0.34
Cl−(mmol/ L)	103.13 ± 1.53	103.56 ± 0.63	103.42 ± 1.14	103.42 ± 1.11	103.74 ± 1.51	103.78 ± 1.12	103.79 ± 0.72	103.44 ± 0.54
Urea(mg/dL)	21.61 ± 5.19	21.61 ± 2.75	22.47 ± 3.53	23.33 ± 3.26	27.39 ± 3.88	29.75 ± 3.10	29.10 ± 7.15	28.68 ± 6.48

Data are presented as mean ± SD. N = 10 animals per group for all parameters. * Statistically significant difference from control (p < 0.05; one-way ANOVA followed by Dunnett’s post hoc test). ↑ / ↓ indicate increase or decrease relative to controls.

TBI, total bilirubin; TP, total protein; GLOB, globulin (calculated); ALP, alkaline phosphatase; TGL, triglycerides; CHOL, cholesterol; PHOS, phosphorus; AST, aspartate aminotransferase; ALB, albumin; ALT, alanine aminotransferase; BUN, blood urea nitrogen; GLUC, glucose; Ca, calcium; CREA, creatinine; Na+, sodium; K+, potassium; Cl−, chloride; Urea, calculated urea concentration; SD, standard deviation; N, number of animals; TOS: Total Organic Solids.

In females, total bilirubin concentrations were significantly decreased at 1000 and 2000 mg TOS/kg bw/day (−55% and −50%, compared to control), phosphorus was slightly increased at 2000 mg TOS/kg bw/day (+9.95% compared to control), and calcium was decreased at 1000 mg TOS/kg bw/day (−4.03% compared to control). These findings were small in magnitude, lacked clear toxicological relevance, and were not accompanied by consistent dose-related changes in liver-associated parameters, protein fractions, or renal biomarkers. No treatment-related effects were observed for total protein, albumin, globulin, triglycerides, cholesterol, glucose, sodium, chloride, blood urea nitrogen, creatinine, or calculated urea in either sex. Absolute bilirubin values in all groups remained within the historical control range (mean 0.10 mg/dL; SD 0.09 mg/dL), which is characterised by a wide dispersion and includes values close to the lower physiological and analytical limits of detection. The control group also showed relatively elevated variability (0.20 ± 0.07 mg/dL), indicating that small absolute differences translate into large apparent percentage changes [21]. Overall, the isolated statistically significant differences were considered incidental, non-adverse, and unrelated to test item administration, since in rats, bilirubin is present at very low basal concentrations and exhibits limited dynamic range, which reduces its sensitivity as a stand-alone marker of hepatobiliary function in the absence of corroborative changes in liver enzymes or histopathology [22].

No test item-related adverse effects were observed in urinalysis parameters following 28 days of administration. In males, urine volume, pH, specific gravity, and urobilinogen concentrations were comparable with controls at all dose levels, with no statistically significant or dose-dependent changes (Table 7). In females, urine volume was increased at 1000 and 2000 mg TOS/kg bw/day, reaching statistical significance at 2000 mg TOS/kg bw/day (10.60 mL vs 8.00 mL in controls, +32.5% compared to control). However, this finding was not accompanied by changes in urine pH, specific gravity, urobilinogen, serum markers of renal function, or microscopic renal findings, and was therefore considered non-adverse.

Table 7.

Urinalysis parameters in male and female rats following 28-day oral administration of test item (Mean ± SD).

Sex	Parameter	Control	500 mg TOS/kg bw/day	1000 mg TOS/kg bw/day	2000 mg TOS/kg bw/day
Male	Urine volume (mL)	11.80 ± 2.66	12.60 ± 2.07	11.50 ± 2.64	12.60 ± 1.84
	pH	6.95 ± 0.37	7.15 ± 0.24	7.30 ± 0.35	7.05 ± 0.16
	Specific gravity	1.018 ± 0.000	1.019 ± 0.000	1.016 ± 0.000	1.016 ± 0.000
	Urobilinogen (µmol/L)	3.20 ± 0.00	3.20 ± 0.00	3.20 ± 0.00	3.20 ± 0.00
Female	Urine volume (mL)	8.00 ± 1.94	8.20 ± 2.49	10.00 ± 1.94	10.60 ± 1.35*
	pH	7.00 ± 0.00	7.05 ± 0.16	6.95 ± 0.37	7.00 ± 0.00
	Specific gravity	1.015 ± 0.000	1.016 ± 0.000	1.015 ± 0.000	1.014 ± 0.000
	Urobilinogen (µmol/L)	3.20 ± 0.00	3.20 ± 0.00	3.20 ± 0.00	3.20 ± 0.00

Statistically significant vs control (p < 0.05). Values are mean ± SD (n = 10/sex/group).

TOS: Total Organic Solids.

Minor qualitative variations were observed in both sexes, including differences in urine colour (light yellow to yellow) and occasional slightly cloudy samples, without a dose-related pattern. Leukocytes were detected as trace amounts in a small number of animals across all groups in males (4/10 animals) at the high dose (compared to 2/10 in controls). This finding was considered not toxicologically relevant due to its minimal severity (trace only), absence of a clear dose–response relationship, and lack of associated changes in nitrites, bacteria, or histopathological findings in the urinary tract. Variations in ketone bodies, protein, and occult blood were sporadic, of low magnitude, and did not show dose-dependency; notably, the incidence and severity of proteinuria were comparable across groups and not associated with microscopic or histopathological correlates. Microscopic examination of urine sediment revealed occasional epithelial cells, red blood cells, and white blood cells at low incidence in both control and treated groups. Triple phosphate crystals were commonly observed in males, including controls, and are considered a background finding in this species, while absent in females. It must be noted that normal rat urine contains varying crystals, and these calcium phosphate-containing precipitates often occur in treated rats or in rats that are frequently handled for purposes such as oral gavage, particularly in males [24], [23]. Crystalluria is a well-documented spontaneous finding in rats and is influenced by urine concentration, pH, dietary mineral composition, and physiological hydration status and are not considered adverse in the absence of associated renal histopathology or functional impairment. No casts, bacteria, yeast, or test item-related material were detected in either sex. Overall, the urinalysis findings were considered within normal biological variation and unrelated to administration of the test item.

No test item-related changes were observed in absolute organ weights or in organ weights relative to terminal body weight or brain weight in males or females following 28 days of administration (data not shown). In males, mean weights of all examined organs, including adrenals, thymus, spleen, heart, brain, kidneys, liver, testes, epididymides, and prostate with seminal vesicles/coagulating glands, were comparable with controls across all dose groups, with no statistically significant differences and no consistent dose-related trends.

In females, a statistically significant increase in absolute ovarian weight was observed at 500 mg TOS/kg bw/day (+30.86% compared to control). At the same dose level, increases were also noted in ovarian weight relative to body weight and brain weight (+29.58% and +32.05% compared to control), together with a slight increase in liver weight relative to body weight (+9.29% compared to control). However, these findings were not present at 1000 or 2000 mg TOS/kg bw/day and therefore lacked a dose–response relationship. In addition, the ovarian and liver values remained within the expected physiological range for rats of this age and strain, and no correlated macroscopic or microscopic findings were identified in the reproductive organs or liver. Ovarian weight is known to fluctuate with the oestrous cycle stage in rats, and therefore females necropsied at non-synchronised cycle stages may exhibit high inter-individual variability. Consequently, isolated statistically significant differences in ovarian weight in the absence of dose–response and histopathological correlates are considered incidental and not toxicologically relevant [26], [25]. Weights of other female organs, including adrenals, thymus, spleen, heart, brain, kidneys, and uterus, were comparable with controls and showed no treatment-related trends. Overall, the isolated statistically significant differences observed in females at the low-dose level were considered incidental and not toxicologically relevant [27]. Therefore, no adverse effects on organ weights were attributed to administration of the test item up to 2000 mg TOS/kg bw/day.

Finally, at terminal sacrifice on microscopic evaluation, no test item-related histopathological changes were noted at highest dose of 2000 mg TOS/kg bw/day in both sexes when compared with control. Two incidences of mineralization of tubules in kidneys, infiltration of mononuclear cells in liver and decreased secretion of sublingual salivary gland were observed in female and male rats respectively at 2000 mg TOS/kg bw/day with one incidence of each in their respective control groups. All these changes were not attributed to the test item and were likely considered as spontaneous and/or incidental as the number of incidences were minimal and there were no clinical pathology correlates. All other microscopic findings observed in the study animals of both the sexes at terminal sacrifice were considered incidental, spontaneous and/or physiological, isolated incidences with minimal severity and not related to the test item, because they were randomly distributed across the test item and concurrent vehicle control groups and/or were generally observed in the rats of this age and strain.

4. Discussion

The bacterial reverse mutation assay was conducted to evaluate the mutagenic potential of Convero® under OECD 471-compliant conditions using multiple S. typhimurium strains and E. coli WP2 uvrA (pKM101), both in the presence and absence of metabolic activation. The test item demonstrated adequate solubility and stability under the experimental conditions, with no precipitation observed at relevant concentrations. Furthermore, no cytotoxicity was detected across the tested concentration range, indicating that bacterial exposure was not limited by toxic effects and that the assay operated within an appropriate concentration window. Convero® did not induce an increase in revertant colony numbers in any tester strain, either with or without S9 metabolic activation, indicating a lack of direct or metabolically activated mutagenic activity. The absence of mutagenic response was consistent across all tested concentrations and strains, and no dose–response relationship was observed. The validity of the study is supported by the performance of all controls. Positive controls produced the expected significant increases in revertant colonies, confirming the responsiveness of the bacterial strains and the metabolic activity of the S9 fraction. In addition, vehicle, basal, and sterility controls were within acceptable historical ranges, ensuring the reliability of the experimental system. Overall, under the conditions of this study, Convero® did not demonstrate mutagenic potential in the bacterial reverse mutation assay. These findings support the conclusion that Convero® is non-mutagenic under the tested conditions.

Additionally, an in vitro micronucleus assay was conducted to evaluate the clastogenicity and aneugenicity potential of Convero® in cultured human peripheral blood lymphocytes under OECD 487-compliant conditions. Cytotoxicity was limited to low or moderate levels across treatment conditions, with the highest reductions in cell proliferation remaining within acceptable ranges for assay interpretation. These findings indicate that sufficient cellular exposure was achieved without excessive toxicity that could compromise the evaluation. Convero® did not induce a statistically significant increase in micronucleated binucleate cells at any tested concentration, either in the presence or absence of metabolic activation. Micronucleus frequencies remained comparable to concurrent vehicle controls, showed no concentration-related trend, and were within the laboratory's historical control ranges. The absence of response under both activated and non-activated conditions indicates that Convero® did not exhibit clastogenic or aneugenic activity, either directly or following metabolic conversion. The validity of the study was confirmed by the appropriate responses of the positive controls. Vinblastine sulphate, cyclophosphamide monohydrate, and mitomycin C each produced clear and statistically significant increases in micronucleus frequency under their respective treatment conditions, demonstrating the sensitivity of the test system and the functional activity of the metabolic activation system where applicable. In addition, vehicle control values were within acceptable historical ranges, supporting the reliability of the assay. Overall, under the conditions of this study, Convero® did not demonstrate genotoxic potential in the in vitro mammalian cell micronucleus test. These findings support the conclusion that Convero® is not clastogenic nor aneugenic under the tested conditions.

These outcomes are consistent with the established safety profile of food enzymes produced via microbial fermentation, where negative results in bacterial reverse mutation and mammalian cell assays are commonly reported across multiple EFSA evaluations of food enzymes, including inulinase from Aspergillus oryzae, β-fructofuranosidase from Trichoderma reesei [15], and alternansucrase from genetically modified E. coli [14], all of which concluded no genotoxic concern under their intended conditions of use. A similar conclusion was reached for proline-specific oligopeptidase (Tolerase® G), an enzyme preparation of prolyl-oligopeptidase produced with a genetically modified Aspergillus niger self-clone strain intended to be used as Novel Food, where EFSA evaluated a Tier 1 genotoxicity testing strategy, including the “treat-and-wash” bacterial reverse mutation assay, and an in vitro chromosome aberration test, and concluded that the data do not indicate genotoxicity [28].

The present 28-day repeated dose oral toxicity study was conducted to evaluate the systemic toxicity potential of Convero® (inulosucrase) in Sprague Dawley rats at dose levels of 500, 1000, and 2000 mg TOS/kg bw/day. Overall, the study demonstrated that oral administration of the test item did not result in any treatment-related adverse effects under the conditions tested. Dose formulation analysis confirmed the stability, homogeneity, and accuracy of dosing, with all analytical results within acceptable limits and no evidence of contamination in vehicle controls. This confirms that animals were exposed to the intended dose levels throughout the study. No mortality, morbidity, or treatment-related clinical signs were observed, indicating an absence of overt systemic toxicity. Similarly, body weight development and feed consumption were unaffected across all dose groups, suggesting no impact on general health status or metabolic performance. Although a statistically significant reduction in body weight gain was observed in males at 500 mg/kg bw/day during the first week, the absence of a dose-response relationship, lack of persistence, and normal terminal body weight indicate that this finding was incidental and not biologically meaningful.

Clinical pathology evaluation, including haematology, coagulation, clinical chemistry, and urinalysis, revealed a few statistically significant differences; however, these changes were minimal in magnitude, lacked dose-dependency, and were within normal biological variation. Importantly, no correlating histopathological or organ weight changes were observed, further supporting their non-adverse nature. No toxicologically relevant changes were detected in organ weights, macroscopic examination, or histopathology, including in target organs such as liver, kidneys, spleen, and reproductive tissues. This strongly supports the absence of systemic organ toxicity. Functional observational battery and ophthalmological assessments further confirmed the absence of neurobehavioral or sensory toxicity. Taken together, the absence of treatment-related effects across all evaluated endpoints demonstrates that Convero® (inulosucrase) did not induce systemic toxicity up to the highest tested dose of 2000 mg TOS/kg bw/day in Sprague Dawley rats under the conditions of this 28-day study.

Overall, considering the nature of the test item as a recombinant protein intended for oral ingestion, the absence of consistent dose–response relationships, and the lack of corroborative changes across clinical pathology, organ weights, and histopathology, the findings observed in this study are not considered toxicologically relevant. The minor variations detected in haematology, clinical chemistry, urinalysis, and organ weight parameters were isolated, of low magnitude, and largely within the range of physiological or historical control values for the species and strain. In particular, the renal-related observations, including sporadic crystalluria, slight changes in urine volume, and isolated microscopic mineralization, are consistent with well-documented background findings in rats and are known to be influenced by physiological variability, urine concentration, handling, and dietary factors, rather than representing treatment-related nephrotoxicity in the absence of supporting functional or histopathological evidence. Similarly, transient and non-dose-dependent fluctuations in electrolyte and bilirubin values are considered reflective of normal biological variability and the inherently low dynamic range of these parameters in rodents. Importantly, no adverse effects were identified in key target organs, including kidney, liver, or hematopoietic tissues, and no consistent systemic pattern indicative of toxicity was observed at any dose level up to 2000 mg TOS/kg bw/day. Taken together, the overall data indicate that administration of the test item did not induce any biologically or toxicologically meaningful effects under the conditions of this study.

The findings of the present study are consistent with the growing body of evidence supporting the favourable toxicological profile of recombinant proteins produced by Komagataella phaffii through precision fermentation. In particular, recent GLP studies on recombinant human lactoferrin reported no treatment-related adverse effects following repeated oral administration for both 14 and 28 days at doses up to 2000 mg/kg bw/day [29], [30]. These findings closely align with the absence of treatment-related toxicity observed in the present 28-day repeated-dose study. Similarly, studies evaluating recombinant proteins produced in K. phaffii using comparable genotoxicity test batteries, have consistently reported no evidence of genotoxic potential [32], [31]. Beyond studies with similar experimental designs, longer-term investigations have also demonstrated favourable safety profiles. For example, brazzein produced in K. phaffii showed no adverse effects in a 90-day oral toxicity study [32], while other recombinant proteins expressed in K. phaffi, including recombinant batroxobin and the fungal immunomodulatory protein GMI, similarly showed no treatment-related systemic toxicity following repeated administration [33], [31]. Collectively, these findings support the favourable toxicological profile of recombinant proteins produced using K. phaffii when appropriately characterised and manufactured under controlled conditions.

In this study, the NOAEL of Convero® (inulosucrase) in rats was established at 2000 mg TOS/kg bw/day in a 28-day repeated-dose toxicity study. This dose could be selected as the high dose for a long-term toxicity study in rats, e.g. a 90-day repeated dose toxicity study according to OECD Test Guideline 408. For human safety considerations, application of standard uncertainty factors (10-fold for interspecies variability and 10-fold for intraspecies variability) results in an overall 100-fold uncertainty factor applied to the NOAEL. Given that the point of departure is derived from a 28-day study, an additional uncertainty factor is required to account for extrapolation to potential chronic exposure. This subacute-to-chronic factor is typically applied in the range of 2–10, depending on the regulatory jurisdiction, the nature of the substance, and the completeness of the toxicity dataset (weight of evidence).

The present study has some limitations that should be considered when interpreting the findings. First, the repeated-dose study was limited to 28 days and did not include a recovery group; therefore, the results are restricted to the exposure duration and endpoints evaluated. However, no treatment-related adverse effects or target organ toxicity were observed at any dose, and therefore there was no indication that additional recovery assessments were warranted. Likewise, dedicated immunotoxicity or allergenicity assessments were not performed, as these are not standard endpoints of OECD Test Guideline 407, no treatment-related findings suggestive of immune system effects were identified, and allergenicity is evaluated through a separate weight-of-evidence approach rather than conventional toxicological testing. Finally, the present work focused on hazard identification and did not include an assessment of human dietary exposure or margins of exposure, as these form part of the overall regulatory safety assessment rather than the toxicological evaluation itself. Accordingly, the conclusions should be interpreted within the scope of the endpoints and experimental conditions investigated.

5. Conclusion

This study evaluated the genotoxicity and 28-day repeated-dose oral toxicity of Convero®, a recombinant inulosucrase enzyme produced by precision fermentation using Komagataella phaffii. All studies were conducted in compliance with GLP and followed relevant OECD test guidelines. The bacterial reverse mutation assay and the in vitro micronucleus test demonstrated that Convero® does not induce gene mutations, chromosomal damage, or aneugenic effects under the conditions tested. In the 28-day repeated-dose oral toxicity study, no treatment-related adverse effects were observed at dose levels up to 2000 mg TOS/kg bw/day, the highest dose tested. Isolated statistically significant changes in clinical pathology, organ weights, or urinalysis parameters were small in magnitude, lacked dose–response relationships, and were not associated with corroborative histopathological findings, and were therefore considered incidental and within the range of normal biological variability.

From a mechanistic perspective, Convero® is an enzyme expected to undergo at least partial proteolytic degradation in the gastrointestinal tract, although its engineered stability may confer some resistance to digestion. Therefore, systemic exposure to intact protein cannot be entirely excluded. Importantly, the absence of treatment-related effects in the in vivo toxicity study demonstrates that any absorbed fraction, if present, does not result in adverse systemic effects under the conditions tested. This is consistent with the absence of systemic toxicity observed in vivo. In addition, the lack of genotoxic activity in both bacterial and mammalian cell systems support the absence of intrinsic hazard potential. Overall, the combined evidence from in vitro genotoxicity testing and in vivo oral toxicity studies supports that Convero® does not present a toxicological concern under the conditions of intended use.

Disclaimer

The authors affiliated with Atova Regulatory Consulting SLU confirm that their involvement in the present work was limited exclusively to the review and critical evaluation of the study documentation and associated reports. They were not involved in the manufacture, production, or supply of the test item, nor in the design, conduct, execution, or direct oversight of the toxicological studies described herein. Accordingly, responsibility for the generation of raw experimental data, study conduct, and compliance with the relevant study protocols and Good Laboratory Practice (GLP) requirements rests solely with the conducting laboratory and the sponsor. The Atova-affiliated authors assume responsibility only for the assessment and review of the provided documentation and do not bear responsibility for the underlying experimental work or data generation.

CRediT authorship contribution statement

Fernando Rivero-Pino: Writing – original draft, Supervision. Hannah Lester: Writing – review & editing, Supervision. Bikram Kumar Pradhan: Writing – review & editing, Investigation. Rangappa Thannirappa: Writing – review & editing, Investigation. Mohan Cheluru Umesh: Writing – review & editing, Investigation. Prathyusha Thippana: Writing – review & editing, Investigation. Justina Daphne: Writing – review & editing, Investigation. Kumar Krishnachari: Writing – review & editing, Investigation. Chandrashekar Mataguru Doreswamy: Writing – review & editing, Supervision. Sajeev Justin Dev: Writing – review & editing, Supervision. Suresh Babu Venkataramaiah: Writing – review & editing, Supervision. Srinivas Seekallu: Writing – review & editing, Supervision, Project administration. Niels Wicke: Writing – review & editing, Supervision, Project administration, Funding acquisition.

Declaration of Competing Interest

Atova Regulatory Consulting SLU declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Zya Enzymes Ltd intends to use Convero® in products for sale and stands to benefit financially if Convero® is determined to be safe. Anthem Biosciences Ltd was contracted by Zya Enzymes Ltd to perform the toxicology studies reported in this paper. Zya Enzymes Ltd did not participate in any of the experimentation, evaluation or analysis. Authors from Zya Enzymes Ltd listed contributed minimal linguistic editing, but final approval of accuracy was given by Anthem Biosciences. Anthem Biosciences has no financial or other interest in Zya Enzymes Ltd or Convero® beyond the contracted work described in this paper and subsequent related toxicology studies.

Data availability

Access to the full dataset may be provided upon reasonable request and where required for regulatory or safety assessment by competent authorities.