Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
according to OECD 421 (GLP)
Justification for type of information:
Following ECHA decision (CCH-D-2114379324-45-01/F) on Benzyl Salicylate it was requested to conduct additional toxicological studies.
The Screening study for reproductive/developmental toxicity (Annex VIII, Sections 8.6.1 and 8.7.1.; test method: OECD TG 421) in rats, oral route, and Pre-natal de-velopmental toxicity study (Annex IX, Section 8.7.2.; test method: EU B.31./OECD 414) in a first species (rat or rabbit), oral route.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 421 (Reproduction/Developmental screening test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Benzyl Salicylate
- Substance type: no data
- Physical state:no data
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: 299171
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: no data
Specific details on test material used for the study:
Benzyl Salicylate (CAS No. 118-58-1)

Test animals

Species:
rat
Strain:
Sprague-Dawley

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: PMI Nutrition International LLC Certified Rodent LabDiet®
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test substance diet formulations have been previously shown to be stable and homogeneous over the range of concentrations used on this study for at least 11 days at room temperature (18°C to 24°C).Therefore, stability of test substance in the test diet will not be assessed on this study. However, homogeneity of the test substance in the dietary preparations will be assessed in this study (prior to the start of dosing, if possible). Representative batches of test diet at each of the selected dosage levels will be mixed in a Hobart blender. Duplicate samples will be taken from the top, middle and bottom strata of each test diet. These samples at each of the selected dosage levels will be analyzed for homogeneity.
The acceptable results for homogeneity assessment include an RSD for the mean concentration within 10% or less at a concentration that is within the acceptable limits (85-115% of the target concentrations).
Details on mating procedure:
After a minimum of 14 days of dosing, the animals were paired on a 1:1 basis within each group. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. Vaginal lavages were performed daily during the mating period until evidence of mating was observed. If evidence of mating was not apparent after 14 days, the animals were separated, with no further opportunity for mating. Animals cohabited over a 12-hour dark cycle were considered to have been paired for 1 day.
Duration of treatment / exposure:
The control and test diets were offered continuously throughout the study. The test substance was administered as a constant concentration (ppm) in the diet. Males were exposed for 14 days prior to mating, throughout mating and continuing until the day prior to euthanasia. Females were exposed for 14 days prior to mating and continuing through Lactation Day 13. Females with no evidence of mating were exposed through the day prior to euthanasia. The F1 animals were not directly exposed to the test substance at any time during the study; the offspring of the F0 parental generation were potentially exposed to the test substance in utero and while nursing.
Frequency of treatment:
Reported previously.
Duration of test:
Reported previously.
Doses / concentrationsopen allclose all
Dose / conc.:
500 ppm
Remarks:
Equivalent to 32 and 34 mg/kg bw/d (males and females respectively)
Dose / conc.:
750 ppm
Remarks:
Equivalent to 48 and 49 mg/kg bw/d (males and females respectively)
Dose / conc.:
2 500 ppm
Remarks:
Equivalent to 158 and 166 mg/kg bw/d (males and females respectively)
No. of animals per sex per dose:
10
Control animals:
yes
yes, plain diet
Details on study design:
he route of administration was oral (dietary), because oral ingestion is a potential route of exposure in humans. Historically, this route has been used extensively for studies of this nature.
Dose selection for this study was based on a previous rat embryo/fetal developmental toxicity study in which benzyl salicylate was administered at doses of 0, 1000, 3000, and 4000 ppm in the diet. Mean absolute body weights in the 4000 ppm group were up to 6.4% lower than the control group during Gestation Days 7–21. Mean body weights in the 3000 ppm group were sporadically lower than the control group; however, the values were ≤ 4.3% lower than the control group. Mean fetal body weights (male, female, and combines sexes) were 6.8% to 7.1% and 10.2% to 10.7% lower in the 3000 and 4000 ppm groups, respectively, compared to the concurrent control group.
Based on these results above and based on the longer exposure that animals were submitted to on this study (at least 28 days for males and approximately 60 days for females), 0, 500, 750, and 2500 ppm dose levels were selected. The high-dose level was expected to produce some toxicity, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level was expected to produce minimal toxic effects. The low-dose level was expected to produce no observable indications of toxicity.

Examinations

Maternal examinations:
Clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen, thyroid hormones, gross necropsy findings, organ weights, and histopathologic examinations.
Ovaries and uterine content:
For all females, vaginal lavages were performed daily for 14 days prior to randomization and continuing until evidence of mating was observed. The slides were microscopically examined to determine the stage of the estrous cycle. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P] for 14 consecutive days before cohabitation and until the detection of evidence of mating). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the individual mean estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Information included in the section 7.8.1 (Toxicity to Reproduction).

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Maternal
Effect level:
158 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No test substance-related effects were noted in general toxicity and repro-developmental toxicity at higher dose tested in 421 study (158 mg/kg bw/d)
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive
Effect level:
158 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No test sunstace-related effects were noted on general toxicity and reproductive
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
158 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No test substance related effects were noted
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Abnormalities:
no effects observed
Localisation:
other: No effects observed

Results (fetuses)

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
158 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall developmental toxicity

Developmental effects observed:
no
Lowest effective dose / conc.:
158 mg/kg bw/day
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of this screening study, no test substance-related effects were noted on reproductive performance in F0 males and females at any exposure concentration. Based on the lack of F0 parental toxicity at any exposure concentration, an exposure concentration of 2500 ppm (the highest dose level tested) was considered to be the no-observed-adverse-effect level (NOAEL) for F0 systemic and reproductive toxicity of benzyl salicylate when administered in the diet to Crl:CD(SD) male and female rats. Based on the lack of test substance-related effects at any dose level, an exposure concentration of 2500 ppm (the highest dose level tested) was considered to be the no-observed-adverse-effect level (NOAEL) for F1 neonatal toxicity.
The 2500 ppm dose level corresponded to actual consumption of 166 mg/kg/day for males during the pre-mating period and 158, 170, and 324 mg/kg/day for females during pre-mating, gestation, and lactation, respectively.
Executive summary:

Information included in the section 7.8.1 (Toxicity to Reproduction).