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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-08-26 - 2017-02-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Benzyltoluene
EC Number:
248-654-8
EC Name:
Benzyltoluene
Cas Number:
27776-01-8
Molecular formula:
C14H14
IUPAC Name:
benzyltoluene
Test material form:
liquid

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 60 days
- Weight at study initiation: 197.2 - 255.2 g
- Fasting period before study: no
- Housing: singly in MAKROLON cages
- Diet (e.g. ad libitum): Commercial diet ssniff® R/Z V1324
- Water (e.g. ad libitum):ad libitum
- Acclimation period:6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): fifteen to twenty air changes per hour
- Photoperiod (hrs dark / hrs light):12 hours dark/12 hours light cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
cotton seed oil
Details on exposure:
Route of administration Oral, via gavage

Frequency of administration Once daily

Treatment period Day 6 to 20 of gestation

Vehicle Cotton seed oil

Administration volume 2 mL/kg b.w./day

Selection of route of administration According to OECD guideline 414

The test item formulations were freshly prepared every day.
The test item was diluted in the vehicle to the appropriate concentrations and was ad-ministered orally at a constant volume once daily from the 6th to the 20th day of gestation.
The amount of the test item was daily adjusted to the current body weight of the animal. The control animals received the vehicle at the same administration volume daily in the same way.
The male rats for mating remained untreated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method (HPLC-UV) was validated by the laboratory. The following parameters were determined:
- Linearity
- Accuracy
- Precision
- Sensitivity
- Specifity
- Stability
Details on mating procedure:
Sexually mature ('proved') male rats of the same breed served as partners. The female breeding partners were randomly chosen.
Mating was monogamous: 1 male and 1 female animal were placed together in one cage during the dark period. Each morning a vaginal smear was taken to check for the presence of sperm. If findings were negative, mating was repeated with the same partner. The day on which sperm was found was considered as the day of conception (day 0 of pregnancy). This procedure was repeated until enough pregnant dams were available for all groups. Rats which did not become pregnant were excluded from the analysis of the results and replaced by other animals. A post-mortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.
Duration of treatment / exposure:
Day 6 to 20 of gestation
Frequency of treatment:
once daily
Duration of test:
Day 6 to 20 of gestation
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
30 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected in agreement with the Sponsor based on the results of a dose-range-finding study in rats. In the dose-range-finding study, the test item Benzyltoluene was administered orally to female rats at dose levels of 100, 300 or 800 mg/kg b.w./day from the 6th to 20th day of pregnancy.
Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 100 mg/kg b.w./day for the dams.
One of 6 dams of the high dose group (800 mg/kg b.w./day) was found dead before the end of the study.
An increase in the incidence, duration and in the number of signs of clinical toxicity (sal-ivation, reduced motility, prone position, piloerection) was noted from the low dose group (100 mg/kg b.w./day) to the high dose group (800 mg/kg b.w./day).
A reduced body weight and body weight gain were noted in the intermediate and high dose level (300 or 800 mg/kg b.w./day) and a reduced food consumption at the high dose level.
No test item-related changes were noted at necropsy.
The no-observed-adverse-effect level (NOAEL) for the fetal organism was also 100 mg/kg b.w./day.
No dead foetuses and no test item-related variations or malformations were noted at any of the tested dose levels during the external examination.
In the intermediate and high dose group (300 or 800 mg/kg b.w./day) a decreased pla-cental and fetal weight was noted.
Together with the fact that no external malformations or variations were noted for the fetuses, dose levels of 30, 100 or 300 mg Benzyltoluene/kg b.w./day were selected in agreement with the Sponsor for the present main study.

Examinations

Maternal examinations:
Clinical signs:
Individual animals were observed daily for behavioural changes, reaction to treatment, or illness.
Viability:
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals.
Body weight:
The weight of each rat was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weighing - always at the same time of the day.
The body weight gain was calculated in intervals (i.e. day 0-3, 3 6, 6-9, 9-12, 12-15, 15-18 and 18-21), for the whole study (gestation day 0 - 21) and for the period after the start of dosing (gestation day 6 to gestation day 21). Furthermore the carcass weight and the net weight gain from day 6 is given.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes / No / No data
- Number of corpora lutea: Yes /
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
Statistics:
Parametrical data: The statistical evaluation of the parametrical values was done by Provantis.
Non-parametrical data: The statistical evaluation of non-parametrical values was done using the FISHER or Chi test.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Slight or moderate - in one dam pronounced - salivation was noted in 16 of 22 high dose dams (300 mg Benzyltoluene/kg b.w./day) on 1 to 7 test days. Salivation started immediately to 5 minutes after administration and disappeared within 20 to 60 minutes after administration. In addition, an increased drinking water consumption was noted (by visual appraisal) in 5 of 22 high dose dams on 1 or 2 test days. Salivation and increased drinking water consumption are considered to be test item-related findings.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 300 mg Benzyltoluene/kg b.w./day, a stagnation in body weight was noted for the dams of the high dose group during the first days of treatment (first dosing on gestation day 6) due to reduced food consumption. Thereafter, the body weight improved but remained below the values of the control group by up to 4% or 5%. Statistically significant differences from control (at p ≤ 0.05) were noted on gestation days 10, 17, 18 and 19. A more distinct reduction of body weight (significant at p ≤ 0.01) was noted towards the end of test on gestation days 20 and 21. The maximal difference (11.1% below the value of the control group) was reached on gestation day 21 (day of laparotomy). This finding was partly due to reduced food intake and partly due to a lower gravid uterus weight
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At the high dose level (300 mg Benzyltoluene/kg b.w./day), the start of treatment evoked slight but statistically significant (p ≤ 0.01) reductions in food consumption on every day between gestation days 7 and 11 (between 17.6% and 26.4% below the values of the control group). Thereafter, the food consumption of the dams from the high dose group recovered. However, marginal reductions (significant at p ≤0.05) in food consumption were noted again on gestation day 16-17 (12.5% below the values of the control group). Severe statistically significant (p ≤ 0.01) reductions in food consumption were noted on gestation day 20-21.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
An increased water consumption was noted for 5 of 22 dams of the high dose group (300 mg Benzyltoluene/kg b.w./day) by visual appraisal on one or two test days and considered as test item-related
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic inspection during necropsy revealed gastric changes (haemorrhagic focus/foci, thin walls) in 4 of 22 high dose dams (300 mg Benzyltoluene/kg b.w./day).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
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
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
water consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 300 mg Benzyltoluene/kg b.w./day, statistically significant decreases (at p ≤ 0.01) were noted for the fetal weights (by approx. 26% below the control) and for the placental weights (by approx. 13% or 14% below the control).
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): At 300 mg Benzyltoluene/kg b.w./day, statistically significant decreases (at p ≤ 0.01) were noted for the fetal weights (by approx. 26% below the control) and for the placental weights (by approx. 13% or 14% below the control).
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
other: skeletal and soft tissue variations (see summary)

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In this prenatal developmental toxicity study, the test item Benzyltoluene was adminis-tered orally to female rats at dose levels of 30, 100 or 300 mg/kg b.w./day from the 6th to 20th day of pregnancy.
Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 100 mg Benzyltoluene/kg b.w./day for the dams.
Treatment with 300 mg Benzyltoluene/kg b.w./day caused salivation and increased intake of drinking water on a few or individual test days as well as reductions in the body weight, the body weight gain and the food consumption.
A few dams with gastric changes (haemorrhagic focus/foci, thin walls) were noted at the high dose level during the macroscopic inspection at necropsy.
The no-observed-adverse-effect level (NOAEL) for the fetal organism was 100 mg Benzyltoluene/kg b.w./day.
At the materno-toxic dose of 300 mg Benzyltoluene/kg b.w./day, pronounced decreases were noted for fetal body weights (by approx. 26%) and placental weights (by approx. 14%).
At 300 mg Benzyltoluene/kg b.w./day, an increased incidence was noted for retarded skeletal ossification (sternum, caudal vertebral bodies, metacarpalia and metatarsalia). These changes are regarded to be a secondary effect related to the maternal toxicity (Carney and Kimmel, 2007).
In addition, an increased incidence was noted for slight soft tissue variations of the kidney (uni- or bilateral dilatation of renal pelvis) and the 4th cerebral ventricle (dilatation). According to the ECETOC guidance document these effects are regarded to represent a 'low level of concern'. Considering the maternal and fetal toxicity at the high dose level the final conclusion according to the ECETOC guidance document for these findings is 'no concern'.
No test item-related malformation was noted at any of the test item treated groups. There was no test item-related increase in the incidence of fetal external / internal or skeletal variations at any tested dose level.
Under the conditions of the study, Benzyltoluene did not show any teratogenic potential.

Executive summary:

In this prenatal developmental toxicity study, the test item Benzyltoluene was administered orally to female rats at dose levels of 30,100or300 mg/kg b.w./day from the 6th to 20th day of pregnancy.

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 100 mg Benzyltoluene/kg b.w./day for the dams.

Treatment with 300 mg Benzyltoluene/kg b.w./day caused salivation and increased intake of drinking water on a few or individual test days as well as reductions in the body weight, the body weight gain and the food consumption.

A few dams with gastric changes (haemorrhagic focus/foci, thin walls) were noted at the high dose level during the macroscopic inspection at necropsy.

The no-observed-adverse-effect level (NOAEL) for the fetal organism was 100 mg Benzyltoluene/kg b.w./day.

At the materno-toxic dose of 300 mg Benzyltoluene/kg b.w./day, pronounced decreases were noted for fetal body weights (by approx. 26%) and placental weights (by approx. 14%).

At 300 mg Benzyltoluene/kg b.w./day, an increased incidence was noted for retarded skeletal ossification (sternum, caudal vertebral bodies, metacarpalia and metatarsalia). These changes are regarded to be a secondary effect related to the maternal toxicity (Carney and Kimmel, 2007).

In addition, an increased incidence was noted for slight soft tissue variations of the kidney (uni- or bilateral dilatation of renal pelvis) and the 4th cerebral ventricle (dilatation). According to the ECETOC guidance document ("Identification and assessment of the effects of chemicals on reproduction and development (Reproductive Toxicology)", Monograph No. 5, December 1983) these effects are regarded to represent a 'low level of concern'. Considering the maternal and fetal toxicity at the high dose level the final conclusion according to the ECETOC guidance document for these findings is 'no concern'. No test item-related malformation was noted at any of the test item treated groups. There was no test item-related increase in the incidence of fetal external / internal or skeletal variations at any tested dose level.

Under the conditions of the study, Benzyltoluene did not show any teratogenic potential.