methyl 2-hydroxybut-3-enoate

Endpoint summary

Description of key information

The tests were conducted with the purified and stabilised (registered) form of the test substance. 

 

In a 14-day oral range-finding study in male and female Wistar rats, the dose levels for a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test (main study) were chosen as 0, 20, 50 and 100 mg/kg bw/day based on dose-dependent test item related local effects in the stomach.

 

In a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD guideline 422, the test item administered at 20, 50 and 100 mg/kg bw/day by oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) and did not cause systemic toxicity in parental male and female Han:WIST rats. Local effects were detected macroscopically and microscopically in the forestomach in male and female animals at 50 and 100 mg/kg bw/day. This local gastric effect did not induce clear adverse systemic changes although early signs (slight effects on body weight, food consumption, salivation) were noted. Therefore, a NOEL/NOAEC was deduced in addition to the NOAEL. Based on these observations the No Observed Adverse Effect Levels (NOAEL), No Observed Effect Level (NOEL) and No Observed Adverse Effective Concentration (NOAEC) were determined as follows:

NOAEL for systemic toxicity of male/ female rats: 100 mg/kg bw/day
NOEL for systemic toxicity of male/ female rats: 20 mg/kg bw/day
NOAEL for reproductive performance of male/ female rats: 100 mg/kg bw/day
NOAEC (local effects stomach): 4 mg/mL

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-07-28 to 2022-03-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Test Guidelines: OPPTS 870.3650 Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening

Version / remarks:

2000-07

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han:WIST

Details on species / strain selection:

The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90. Hungary
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 80-85 days (P, males), 70-75 days (P, females)
- Weight at study initiation: 355-414 g (P, males), 178-226 g (P, females)
- Fasting period before study: No
- Housing: 2 animals of the same sex/cage (before mating), 1 male and 1 female / cage (during mating), 2 animals / cage (males after mating), individually (mated females) in type III polypropylene/polycarbonate cages
- Diet: Ad libitum (ssniff® SM R/M-Z+H complete diet for rats and mice)
- Water: Ad libitum
- Acclimation period: 20 days

DETAILS OF FOOD AND WATER QUALITY: The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used. Animals received tap water from watering bottles. Water quality control analysis and microbiological assessment are performed once in every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service (Váci út 172-174. Budapest, H-1138 Hungary). The quality control results are available at the laboratory's archives

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): Above 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 0 to day 49 (males), from day 0 to day 50-68, depending on the duration of the mating period (females), from day 0 to day 64 (males/females of the recovery group), from birth (day 0) to post-natal day 13 (offspring)

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered orally via gavage. The route of application was selected in compliance with international guidelines. The oral route is the anticipated route of human exposure to the test item.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (distilled water) in concentrations of 4, 10 and 20 mg/mL. Formulations were prepared in the formulation laboratory of the test facility not longer than three days beforehand and stored at 2 - 8°C until use.

VEHICLE
- Concentration in vehicle: 4, 10 and 20 mg/mL
- Amount of vehicle: 5 mL/ kg bw
- Batch No: 202104026, 202105039, 202107056

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Five aliquots of 10 mL of each formulation and five aliquots of control substance (vehicle) were taken two times and were analysed. Concentration of the test item in the dosing formulations varied between the range of 95 % and 103 % in comparison to the nominal values. The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front in a GLP compliant study. The recovery of the test item from the vehicle was within the acceptance criteria (102 and 101 % relative to nominal concentrations of 1 mg/mL and 200 mg/mL, respectively). The test item proved to be stable in distilled water at the intended concentrations at 5 ± 3 °C for three days.

Duration of treatment / exposure:

49 days (males), 50 days (females)

Frequency of treatment:

Daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control group

No. of animals per sex per dose:

12 animals per sex per dose (main study), 5 animals per sex of the control and 100 mg/kg bw/day group (recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen on the basis of the results of preliminary studies: Acute Oral Toxicity Test (see IUCLID section 2.1), Comet Assay (see IUCLID section 7.6.2) and 14-Day Oral Gavage Dose Range Finding Study (see IUCLID section 7.5). The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The changes in the forestomach (detected macroscopically and microscopically) in combination with effects on feed intake, body weight and body weight gain, behavioral changes and changes in hematological parameters observed above 100 mg/kg bw/day in previous studies were considered excessive and thus, 100 mg/kg bw/day was chosen as the highest dose. The low dose was chosen to induce no toxic effect. The mid dose is interpolated geometrically.
- Fasting period before blood sampling for clinical biochemistry: Approx. 16 hours (overnight)

Positive control:

Not required

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily (after the administration at approximately the same time and twice daily (signs of morbidity and mortality)
- Cage side observations checked in table 1 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the first exposure and once weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: On the first day of dosing and weekly thereafter and on the day of the necropsy (P, males); on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum (P, females). Body weight was measured on the day of necropsy for female animals subjected to organ weighing.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes (in g/animal/day)

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: One day after the last treatment or 15 days after the last treatment (recovery group)
- Anaesthetic used for blood collection: Isofluran CP®
- Animals fasted: Approx. 16 hours (overnight)
- How many animals: 5 animals per sex randomly selected from each group
- Parameters checked in table 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: One day after the last treatment or 15 days after the last treatment (recovery group)
- Anaesthetic used for blood collection: Isofluran CP®
- Animals fasted: Approx. 16 hours (overnight)
- How many animals: 5 animals per sex randomly selected from each group
- Parameters checked in table 3 were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: On post-partum/ post-natal day 1 (females), one day after the last treatment (males) or 15 days after the last treatment (recovery group)
- Animals fasted: Approx. 16 hours (overnight)
- How many animals: From all dams on post-partum/ post-natal day 1, from all parent male animals at termination, from animals in the recovery groups at termination (15th day of recovery period)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During the last exposure week
- Dose groups that were examined: 5 animals per sex randomly selected from each group
- Battery of functions tested: Sensory activity / grip strength / motor activity. A modified Irwin test was performed. (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968)

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded.

HISTOPATHOLOGY: Yes. At the time of termination, body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all male adult animals were determined. Paired organs were weighed together. In addition, for five males and females randomly selected from each group, adrenal glands, brain, heart, kidneys, liver, spleen and thymus were weighed. The weight of thyroid gland was determined in all male and female animals at the termination of the treatment and at the end of recovery period. Absolute organ weight was recorded. Relative organ weights (to body and brain weight) were calculated and reported.

The tissues indicated in Table 4 were preserved for five male and five female animals randomly selected from each group. Full histopathology examinations were performed on these preserved organs and tissues of the randomly selected animals in the control and high dose group. Based on macroscopic findings in high dose treated animals, the stomach was preserved and histologically processed and evaluated in all animals (male and female, control, 20, 50 and 100 mg/kg bw/day). Additionally, lungs, kidneys, uterus and skin with necropsy findings were processed and evaluated histologically in some parental animals and offspring.

Detailed histological examination was performed on the ovaries, uterus with cervix, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals of control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Optional endpoint(s):

Optional endpoints: Yes (see below)

Other examinations:

OESTROUS CYCLICITY
Oestrous cycle was monitored by examining vaginal smears each day before the treatment started from each animal being considered for study for two weeks. Estrous cycle was evaluated and considered at randomization. Vaginal smears were also prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating. Vaginal smears were prepared and evaluated in each female animal on the day of necropsy. Vaginal smears were stained with 1 % aqueous methylene blue solution then were examined with a light microscope.

SPERM PARAMETERS
Parameters examined in all P male parental generation:
Weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole, sperm count in testes, sperm morphology, histology of epididymides, prostate, seminal vesicles, and coagulating glands

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible. For the evaluation of data in the recovery group, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed. Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment period:
Test item related findings (salivation, nuzzling up the bedding material) were detected in male and female animals at 50 and 100 mg/kg bw/day immediately after the administration and signs ceased after a short duration. The effects were considered to be linked to the irritation noted in the stomach and considered a systemic effect due to local tissue damage but, because of the short duration, not considered adverse.

Noisy breathing in one male animal (1/5 at 50 mg/kg bw/day) and alopecia on the abdomen in one female animal (1/5 at 50 mg/kg bw/day) were not considered to be treatment-related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male animal was found dead at 100 mg/kg bw/day on Day 9. Necropsy and histopathologic observations indicate that gavage treatment resulted in suffocation and death of this animal. There was no mortality in control, 20 or 50 mg/kg bw/day groups during the course of study (male and female).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment period:
Slightly reduced mean body weight gain resulted in minor changes in the mean body weight in male animals in each dose group (less than 6 % relative to control). The body weight gain of these animals was depressed significantly during week 1 then recovered.

No effects were detected in females. Findings were considered to be linked to the local irritative effect in the stomach.

Recovery period:
No differences in mean body weights were detected after the recovery period in male and female animals.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A slight, toxicologically not relevant reduction in the mean daily food intake was in accordance with the minor body weight depression in male animals at 100 mg/kg bw/day during week 1 and was related to the local irritative effect in the stomach.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment period:
Some statistically significant difference with respect their control was detected at the lower mean corpuscular hemoglobin concentration (MCHC) at 20 and 100 mg/kg bw/day and at the higher mean platelet count (PLT) at 100 mg/kg bw/day in the male animals. These effects were not considered adverse.

There were no statistically or biologically significant differences in the examined hematology and blood coagulation parameters between the control and test item administered female animals at the end of the treatment period.

Recovery period:
In the male animals at 100 mg/kg bw/day, statistical significance with respect to the control was observed at the slightly lower mean percentage of reticulocytes (RET).

In the female animals at 100 mg/kg bw/day, elevated mean percentage of lymphocytes (LYM), elevated mean concentration of hemoglobin (HGB) and elevated mean hematocrit value (HCT) were detected when compared to the control at the end of the recovery period.

All these changes were considered to be variations and toxicologically not relevant due to the minor degree or the lack of related findings. Individual values met well (i.e., were within or marginal to) the historical control ranges except for PLT of two male animals at 100 mg/kg bw/day. As no effects were noted at related hematological parameters or histopathology, the variation noted for PLT was not considered biologically relevant.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment period and recovery period:
Slight elevation in the alanine amino transferase activity, total bilirubin, cholesterol, urea and albumin at 100 mg/kg bw/day (male or female) might be related to the enhanced renal or hepatic function but were not rated adverse as there were no accompanying histological alterations and were within or close to the historical control range.

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Sporadic differences in organ weights were not dose-dependent, within the historical control range or not related to other effects like histopathological changes.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment period:
Dose-dependent test item related changes in the stomach (thickening of the wall, hemorrhage or congestion) at 50 mg/kg bw/day (male) and at 100 mg/kg bw/day (male and female) were observed. These effects were linked to local effects of the test item and were supported by histopathological investigations.

Other findings occurred at low frequencies without dose dependence and/or are common observations in untreated experimental rats of this strain with similar age and similar occurrence (e.g. pyelectasia and hydrometra).

Recovery period:
No treatment-related effects were observed after the recovery period.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histological examination revealed squamous cell hyperplasia in the stomach in male and female animals in connection with the local effect of 50 and 100 mg/kg bw/day of test item. Squamous cell hyperplasia is indicative of a restorative reaction following the irritative effect of the test item on the mucous membrane. Squamous cell hyperplasia in the stomach was partially reversible as it was detected in some male and female animals at the end of the recovery period.

Other findings occured sporadically, were not dose-dependent and/or within the historical control range.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: The NOEL was was based on slight systemic effects at 50 and 100 mg/kg bw/day which were considered to be secondary to local effects in the stomach.

Key result

Dose descriptor:

conc. level: NOAEC (local effects)

Effect level:

4 other: mg/mL

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: The NOAEC was based on local effects on the stomach at 10 and 20 mg/mL dosing concentrations.

Key result

Critical effects observed:

no

Conclusions:

In a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD guideline 422, the test item administered at 20, 50 and 100 mg/kg bw/day by oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition).

Adverse local effects were detected macroscopically and microscopically in the forestomach in male and female animals at 50 and 100 mg/kg bw/day. These local gastric effects induced early signs of systemic toxicity in parental male and female rats (slight effects on body weight, food consumption, salivation). As higher substance concentrations induced excessive local effects in the dose-range finding test, acute oral toxicity test and in vivo genotoxicity studies, 100 mg/kg bw/day was considered the maximum tolerated dose. As systemic effects were considered secondary to the local effects, a local NOAEC was deduced in addition to the systemic NOAEL/ NOEL. The development of the F1 offspring was not impaired from birth up to post-natal day 13 as far as investigated in this study after repeated oral administration of dams at 20, 50 and 100 mg/kg bw/day. Based on these observations the No Observed Adverse Effect Levels (NOAEL), No Observed Effect Level (NOEL) and No Observed Adverse Effective Concentration (NOAEC) were determined as follows
NOAEL for systemic toxicity of male/ female rats: 100 mg/kg bw/day
NOEL for systemic toxicity of male/ female rats: 20 mg/kg bw/day
NOAEL for reproductive performance of male/ female rats: 100 mg/kg bw/day
NOAEC (local effects stomach): 4 mg/mL

Executive summary:

The purpose of this study was to obtain initial information on the systemically toxic potential of test item and on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 20, 50 and 100 mg/kg bw/day compared to control animals according to OECD 422 and GLP. As a screening test, it was intended to provide initial information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time and on the possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 13 post-partum associated with administration of repeated maternal doses. Satellite animals were included in the control and in the high dose groups for follow-up observations without treatment to detect the potential reversibility or persistence of possible toxic effects for 14 days. Methyl Vinyl Glycolate (MVG) was administered orally (by gavage) once daily at 0 (vehicle only), 20, 50 and 100 mg/kg body weight (mg/kg bw/day) doses to four groups of Han:WIST rats consisting of 17 animals per sex in the control and high dose groups and 12 animals per sex in the low and mid dose groups in concentrations of 4, 10 and 20 mg/mL corresponding to a 5 mL/kg bw dosing volume. A group of vehicle (distilled water) treated animals served as a control. 5 animals/ sex in the control and high dose groups were observed to detect the potential reversibility or persistence of possible toxic effects for 14 days. The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Methyl Vinyl Glycolate (MVG) was stable in the vehicle in concentrations of 1 mg/mL and 200 mg/mL for three days in a refrigerator (at 5 ± 3 °C). The concentration of the test item in the dosing formulations administered to the animals was checked once during the study. Methyl Vinyl Glycolate (MVG) concentrations in the dosing formulations varied within the range of 95 % and 103 % (in comparison to the nominal values) and confirmed the proper preparation of the dosing formulations. All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 49 days). Dams were additionally exposed through the gestation period and up to lactation days 13-16, i.e., up to the day before necropsy (altogether for 50-68 days). Animals in the recovery groups were administered up to and including Day 49 (altogether for 50 days). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period and during the mating period until evidence of copulation. Vaginal smears were also prepared and investigated on the day of the necropsy for each dam. The dams were allowed to litter and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on postnatal day 13 or shortly thereafter. Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4 and TSH) from 2-6 pups per litter (in litters with at least 10 pups) on post-natal day (PND) 4, from all dams and from 4-7 pups per litter at termination on post-partum/post-natal day 13 and from all parent male animals at termination. Serum level of FT3, FT4 and TSH were determined in the samples of parental male and female animals and PND 13 offspring. Five dams and their male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology and blood coagulation, clinical chemistry, gross necropsy, organ weighing and histopathology examination. All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight and weight of the testes and epididymides and prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined. In addition, for five males and females randomly selected from each group, adrenal glands, brain, heart, kidneys, liver, spleen, thymus and thyroid glands were weighed. Thyroid glands were preserved from all adult males and females and one male and one female pup per litter for the intended subsequent histopathological examination. Histopathology examination was performed on ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals (male and female) in the control and high dose groups. Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose groups (main and recovery). In addition, the stomach was processed and evaluated histologically in all male and female animals in control, low, mid and high dose groups based on necropsy findings. Organs showing macroscopic findings at the necropsy were also processed and examined histologically (lungs, kidneys, skin and uterus) in parental animals and offspring in the low and mid dose groups. The results of this study were summarized as follows:

 

Mortality

One male animal was found dead at 100 mg/kg bw/day on Day 9 presumably due to the gavage treatment. Necropsy and histopathology observations revealed signs of suffocation causing death of this animal.

 

Clinical and functional observation

Treatment or test item related findings (salivation, nuzzling up the bedding material) were detected in male and female animals at 50 and 100 mg/kg bw/day immediately after the administration. There were no clinical signs during the recovery period in male or female animal in the control and 100 mg/kg bw/day. Functional observation battery did not demonstrate any alterations in the behavior or reactions to different type of stimuli of selected male or female animals in the control, 20, 50 or 100 mg/kg bw/day groups at the end of the treatment period.

 

Body weight and body weight gain

The body weight development was not influenced in male or female animals at 20 and 50 mg/kg bw/day during the entire treatment period. Slightly reduced mean body weight gain resulted in minor changes in the mean body weight (less than 6 % relative to control) in male animals at 100 mg/kg bw/day. This finding was not considered adverse but was regarded as secondary to the pronounced adverse local effects on the stomach at this dose.

 

Food consumption

The mean daily food consumption was not adversely affected at 20, 50 or 100 mg/kg bw/day dose levels during the entire treatment period (pre-mating and post-mating periods in male animals; during the pre-mating, gestation and lactation periods in female animals). The mean daily food consumption was slightly reduced in male animals at 100 mg/kg bw/day during the first week of observation period and being in accordance with slightly reduced body weight gain in this group.

 

Estrous cycle

A test item influence on the estrous cycle was not found at any dose level (20, 50 or 100 mg/kg bw/day).

 

Delivery and pregnancy data of female animals

There were no toxicologically relevant differences in the evaluated parameters of delivery between the control and test item treated groups (20, 50 or 100 mg/kg bw/day).

 

Reproductive performance

The examined parameters of reproductive performance were not affected by the test item at 20, 50 or 100 mg/kg bw/day in male or female animals.

 

Clinical pathology – Hematology, blood coagulation, clinical chemistry

Clinical pathology investigation did not reveal test item related changes in the examined hematology, blood coagulation or clinical chemistry parameters at 20, 50 or 100 mg/kg bw/day.

 

Serum thyroid hormones

The thyroid hormone (FT3, FT4 and TSH) levels were not affected by the test item in parental male and female animals (20, 50 and 100 mg/kg bw/day) and in offspring sampled on postnatal day 13.

 

Necropsy

Gross necropsy observations revealed test item related changes in the stomach (thickening of the wall, hemorrhage or congestion) at 50 mg/kg bw/day (male) and at 100 mg/kg bw/day (male and female), which were linked to the local effect of the test item.

 

Organ weight

There were no adverse test item related alterations in the weights of the examined organs in male or female animals at 20, 50 or 100 mg/kg bw/day.

 

Histopathology

Histological examination revealed squamous cell hyperplasia in the stomach in male and female animals in connection with the local effect of 50 and 100 mg/kg bw/day of test item. Squamous cell hyperplasia in the stomach was partially reversible as it was detected in some male and female animals at the end of the recovery period.

 

Offspring

The offspring’s development was undisturbed at 20, 50 and 100 mg/kg bw/day from birth to post-natal day 13. No effect on the mortality, clinical signs, body weight development, anogenital distance (male and female) or nipple retention (male) were detected.

 

Conclusion

Under the conditions of the present study, Methyl Vinyl Glycolate (MVG) administered at 20, 50 and 100 mg/kg bw/day by oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition). Adverse local effects were detected macroscopically and microscopically in the forestomach in male and female animals at 50 and 100 mg/kg bw/day. These local gastric effects induced early signs of systemic toxicity in parental male and female rats (slight effects on body weight, food consumption, salivation). As higher substance concentrations induced excessive local effects in the dose-range finding test, acute oral toxicity test and in vivo genotoxicity studies, 100 mg/kg bw/day was considered the maximum tolerated dose. As systemic effects were considered secondary to the local effects, a local NOAEC was deduced in addition to the systemic NOAEL/ NOEL. The development of the F1 offspring was not impaired from birth up to post-natal day 13 as far as investigated in this study after repeated oral administration of dams at 20, 50 and 100 mg/kg bw/day. Based on these observations the No Observed Adverse Effect Levels (NOAEL), No Observed Effect Level (NOEL) and No Observed Adverse Effective Concentration (NOAEC) were determined as follows:

 

NOAEL for systemic toxicity of male/ female rats: 100 mg/kg bw/day

NOEL for systemic toxicity of male/ female rats: 20 mg/kg bw/day

NOAEL for reproductive performance of male/ female rats: 100 mg/kg bw/day

NOAEL for F1 Offspring: 100 mg/kg bw/day

NOAEC (local effects stomach): 4 mg/mL

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2021-02-26 to 2021-06-02

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

14-day range-finding study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OPPTS 870.3050 Repeated Dose 28–Day Oral Toxicity Study in Rodents, EPA Health Effects Test Guidelines.

Version / remarks:

2000-07

Deviations:

yes

Remarks:

Range-finding study: Shorter time period and less parameters investigated.

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

2008-05-31

Deviations:

yes

Remarks:

Range-finding study: Shorter time period and less parameters investigated.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008-10-03

Deviations:

yes

Remarks:

Range-finding study: Shorter time period and less parameters investigated.

GLP compliance:

no

Remarks:

This study was not performed according to GLP compliances, however the principles of GLP were followed and all data were recorded and retained. All procedures were performed according to the laboratory's SOP´s.

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han:WIST

Details on species / strain selection:

The rat is a commonly used species for toxicological studies in accordance with international recommendations. The Wistar rat was the system of choice because it has been the preferred and most commonly used species for oral toxicity tests and is a well-known laboratory model with sufficient historical data.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 42 – 45 days (both males and females)
- Weight at study initiation: 201 – 232 g (male animals), 140 – 160 g (female animals)
- Fasting period before study: No
- Housing: 5 animals of the same sex/ cage were housed in type IV polypropylene/ polycarbonate cages with certified laboratory wood bedding.
- Diet: Ad libitum (except for overnight food deprivation before the blood sampling)
- Water: Ad libitum
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY: The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used. Water quality control analysis and microbiological assessment are performed once every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service. The quality control results are available at the laboratory's archives.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): More than 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 0 to day 14

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered orally via gavage. The route of application was selected in
compliance with international guidelines. The oral route is the anticipated route of human exposure to the test item.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle (distilled water) in concentrations of 5, 10, 18 and 36 mg/mL in the formulation laboratory of Test Facility beforehand not longer than for three days and stored at 5 ± 3 °C until use.

VEHICLE
- Concentration in vehicle: 5, 10, 18 and 36 mg/mL
- Amount of vehicle: 5 mL/kg bw/day
- Lot/batch no.: 202010099 (expiry date: April 19, 2021), 202010095 (expiry date: April 01, 2021)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical dose verification of the formulations was performed once during this DRF study.
Five aliquots of 10 mL of each formulation administered to the animals (5, 10, 18 and
36 mg/mL) and five aliquots of 10 mL control substance (vehicle) were taken. Measured concentrations of formulations applied in the study varied in the acceptable range – between 93% and 102% of the nominal concentrations (by sample results), thereby confirming proper dosing. The suitability of the chosen vehicle (recovery and stability) for the test item at the intended
concentrations was analytically verified up front. The recovery of the test item from the vehicle was within the acceptance criteria (102 and 101% relative to nominal concentrations of 1 mg/mL and 200 mg/mL, respectively). The test item proved to be stable in distilled water at the intended
concentrations at 5 ± 3 °C for three days. A separate analytical report (Study no. 941-100-5436) is available.

Duration of treatment / exposure:

14 days

Frequency of treatment:

Daily

Dose / conc.:

180 mg/kg bw/day (nominal)

Dose / conc.:

90 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose setting with 0, 25, 50, 90 and 180 mg/kg bw/day was based on the results of an acute oral toxicity test (see IUCLID section 7.2.1) and Comet assay (see IUCLID section 7.6.2). Doses were selected with the aim of inducing toxic effects but no mortality or suffering at the highest dose and a NOAEL at the lowest dose. In addition, the study should allow the dose setting for the planned OECD 422 study.
- Fasting period before blood sampling for clinical biochemistry: Yes, overnight

Positive control:

Not needed

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (at the beginning and end of each working day). On Day 0, animals were observed continuously for 30 minutes then 1, 2, 3 and 5 hours after the administration to estimate the peak time window of effects.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a day, after treatment at approximately the same time.
- Detailed clinical observations checked in table 1 were included.

BODY WEIGHT: Yes
- Time schedule for examinations: On Day 0 (prior to study start) and twice weekly – on Days 0, 3, 7, 10 and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, calculated as g food/animal/day
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No, body weight gain (g) between days was calculated.

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On day 14
- Anaesthetic used for blood collection: Yes (Isofluran CP®)
- Animals fasted: Yes, for approx. 16 hours before blood collection
- How many animals: All surviving animals
- Parameters checked in table 2+3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On day 14
- Animals fasted: Yes, for approx. 16 hours before blood collection
- How many animals: All surviving animals
- Parameters checked in table 4 were examined.

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. The external appearance (surface of the body, all orifices) was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically for each animal. All observations were recorded with details of the location, color, shape and size. Organs/tissues preserved and weighted are shown in table 5.

HISTOPATHOLOGY: Yes. Histopathological examinations were performed on the kidneys and liver in male and female animals in the control and high dose groups. Additionally, the stomach was processed and investigated histologically in all male and female animals in all groups based on the macroscopic findings.

Optional endpoint(s):

Optional endpoints: No

Statistics:

Statistical analysis was done with SPSS PC+ software for the following data: Body weight, Hematology, Blood coagulation, Clinical chemistry, Organ weights.

The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.

Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test. Frequency of clinical signs, ophthalmoscopy, pathological and histopathological findings by sex and dose was calculated.


Food consumption was calculated according to the following formula: Food consumption (g/animal/day) = Given food (g) - Remaining food (g) / Number of days * Number of animals per cage

The use of the word “significant” or “significantly” indicates a statistically significant difference between the control and the experimental groups. Significance was judged at a probability value of p < 0.05 and < 0.01. Male and female rats were evaluated separately.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Salivation before the treatment and nuzzling up the bedding material were only detected at 50, 90 or 180 mg/kg bw/day in relation to the administration procedure. These signs ceased shortly after the treatment (5 and 1 minutes, respectively).

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

180 mg/kg bw/day: Significantly reduced body weight gain in male animals between days 0-3 and a significantly higher body weight gain between days 7-10 was noted and resulted in about 7% reduction in body weight gain for the entire study period. A statistically significant reduced body weight compared to the concurrent control was observed in the male animals on day 7 (7% reduction) as a result of the lower mean body weight gain between Days 0-3.

The mean body weight was comparable in male animals in the control, 25, 50 and 90 mg/kg bw/day. No statistically significant or biologically relevant differences were noted during the entire observation period in females in all treatment groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

180 mg/kg bw/day: The mean daily food consumption was slightly reduced in male animals.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Elevated white blood cell count (males), elevated percentage of reticulocytes and platelet count (males and females) at 180 mg/kg bw/day and elevated platelet count in male animals at 90 mg/kg bw/day were considered to be related to the gastric effect and the noted local inflammation caused by the test item.

A slightly shorter mean prothrombin time (PT) was noticed in male animals treated with 50 mg/kg bw/day and shorter mean PT and higher mean platelet count (PLT) was noted in male animals treated with 90 mg/kg bw/day. At 180 mg/kg bw/day, a higher mean white blood cell count (WBC), shorter mean PT, shorter mean activated partial thromboplastin time (APTT), higher mean percentage of reticulocytes (RET) and higher mean platelet count were noted in male animals.

In the female animals at 50 mg/kg bw/day, slightly higher mean corpuscular volume (MCV) and lower mean corpuscular hemoglobin concentration (MCHC) were detected when compared to the control. This effect was however, not seen at higher doses and therefore considered to be accidental. Higher mean hematocrit (HCT), higher mean percentage of reticulocytes and higher mean platelet count was observed in the female animal at 180 mg/kg bw/day.

Changes in WBC, RET and PLT might be secondary to the gastric lesions in male or female animals at 90 or 180 mg/kg bw/day.

The statistically significant differences in other hematology parameters – MCV, MCHC, PT, APTT – were with low degree and were within of historical control ranges. In addition, in most cases no dose-response relationship was noted. Therefore, these effects were judged as natural variations or accidental without toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Sporadic statistically significant differences with respect to the control was probably indicative of biological variation and not related to the test item. In the lack of dose dependency, these changes are evaluated as accidental ones and not test item related findings.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Changes in the mean weights of some organs were not related to doses (liver, prostate, seminal vesicle and coagulating gland, as a whole, spleen in male animals) or were with minor degree (kidneys in female animals). Therefore, these findings were judged to be toxicologically not relevant.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Dose-dependent test item related changes in the stomach (thickening of the wall, hemorrhage, erosion, congestion, adhesion to diaphragm) were observed at 50 mg/kg bw/day (male) and at 90 and 180 mg/kg bw/day (male and female).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histological investigations revealed squamous cell hyperplasia accompanied with hemorrhages, occasionally with erosion and inflammatory cell infiltration in the nonglandular part of stomach (forestomach) in a dose related manner at 50 mg/kg bw/day (one male) and at 90 and 180 mg/kg bw/day (male and female).

In this study the development of squamous cell hyperplasia is likely to be linked to the local effect of 50, 90 mg/kg bw/day and 180 mg/kg bw/day of test item (as similar findings were noted already in the acute oral study and the Comet assay in vivo, see IUCLID section 7.6.2).

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

Remarks on result:

not determinable because of methodological limitations

Remarks:

This study was designed as a dose-range finding study for a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test. 100 mg/kg bw/day was chosen as the highest dose level for the main study based on pronounced local effects at 90 and 180 mg/kg bw/day which were accompanied with the onset of clinical signs at 180 mg/kg bw/day. 50 mg/kg bw/day was chosen as the mid-dose for the main study based on the local effect detected in the stomach of one male animal administered with 50 mg/kg bw/day. There were no test item related local or systemic effects at 25 mg/kg bw/day dose, thus, a dose of 20 mg/kg bw/day was chosen as the lowest dose in the main test.

Key result

Critical effects observed:

no

Conclusions:

Based on the observations made in this toxicity study, the dose levels for a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test (main study) were chosen as 0, 20, 50 and 100 mg/kg bw/day.

Executive summary:

The purpose of this study was to obtain first information on the toxic potential of the test item in rats after repeated administration at four dose levels to allow a dose-setting for a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test in Rats (main study according to TG OECD 422). The dose setting with 0, 25, 50, 90 and 180 mg/kg bw/day was based on the results of an acute oral toxicity test (Toxi-Coop study no. 941-423-5434) and Comet assay (Toxi-Coop study no. 941-489-5435) with Methyl Vinyl Glycolate (MVG). Doses of 0 (vehicle only), 25, 50, 90 and 180 mg/kg bw/day were orally administered (by gavage) to five groups of Han:WIST rats consisting of five animals per group and sex at a dosing volume of 5 mL/kg in concentrations of 5, 10, 18 and 36 mg/mL. A group of vehicle (distilled water) treated animals (n= 5/sex) served as a control. The suitability of the chosen vehicle for the test item was analytically verified based on a validated GLP compliant method. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. Measured concentrations of formulations applied in the study varied in the acceptable range (between 93 % and 102 % of the nominal concentrations), thereby confirming proper dosing. Detailed clinical observations were performed daily after the treatment. On Day 0, animals were observed continuously for 30 minutes then 1, 2, 3 and 5 hours after the administration to estimate the peak time window of effects. Body weights were recorded twice weekly. The food consumption was determined weekly to coincide with body weight measurements during the study. Clinical pathology (hematology, blood coagulation and clinical chemistry) and gross pathology examinations were conducted one day after the last treatment (on Day 14). Selected organs were weighed. The kidneys and liver were processed and examined histologically in all male and female animals in the control and high dose groups. Additionally, the stomach of all male and female animals was processed and investigated histologically in all groups based on the macroscopic findings and to facilitate the correct dose level selection for the main study. The results of this study were summarized as follows:

Mortality: There were no mortalities in the control, 25, 50, 90 or 180 mg/kg bw/day groups (male or female).

Clinical observations: The test item did not induce adverse clinical signs at 25 mg/kg bw/day. At 50, 90 and 180 mg/kg bw/day dose levels, detected clinical signs (salivation before the treatment and nuzzling up the bedding material) were timely closely linked to the administration procedure and were considered to result from the local effects on the stomach.

Body weight and body weight gain: The body weight development was not adversely influenced in male or female animals at 25, 50, 90 or 180 mg/kg bw/day by during the entire treatment period. The slightly depressed mean body weight gain in male animals at 180 mg/kg bw/day (about 7% less than the concurrent control) resulted also in minor changes in the mean body weight.

Food consumption: The mean daily food consumption was slightly reduced in male animals at 180 mg/kg bw/day during the first week of observation period and being in accordance with slightly reduced body weight gain in this group.

Hematology and blood coagulation: Elevated white blood cell count (male), elevated percentage of reticulocytes and platelet count (male and female) at 180 mg/kg bw/day and elevated platelet count in male animals at 90 mg/kg bw/day were probably related to the gastric effect (irritation and inflammation) of the test item.

Clinical chemistry: Clinical chemistry evaluation did not reveal pathological changes in the investigated parameters at 25, 50, 90 or 180 mg/kg bw/day (male and female).

Necropsy: Gross necropsy observations revealed test item related changes in the stomach (thickening of the wall, hemorrhage, erosion, congestion, adhesion to diaphragm) at 50 mg/kg bw/day (one male) and at 90 and 180 mg/kg bw/day (male and female).

Organ weight: The weights of investigated organs were not adversely affected by the test item at 25, 50, 90 or 180 mg/kg bw/day (male and female).

Histopathology: Histological examination revealed squamous cell hyperplasia accompanied with hemorrhages, occasionally with erosion and inflammatory cell infiltration in the nonglandular part of stomach (forestomach) in a dose related manner at 50 mg/kg bw/day (one male) and at 90 and 180 mg/kg bw/day (male and female).

Conclusion

Under the conditions of the present study, the test item induced changes in the forestomach (detected macroscopically and microscopically) after the consecutive 14-day oral (by gavage) administration of 90 mg/kg bw/day and 180 mg/kg bw/day doses to Han:WIST rats (male and female). Effects on feed intake, body weight and body weight gain were noted at 180 mg/kg bw /day. As a consequence of the local gastric effects, some hematological parameter also changed mainly in 180 mg/kg bw/day animals (male and female) and in male animals at 90 mg/kg bw/day. Local effect was detected in the stomach in one male animal administered with 50 mg/kg bw/day. There were no test item related local or systemic effects at 25 mg/kg bw/day dose (male or female).

Based on the marked local effects observed macroscopically and microscopically at 180 mg/kg bw/day, accompanied by hematological effects, the onset of clinical signs and decreased body weight gain, this dose was considered to cause excessive toxicity (adverse local effects followed by the onset of systemic signs of toxicity). Thus, the dose levels for a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test (main study) are as follows:

Group 1: Control

Group 2: 20 mg/kg bw/day

Group 3: 50 mg/kg bw/day

Group 4: 100 mg/kg bw/day

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Reliable without restrictions (RL1)

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

14-day range-finding study, RL2

The purpose of this study was to obtain first information on the toxic potential of the test item in rats after repeated administration at four dose levels to allow a dose-setting for a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test in Rats (main study according to TG OECD 422). The dose setting with 0, 25, 50, 90 and 180 mg/kg bw/day was based on the results of an acute oral toxicity test (Toxi-Coop study no. 941-423-5434) and Comet assay (Toxi-Coop study no. 941-489-5435) with Methyl Vinyl Glycolate (MVG) and has been chosen in agreement with the Sponsor. Doses of 0 (vehicle only), 25, 50, 90 and 180 mg/kg bw/day were orally administered (by gavage) to five groups of Han:WIST rats consisting of five animals per group and sex at a dosing volume of 5 mL/kg in concentrations of 5, 10, 18 and 36 mg/mL. A group of vehicle (distilled water) treated animals (n= 5/sex) served as a control. The suitability of the chosen vehicle for the test item was analytically verified based on a validated GLP compliant method. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. Measured concentrations of formulations applied in the study varied in the acceptable range (between 93 % and 102 % of the nominal concentrations), thereby confirming proper dosing. Detailed clinical observations were performed daily after the treatment. On Day 0, animals were observed continuously for 30 minutes then 1, 2, 3 and 5 hours after the administration to estimate the peak time window of effects. Body weights were recorded twice weekly. The food consumption was determined weekly to coincide with body weight measurements during the study. Clinical pathology (hematology, blood coagulation and clinical chemistry) and gross pathology examinations were conducted one day after the last treatment (on Day 14). Selected organs were weighed. The kidneys and liver were processed and examined histologically in all male and female animals in the control and high dose groups. Additionally, the stomach of all male and female animals was processed and investigated histologically in all groups based on the macroscopic findings and to facilitate the correct dose level selection for the main study. The results of this study were summarized as follows:

Mortality: There were no mortalities in the control, 25, 50, 90 or 180 mg/kg bw/day groups (male or female).

Clinical observations: The test item did not induce adverse clinical signs in at 25, 50, 90 or 180 mg/kg bw/day dose levels (male or female animals) during the two weeks treatment period. The behavior and physical condition of all surviving animals were considered to be normal. Detected clinical signs (salivation before the treatment and nuzzling up the bedding material at 50, 90 or 180 mg/kg bw/day) were timely closely linked to the administration procedure and were with short duration (5 and 1 minutes, respectively).

Body weight and body weight gain: The body weight development was not adversely influenced in male or female animals at 25, 50, 90 or 180 mg/kg bw/day by during the entire treatment period. The slightly depressed mean body weight gain in male animals at 180 mg/kg bw/day (about 7% less than the concurrent control) resulted also in minor changes in the mean body weight.

Food consumption: The mean daily food consumption was slightly reduced in male animals at 180 mg/kg bw/day during the first week of observation period and being in accordance with slightly reduced body weight gain in this group.

Hematology and blood coagulation: Elevated white blood cell count (male), elevated percentage of reticulocytes and platelet count (male and female) at 180 mg/kg bw/day and elevated platelet count in male animals at 90 mg/kg bw/day were probably related to the gastric effect (irritation and inflammation) of the test item.

Clinical chemistry: Clinical chemistry evaluation did not reveal pathological changes in the investigated parameters at 25, 50, 90 or 180 mg/kg bw/day (male and female).

Necropsy: Gross necropsy observations revealed test item related changes in the stomach (thickening of the wall, hemorrhage, erosion, congestion, adhesion to diaphragm) at 50 mg/kg bw/day (one male) and at 90 and 180 mg/kg bw/day (male and female).

Organ weight: The weights of investigated organs were not adversely affected by the test item at 25, 50, 90 or 180 mg/kg bw/day (male and female).

Histopathology: Histological examination revealed squamous cell hyperplasia accompanied with hemorrhages, occasionally with erosion and inflammatory cell infiltration in the nonglandular part of stomach (forestomach) in a dose related manner at 50 mg/kg bw/day (one male) and at 90 and 180 mg/kg bw/day (male and female).

Conclusion

Under the conditions of the present study, the test item induced changes in the forestomach (detected macroscopically and microscopically) after the consecutive 14-day oral (by gavage) administration of 90 mg/kg bw/day and 180 mg/kg bw/day doses to Han:WIST rats (male and female). Slight effects on feed intake, body weight and body weight gain were noted at 180 mg/kg bw /day. As a consequence of the local gastric effects, some hematological parameter also changed mainly in 180 mg/kg bw/day animals (male and female) and in male animals at 90 mg/kg bw/day. Local effect was detected in the stomach in one male animal administered with 50 mg/kg bw/day.

There were no test item related local or systemic effects at 25 mg/kg bw/day dose (male or female). Based on the observations made in this toxicity study, the dose levels for a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test (main study) are as follows:

Group 1: Control

Group 2: 20 mg/kg bw/day

Group 3: 50 mg/kg bw/day

Group 4: 100 mg/kg bw/day

 

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, RL1

The purpose of this study was to obtain initial information on the systemically toxic potential of test item and on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 20, 50 and 100 mg/kg bw/day compared to control animals according to OECD 422 and GLP. As a screening test, it was intended to provide initial information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time and on the possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 13 post-partum associated with administration of repeated maternal doses. Satellite animals were included in the control and in the high dose groups for follow-up observations without treatment to detect the potential reversibility or persistence of possible toxic effects for 14 days. Methyl Vinyl Glycolate (MVG) was administered orally (by gavage) once daily at 0 (vehicle only), 20, 50 and 100 mg/kg body weight (mg/kg bw/day) doses to four groups of Han:WIST rats consisting of 17 animals per sex in the control and high dose groups and 12 animals per sex in the low and mid dose groups in concentrations of 4, 10 and 20 mg/mL corresponding to a 5 mL/kg bw dosing volume. A group of vehicle (distilled water) treated animals served as a control. 5 animals/ sex in the control and high dose groups were observed to detect the potential reversibility or persistence of possible toxic effects for 14 days. The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Methyl Vinyl Glycolate (MVG) was stable in the vehicle in concentrations of 1 mg/mL and 200 mg/mL for three days in a refrigerator (at 5 ± 3 °C). The concentration of the test item in the dosing formulations administered to the animals was checked once during the study. Methyl Vinyl Glycolate (MVG) concentrations in the dosing formulations varied within the range of 95 % and 103 % (in comparison to the nominal values) and confirmed the proper preparation of the dosing formulations. All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 49 days). Dams were additionally exposed through the gestation period and up to lactation days 13-16, i.e., up to the day before necropsy (altogether for 50-68 days). Animals in the recovery groups were administered up to and including Day 49 (altogether for 50 days). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period and during the mating period until evidence of copulation. Vaginal smears were also prepared and investigated on the day of the necropsy for each dam. The dams were allowed to litter and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on postnatal day 13 or shortly thereafter. Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4 and TSH) from 2-6 pups per litter (in litters with at least 10 pups) on post-natal day (PND) 4, from all dams and from 4-7 pups per litter at termination on post-partum/post-natal day 13 and from all parent male animals at termination. Serum level of FT3, FT4 and TSH were determined in the samples of parental male and female animals and PND 13 offspring. Five dams and their male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology and blood coagulation, clinical chemistry, gross necropsy, organ weighing and histopathology examination. All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight and weight of the testes and epididymides and prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined. In addition, for five males and females randomly selected from each group, adrenal glands, brain, heart, kidneys, liver, spleen, thymus and thyroid glands were weighed. Thyroid glands were preserved from all adult males and females and one male and one female pup per litter for the intended subsequent histopathological examination. Histopathology examination was performed on ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals (male and female) in the control and high dose groups. Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose groups (main and recovery). In addition, the stomach was processed and evaluated histologically in all male and female animals in control, low, mid and high dose groups based on necropsy findings. Organs showing macroscopic findings at the necropsy were also processed and examined histologically (lungs, kidneys, skin and uterus) in parental animals and offspring in the low and mid dose groups. The results of this study were summarized as follows:

 

Mortality

One male animal was found dead at 100 mg/kg bw/day on Day 9 presumably due to the gavage treatment. Necropsy and histopathology observations revealed signs of suffocation causing death of this animal.

 

Clinical and functional observation

There were no adverse clinical signs related to the test item at the daily or detailed weekly clinical observations. Treatment or test item related findings (salivation, nuzzling up the bedding material) were detected with variable incidence in male and female animals at 50 and 100 mg/kg bw/day immediately after the administration and signs ceased after a short duration. There were no clinical signs during the recovery period in male or female animal in the control and 100 mg/kg bw/day. Functional observation battery did not demonstrate any alterations in the behavior or reactions to different type of stimuli of selected male or female animals in the control, 20, 50 or 100 mg/kg bw/day groups at the end of the treatment period.

 

Body weight and body weight gain

The body weight development was not adversely influenced in male or female animals at 20, 50 or 100 mg/kg bw/day during the entire treatment period. Slightly reduced mean body weight gain resulted in minor changes in the mean body weight (less than 6 % relative to control) in male animals at 100 mg/kg bw/day therefore, this finding was considered to have little or no toxicological relevance.

 

Food consumption

The mean daily food consumption was not adversely affected at 20, 50 or 100 mg/kg bw/day dose levels during the entire treatment period (pre-mating and post-mating periods in male animals; during the pre-mating, gestation and lactation periods in female animals). The mean daily food consumption was slightly reduced in male animals at 100 mg/kg bw/day during the first week of observation period and being in accordance with slightly reduced body weight gain in this group and having little or no toxicological relevance.

 

Estrous cycle

A test item influence on the estrous cycle was not found at any dose level (20, 50 or 100 mg/kg bw/day).

 

Delivery and pregnancy data of female animals

There were no toxicologically relevant differences in the evaluated parameters of delivery between the control and test item treated groups (20, 50 or 100 mg/kg bw/day).

 

Reproductive performance

The examined parameters of reproductive performance were not affected by the test item at 20, 50 or 100 mg/kg bw/day in male or female animals.

 

Clinical pathology – Hematology, blood coagulation, clinical chemistry

Clinical pathology investigation did not reveal test item related changes in the examined hematology, blood coagulation or clinical chemistry parameters at 20, 50 or 100 mg/kg bw/day.

 

Serum thyroid hormones

The thyroid hormone (FT3, FT4 and TSH) levels were not affected by the test item in parental male and female animals (20, 50 and 100 mg/kg bw/day) and in offspring sampled on postnatal day 13.

 

Necropsy

Gross necropsy observations revealed test item related changes in the stomach (thickening of the wall, hemorrhage or congestion) at 50 mg/kg bw/day (male) and at 100 mg/kg bw/day (male and female), which were linked to the local effect of the test item.

 

Organ weight

There were no adverse test item related alterations in the weights of the examined organs in male or female animals at 20, 50 or 100 mg/kg bw/day.

 

Histopathology

Histological examination revealed squamous cell hyperplasia in the stomach in male and female animals in connection with the local effect of 50 and 100 mg/kg bw/day of test item. Squamous cell hyperplasia in the stomach was partially reversible as it was detected in some male and female animals at the end of the recovery period.

 

Offspring

The offspring’s development was undisturbed at 20, 50 and 100 mg/kg bw/day from birth to post-natal day 13. No effect on the mortality, clinical signs, body weight development, anogenital distance (male and female) or nipple retention (male) were detected.

 

Conclusion

Under the conditions of the present study, Methyl Vinyl Glycolate (MVG) administered at 20, 50 and 100 mg/kg bw/day by oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) and did not cause systemic toxicity in parental male and female Han:WIST rats as far as investigated in this study. Local effect was detected macroscopically and microscopically in the forestomach in male and female animals at 50 and 100 mg/kg bw/day. This local gastric effect did not induce clear adverse systemic changes although early signs (slight effects on body weight, food consumption, salivation) were noted. Therefore, a NOEL/NOAEC was deduced in addition to the NOAEL. The development of the F1 offspring was not impaired from birth up to post-natal day 13 as far as investigated in this study after repeated oral administration of dams at 20, 50 and 100 mg/kg bw/day. Based on these observations the No Observed Adverse Effect Levels (NOAEL), No Observed Effect Level (NOEL) and No Observed Adverse Effective Concentration (NOAEC) were determined as follows:

 

NOAEL for systemic toxicity of male/ female rats: 100 mg/kg bw/day

NOEL for systemic toxicity of male/ female rats: 20 mg/kg bw/day

NOAEL for reproductive performance of male/ female rats: 100 mg/kg bw/day

NOAEL for F1 Offspring: 100 mg/kg bw/day

NOAEC (local effects stomach): 4 mg/mL

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on repeated dose toxicity, the test item is not classified and labelled according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.