Methyl benzoate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 15, 2022 - January 10, 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 25, 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methyl benzoate

EC Number:

202-259-7

EC Name:

Methyl benzoate

Cas Number:

93-58-3

Molecular formula:

C8H8O2

IUPAC Name:

methyl benzoate

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Breeding Facility, Jai Research Foundation
- Age at study initiation: approximately 13 weeks at start of acclimatisation
- Weight at study initiation: 219.3 ± 11.0 g (198.3 – 239.7 g) at start of acclimatisation
- Housing: individually except for the mating period
- Diet (e.g. ad libitum): standard rodent pelleted diet (Certified Teklad Global 14% Protein Rodent Diet, Batch No 2014SC-010922MA, procured from Envigo, Inc., USA), ad libitum
- Water (e.g. ad libitum): Reverse Osmosis (RO) water, ad libitum
- Acclimation period: 6 days before cohabitation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 65-67
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12/12 (156 - 167 lux)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was received for each dose level separately from the Test Item Control Office (TICO). The test item was mixed with vehicle to prepare dose formulations of required concentrations. The prepared dose formulations were thoroughly mixed using a magnetic stirrer before dosing and with cannula intermittently during dosing. The dose formulations were prepared on each day of dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on solubility
- dose volume: 4 mL/kg bw
- Lot/batch no.: MKCP9878

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of test item in the vehicle was established in JRF Study N° 228-2-13-30087. The test item was found stable in the vehicle (corn oil) up to 8 days from the time of preparation. Active ingredient (a.i.) concentration and homogeneity of dose formulations were analysed by the Department of Chemistry, using a validated analytical method. The active ingredient (A.I.) concentration and homogeneity of the test item in dose formulations were analyzed once before initiation of treatment and once during the treatment period. Duplicate samples of control (one layer) and the dose formulations (upper, middle, and lower layers) of each treatment group were taken to determine the homogeneity and concentration of the test item. A set of samples were sent for analysis and a second set were stored at frozen condition (-20 ± 5°C).
 
The mean concentration was determined and compared to the nominal value, and the coefficient of variation was calculated. The acceptance criteria was ± 10% deviation from the nominal value and %CV < 10. Samples were analysed at JRF using a validated analytical method.
Samples were analyzed by the following instrumental parameters:
Instrument : GC-FID (Agilent Technologies, 8890 with OpenLab Software)
Detector : FID
Column : DB Wax (30 m X 250 µm x 0.25 µm) or equivalent
Carrier Gas : N2
Injection Volume (µL) : 2.0 µL
Injector Temperature : 240 °C
Detector Temperature : 240 °C
Flow Rate (mL/minute) : 1.0
Split Ratio : Splitless
H2 Flow (mL/minute) : 35.0
Air Flow (mL/minute) : 350
Oven Temperature : 60 °C (hold 2 min) to 180 °C @ 20 °C/min (hold 5 min) to 220 °C @ 10 °C/min (hold 5 min).
Retention Time : 7.2 (approximately)

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

GD 3 through 19

Frequency of treatment:

daily for 17 days (GD 3-19)

Duration of test:

20 d

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

750 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on the results of the dose range finding study “Prenatal Developmental Oral Toxicity Study of Methyl benzoate in Wistar Rats (Dose Range Finding)’’ (JRF Study N°: RES-1-04-30089). Treatment related morality was observed in the 1000 mg/kg b. wt./day dose group.

Examinations

Maternal examinations:

MORTALITY AND MORBIDITY: Yes
- Time schedule: twice daily

CLINICAL OBSERVATIONS: Yes
- Time schedule: visible clinical signs, such as changes in the skin, fur, eye, mucous membranes, and the behavioral pattern of the mated female rats, were recorded twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: individually on days 0, 3, 5, 8, 11, 14, 17, and 20 of gestation
- body weight gain was determined for the following intervals: Days 0–3, 3–5, 5–8, 8–11, 11–14, 14–17, 17-20, and 5-20

FOOD CONSUMPTION: Yes
- determined by differentiating the weight of feed input and leftover on gestation day 0, 3, 5, 8, 11, 14, 17 and 20
- food consumption calculated for female rats for the following intervals: days 0 - 3, 3 - 5, 5 - 8, 8 - 11, 11 - 14, 14 - 17, 17 – 20, and 5-20

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Ovaries and gravid uteri, including the cervix
- thyroid glands (post-fixation) and brain were collected and weighed from all female rats; thyroid glands were preserved for histopathology

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Number of live fetuses , Number of dead fetuses

Blood sampling:

- Plasma: No
- Serum: Yes
- Volume collected: approximately 1.0 mL

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes:
- Anogenital distance of all live rodent pups: yes

Statistics:

Parametric:
- Body weight, body weight change/gain, food consumption, corrected body weight/carcass weight, absolute and relative gravid uterine weights, organ weight, organ weight ratio, male sex ratio/male percent, pre-and postnatal loss (%), live fetus (%), and resorptions (%), ano-genital distance (normalized), fetal observations (percent fetal)
- 5% and 1% levels between control and treated groups
Non-parametric:
- Reproductive performance/Pregnancy rate, fetal observations (percent litter), fetal count, number of corpora lutea, number of implants, number of live fetus, number of dead fetus, number of pre-and postnatal losses, number of resorptions
- 5% level between control and treated groups

Historical control data:

refer to attached material

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical sign such as salivation was observed in pregnant rats of the groups treated with 500 and 750 mg/kg b. wt./day. Salivation was not observed during the morning clinical observation. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test article rather than an indication of toxicity (Wook-Joon Yu et al., 2011). Therefore, salivation observed in the study was considered a physiological response to the dose formulation and toxicologically non-adverse in nature.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean food consumption of the pregnant female rats was comparable between the control and the test item treated group up to the dose level of 500 mg/kg bw/day.
Statistically, a significant increase in mean food consumption during gestation days 11-14, 14-17, and 5-20 was observed in pregnant female rats in the group treated with 750 mg/kg bw/day when compared with that of the control group. However, this increase in food consumption did not cause an increase in the body weight and body weight change/gain. These opposite effects in food consumption and body weight were due to decreased feed efficiency and considered as an adverse effect of the test item.

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

Endocrine findings:

no effects observed

Description (incidence and severity):

The serum level of thyroid hormones (T3, T4, and TSH) were comparable between control and the test item treated group up to the dose level of 750 mg/kg bw/day .

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Description (incidence and severity):

The mean absolute and relative weights of brain and thyroid gland of pregnant rats were comparable between the control and treatment groups up to the dose level of 750 mg/kg b. wt./day.

Gross pathological findings:

no effects observed

Description (incidence and severity):

External and internal (gross) examination of terminally sacrificed female rats did not reveal any macroscopic lesion up to the dose level of 750 mg/kg bw/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Congenital lesions, namely ectopic thymus tissue (Rat N° 99) was observed in the group treated with 750 mg/kg b. wt./day, which was considered as unrelated to the test item treatment due to being present in only 1 out of 25 rats in the respective group. Ultimobranchial cyst {Rat N° 1, 15 (G1) and 32 (G2)} was observed in the control and the group treated with 250 mg/kg bw/day, which were considered as unrelated to the test item due to being a congenital lesion.

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Statistically, a significant decrease in the mean absolute and relative (relative to both terminal body weight and brain weight) gravid uterus weight of pregnant female rats in the group treated with 750 mg/kg bw/day was observed when compared with that of the control group. This effect was considered as a treatment-related adverse effect of the test item.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

other: gravid uterine weight

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean litter weight, mean weight of male, female, and total fetuses (male + female) was comparable between the control and the test item treated groups up to the dose level of 500 mg/kg bw/day.
Statistically, a significant decrease in the mean litter weight, mean fetus weight of male, female and for composite of both sexes were observed in pregnant female rats in the group treated with 750 mg/kg bw/day when compared to the control group. These effects were considered as an adverse effect of test item treatment.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Statistically, a significant decrease in the mean litter weight, mean fetus weight of male, female and for composite of both sexes were observed in pregnant female rats in the group treated with 750 mg/kg bw/day when compared to the control group. These effects were considered as an adverse effect of test item treatment.

Anogenital distance of all rodent fetuses:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related external anomalies were observed in fetuses of the control and the treatment groups up to the dose level of 750 mg/kg b. wt./day. However, fetuses with hemorrhage and runt fetuses were observed in the control and the treatment groups as specified in the attached information.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Statistically, a significant increase in the percent litter with 14th rib: extra ossification center (left) was observed in the group treated with 250 mg/kg bw/day. This finding was considered as incidental due to the lack of dose dependency.
Statistically, a significant increase in the percent litter affected with xiphisternum: bipartite ossification was observed in the group treated with 500 mg/kg bw/day. This finding was considered as incidental due to the lack of dose dependency.
Statistically, a significant increase in the percent of fetuses with various anomalies was observed in the group treated with 750 mg/kg bw/day as specified in the attached information.

A significant increase in the percent litter affected with xiphisternum: unossified was considered as non-adverse effect of the test item caused by a mild; non-statistically significant decrease of the fetal mean body weight in the group treated with 500 mg/kg bw/day.
The increased incidences of percent fetus and percent litter affected with various anomalies were well correlated with the decreased fetal mean body weight in the group treated with 750 mg/kg bw/day.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related visceral anomalies were observed in fetuses of the control and the treatment groups up to the dose level of 750 mg/kg bw/day. However, fetuses with hemorrhagic adrenals, dilated renal pelvis, hemorrhagic kidneys, malposition liver, hemorrhagic lungs, elongated thymus, and convoluted ureter were observed in the control and the treatment groups as specified in the attached information.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Head Razor Observations:
No treatment related anomalies were observed in fetuses of the control and treatment groups up to the dose level of 750 mg/kg bw/day. However, fetuses with dilated 3rd and lateral ventricle were observed in the control and the treatment groups as specified in the attached information.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

other: skeletal variations

Fetal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: vertebra

Description (incidence and severity):

The increased incidences of percent fetus and percent litter affected with unossified or incompletely ossified sternebrae in the group treated with 750 mg/kg bw/d were correlated with decreased maternal body weight gain and uterus weight (absolute and relative) as well as a reduction in mean fetal weight.

The increased incidences of percent fetus and percent litter affected with absent 1st, 2nd and 3rd caudal vertebrae in the group treated with 750 mg/kg bw/d were considered as treatment-related adverse effect.

Overall developmental toxicity

1

Developmental effects observed:

yes

Lowest effective dose / conc.:

750 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

2

Developmental effects observed:

yes

Lowest effective dose / conc.:

750 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Please refer to the attached information.