Benzyltoluene

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 8 Nov 2021 to 30 Nov 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The objective of this study was to determine the potential toxicity of the test item, following daily oral administration (gavage) in the non-pregnant female rabbit, and to establish a Maximum Tolerated Dose (MTD), in order to assist the selection of dose levels for a further dose-range finding toxicity study in the pregnant rabbit.
Three non-pregnant female New Zealand White rabbits were orally dosed (gavage) with the test item once daily at 100 mg/kg bw/day for 7 days then at 300 mg/kg bw/day for 7 days. There was no washout period between the dose levels.
The following parameters and end points were evaluated in this study: mortality, clinical observations, body weights, food consumption and macroscopic examination.

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Benzyltoluene

EC Number:

248-654-8

EC Name:

Benzyltoluene

Cas Number:

27776-01-8

Molecular formula:

C14H14

IUPAC Name:

1-benzyl-2-methylbenzene; 1-benzyl-3-methylbenzene; 1-benzyl-4-methylbenzene

Test material form:

liquid

Test animals

Species:

rabbit

Strain:

New Zealand White

Remarks:

KBL, Specific Pathogen Free.

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 18 to 19 weeks old.
- Weight at study initiation: Between 4.105 and 4.210 kg
- Fasting period before study: no.
- Housing: Single housed in Noryl cages. Each cage was identified with a color-coded card indicating study, group, animal number and sex. For psychological / environmental enrichment, animals were provided with dumbbell, hay and music.
- Diet: Pelleted complete diet (Diet Reference No. 3409, KLIBA), and compacted hay pellets (SSNIFF). Ad libitum.
- Water: Municipal tap water filtered with a 0.22 µm filter. Ad libitum.
- Acclimation period: 7 days.

Analysis of the feed and water are on file at the Test Facility. It was considered that there were no known contaminants in the feed or water that interfered with the objectives of the study.

ENVIRONMENTAL CONDITIONS (targeted conditions)
- Temperature (°C): 15°C to 21°C.
- Humidity (%): 30% to 70%.
- Air changes (per hr): Approximately 5 to 15 filtered, non-recycled air changes
- Photoperiod (hrs dark / hrs light): 8 / 16

IN-LIFE DATES: From: 16 Nov 2021 to 30 Nov 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% (w/v) Carboxymethylcellulose (400-800 cps) + 0.5% (w/v) Tween 80 in drinking water treated by reverse osmosis.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were stirred continuously for at least 15 minutes before dosing and during dosing and were maintained under delivery conditions (at room temperature).

VEHICLE
- Justification for use and choice of vehicle: not specified.
- Concentration in vehicle: 20 mg/mL and 60 mg/mL.
- Amount of vehicle: 5 mL/kg.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

7 days at 100 mg/kg bw/day followed by 7 days at 300 mg/kg bw/day.

Frequency of treatment:

Daily

Duration of test:

14 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

From days 1 to 7 (both included)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Remarks:

From days 8 to 14 (both included)

No. of animals per sex per dose:

3 females comprised the totality of the animals used in this study, they received the test item at increasing dose levels of 100 and 300 mg/kg bw/day

Control animals:

no

Details on study design:

- Dose selection rationale: Dose levels were selected based on the results of two previous studies:
1) a 2-Week Dose-Range Finding Study by the Oral Route (Gavage) in male and female Rats (Study No. 48405 TSR, 2021). This study was conducted with dose levels of 0, 100, 300, 600 and 800 mg/kg bw/day. Due to adverse effects observed, the dose levels of 600 and 800 mg/kg bw/day were considered to have exceeded the Maximum Tolerated Dose (MTD) in males and females.
2) a Reproduction/Developmental Toxicity Screening Test (Study No. 48406, 2022) in which the test item was administered orally to Sprague-Dawley rats at 0, 30, 100 or 300 mg/kg bw/day. Signs of poor clinical condition were observed in both sexes at = 100 mg/kg bw/day which led to the premature death of several females at 300 mg/kg bw/day. The Low Observed Adverse Effects (LOAEL) was set at 30 mg/kg bw/day in males based on T4 decrease at all dose levels.
For the present study, the initial dose level was selected at 100 mg/kg bw/day in order to achieve a no or minor effect dose level, taking into account the fact that rabbits can be more sensitive to certain toxicity than rats.

- Rationale for animal assignment (if not random): All animals received the same treatment.

Examinations

Maternal examinations:

MORTALITY: Yes
- Time schedule: At least twice daily (at the beginning and end of the working day) beginning upon arrival through termination/release.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily beginning upon arrival through termination, in addition animals were observed between 1 and 3 hours post-dose on the days of dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least weekly (at least once on the last day of dosing at each dose level).

BODY WEIGHT: Yes
- Time schedule for examinations: Once before the start of dosing, then on days 1, 3, 5, 8, 10, 12 and on day 15 before necropsy.

FOOD CONSUMPTION : Yes
- Food consumption of each animal was recorded on the following intervals: Days -5 to 1, 1 to 3, 3 to 5, 5 to 8, 8 to 10, 10 to 12 and 12 to 15 .

POST-MORTEM EXAMINATIONS: Yes
- Macroscopic observations were conducted at necropsy.
- Sacrifice on day 15 by an intravenous injection of sodium pentobarbital followed by
exsanguination. Non-fasted animals
- Organs examined: principal thoracic and abdominal organs.
- No histology and microscopic evaluation was performed.

Statistics:

Due to the small number of animals, no statistical analysis was performed.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Absence of urine or abnormal color of urine was observed in females treated at 300 mg/kg bw/day, on several occasions (usually starting after 3 administrations and up to 4 days).

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg bw/day, a body weight loss was recorded in all females after 7 days of treatment, down to approximately -11% in one female on Day 15 vs. Day 8 values
At 100 mg/kg bw/day, there were no changes in body weight after 7 days of treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg bw/day, low food consumption values were recorded in all females after 7 days of
treatment, which do not correspond to physiological values for animals of this age. This effect on
food consumption correlated with the effect observed on body weight.
At 100 mg/kg bw/day, there were no relevant changes in food consumption after 7 days of treatment.

Gross pathological findings:

no effects observed

Effect levels (maternal animals)

Dose descriptor:

other: Maximum Tolerated Dose (MTD)

Effect level:

> 300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: Based on these results, it is considered that 300 mg/kg bw/day was below the Maximum Tolerated Dose (MTD) and that a higher dose level could be tested in a further toxicology study performed in pregnant rabbits.

Maternal abnormalities

Abnormalities:

no effects observed

Any other information on results incl. tables

Mean body weights:

	Day(s) relative to starting date
Body weight (kg)	-7	1	3	5	8	10	12	15
Mean	3.921	4.142	4.113	4.178	4.233	4.107	4.023	3.903
SD	0.0651	0.0592	0.0666	0.0889	0.1249	0.1617	0.1229	0.1150
N	3	3		3	3	3	3	3

 

Mean body weight gain:

	Day(s) relative to starting date
Body weight gain(kg)	1-->3	3-->5	5-->8	1-->8	8-->10	10-->12	12-->15	8-->15
Mean	-0.028	0.065	0.055	0.092	-0.127	-0.083	-0.120	0.330
SD	0.0076	0.0278	0.0409	0.0729	0.1049	0.0501	0.0150	0.0966
N	3	3	3	3	3	3	3	3

 

Mean food consumption:

	Day(s) relative to starting date
Food Consumption (g/day)	-5-->1	1-->3	3-->5	5-->8	1 -->8	8-->10	10-->12	12 -->15	8-->15
Mean	229.3	215.0	209.2	210.6	211.4	101.7	65.0	46.1	67.4
SD	19.35	17.50	17.56	25.95	18.94	49.07	27.04	8.39	22.59
3	3	3	3	3	3	3	3	3	3

 

Applicant's summary and conclusion

Conclusions:

Administration of Benzyltoluene by once daily oral gavage was well tolerated at 100 mg/kg bw/day for 7 days. At 300 mg/kg bw/day, slight body weight loss and low food consumption were observed in females. Based on these results, it was considered that 300 mg/kg bw/day was below the Maximum Tolerated Dose (MTD) and that a higher dose level could be tested in a further toxicology study performed in pregnant rabbits.

Executive summary:

The objective of this study was to determine the potential toxicity of Benzyltoluene, following daily oral administration (gavage) in the non-pregnant female rabbit, and to establish a Maximum Tolerated Dose (MTD), in order to assist the selection of dose levels for a further dose-range finding toxicity study in the pregnant rabbit.

The study design was as follows:

Group No.	Test Material	Days of Treatment	Dose Level (mg/kg bw/day)	Dose Volume (mL/kg)	Dose Concentration (mg/mL)	No. of Females
1	Benzyltoluene	1 to 7	100	5	20	3
		8 to 14	300	5	60

 

The following parameters and end points were evaluated in this study: mortality, clinical observations, body weights, food consumption and macroscopic examination.

There were no unscheduled deaths during the study. Clinical observations were observed in females treated at 300 mg/kg bw/day which consisted in abnormal color of urine or absence of urine, for which an effect of the test item could not be excluded. Body weight losses and low food intake were observed in females treated at 300 mg/kg bw/day (e.g., body weight loss of 11% in one female on Day 15 vs. Day 8 associated with a food consumption of approximately 42 g/day over the same period).

There were no macroscopic observations noted at the end of the study.

In conclusion, administration of Benzyltoluene by once daily oral gavage was well tolerated at 100 mg/kg bw/day for 7 days. At 300 mg/kg bw/day, slight body weight loss and low food consumption were observed in females. Based on these results, it was considered that 300 mg/kg bw/day was below the Maximum Tolerated Dose (MTD) and that a higher dose level could be tested in a further toxicology study performed in pregnant rabbits.