methyl 2-hydroxybut-3-enoate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000-07

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

methyl 2-hydroxybut-3-enoate

EC Number:

831-109-9

Cas Number:

5837-73-0

Molecular formula:

C5H8O3

IUPAC Name:

methyl 2-hydroxybut-3-enoate

Test animals

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

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)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on 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

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: Vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing: Replacement of first male by another male.
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged individually

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

20 mg/kg bw/day (nominal)

Doses / concentrations 4

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

Examinations

Parental animals: Observations and examinations:

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)

WATER CONSUMPTION AND COMPOUND INTAKE: 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)

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).

Oestrous cyclicity (parental animals):

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 (parental animals):

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

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- Pups/sex/litter: 4, if feasable. Excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups. Particular attention was paid to the external reproductive genitals which should be examined for signs of altered development; gross evaluation of external genitalia.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external abnormalities; possible cause of death was not determined for pups born or found dead. On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn; negative lung flotation test) from pups died after the birth (dead pups; positive lung flotation test).

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on day 49. Recovery animals were sacrified on day 64.
- Maternal animals: All surviving animals on post-partum day 14 or shortly thereafter (days 50, 51, 54, 55, 56, 57, 68). Recovery animals were sacrified on day 64. Dams selected for toxicology examinations were sacrificed on day 55.

GROSS NECROPSY
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 / ORGAN WEIGHTS
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.

Postmortem examinations (offspring):

SACRIFICE
F1 animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
Pups euthanized on day 13 post-partum, or shortly thereafter, were carefully examined for gross abnormalities. The appearance of the tissues and organs was observed in pups selected for thyroid gland preservation.

HISTOPATHOLOGY / ORGAN WEIGTHS
Thyroid gland was preserved from one male and one female pup per litter for the intended subsequent histopathological examination. Thyroid and parathyroid were preserved together with larynx.

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.

Reproductive indices:

MALES:
Male copulatory index= Number of males with confirmed mating / Total Number of males cohabited x 100
Male fertility index= Number of males impregnating a female / Total Number of males with confirmed mating x 100

FEMALES:
Female copulatory index= Number of sperm positive females / Total Number of females cohabited x 100
Female fertility index= Number of pregnant females / Number of sperm positive females x 100
Gestation index= Number of females with live born pups / Number of pregnant females x 100

Offspring viability indices:

Post-implantation mortality (intrauterine mortality): Number of implantations - Number of liveborns / Number of implantations x 100
Post-natal mortality= Number of liveborns - Number of live pups on PN13 / Number of liveborns x 100
Survival Index= Number of live pups on postnatal day 13 / Number of pups born x 100
Sex ratio= Number of pups examined − Number of males (females) / Number of pups examined x 100
Normalized anogenital distance= Absolute anogenital distance / (Body weight)^1/3

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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.

Dermal irritation (if dermal study):

not examined

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

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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histologically normal and characteristic on the sexually mature organism in the male animals in control (12/12) and 100 mg/kg bw/day (11/11, including not mated male animal) groups. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa); representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals. The histological picture of epididymides, prostate, seminal vesicles, and coagulating glands was normal in all cases as well.

Reproductive performance:

no effects observed

Description (incidence and severity):

Statistical significance with respect to the control was detected at the lower mean number of implantation sites at 20 mg/kg bw/day and at the shorter mean duration of pregnancy at 50 mg/kg bw/day. These variations were not dose-related.

Statistical significance with respect to the control was detected at the slightly lower copulatory index (lower percentage of mated male animals) at 100 mg/kg bw/day as one pair failed to mate. Test item effect was not presumed because data met well historical control value and in the lack of related findings in the reproductive parameters or reproductive organs There was no difference between the control and test item treated male animals in fertility indices. The copulatory, fertility and gestation indices were 100 % in the female animals in the control, 20, 50 and 100 mg/kg bw/day. That indicates, all female animals mated, achieved pregnancy and gave live birth pups in each group.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

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.

3

Key result

Dose descriptor:

NOAEC

Remarks:

(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.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

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