Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

F0 male and female mating and fertility, male copulation and female conception indices, estrous cycle lengths, mean number of days between pairing and coitus, gestation lengths, and the

process of parturition were unaffected by test substance administration at all dietary concentrations.

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.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / 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.
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
The 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 c ntrol 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.
Specific details on test material used for the study:
Benzyl Salicylate (CAS No. 118-58-1)
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague-Dawley rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source : Charles River Laboratories, Inc., Raleigh, NC
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: (P) 7-9 wks
- Weight at study initiation: Males:366 g; Females: 211 g;
- Fasting period before study:
- Housing: On arrival, animals were group housed (up to 3 animals of the same sex) until cohabitation. During cohabitation, F0 animals were paired for mating in the home cage of the male. Following the breeding period, F0 animals were individually housed. Animals were housed in solid-bottomcages containing appropriate bedding equipped with an automatic watering valve throughout the study. Each cage was clearly labeled with a color-coded cage card indicating study, group, animal, cage number(s), dosage level, and sex. Cages were arranged on the racks in group order. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals. The animal facilities at Charles River Ashland are accredited by AAALAC
International.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: prior to initiation of dosing. The testes were palpated at least once for all males during acclimation.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26
- Humidity (%): 30 to 70%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle
IN-LIFE DATES: From: To: 30 May 2019 to 27 Aug 2019
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Animals were administered the test substance continuously in the diet. F0 males were dosed for 14 days prior to mating and continuing through the day of euthanasia. F0 females were dosed for 14 days prior to mating and continuing through Lactation Day 13.
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.
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).
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.
Details on study schedule:
Reported previously.
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:
The 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.
Positive control:
No.
Parental animals: Observations and 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.
Oestrous cyclicity (parental animals):
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.
Sperm parameters (parental animals):
Testes weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- 8 pups/litter; excess pups were euthanized via an intraperitoneal injection of sodium pentobarbital 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.

GROSS EXAMINATION OF DEAD PUPS:
External and internal abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Following completion of the mating period (a minimum of 28 days of dose administration), adult males were euthanized by carbon dioxide inhalation (following blood collection for thyroid hormone assessments). Organ weights were collected and tissues preserved.
- Maternal animals:
- Females Which Deliver: On LD 13, all adult females that delivered will be euthanized by carbon dioxide inhalation following blood collection for thyroid hormone assessments. The abdominal, pelvic, and thoracic cavities were opened and the contents examined. The number of former implantation sites was recorded. Organ weights were collected and tissues preserved.
- Females Which Fail to Deliver On Post-Mating day 25 (females with evidence of copulation) or Post-cohabitation Day 25 (females without evidence of copulation), the females which fail to deliver were euthanized by carbon dioxide inhalation. The abdominal, pelvic, and thoracic cavities were opened and the contents examined. Uteri which appear nongravid by macroscopic examination were opened and placed in a 10% ammonium sulfide solution for detection of early implantation loss. Organ weights will be collected and tissues preserved, with the exception of any ammonium sulfide stained uterus, which will be discarded. If evidence of macroscopic implantations is present, the number of implantation sites and corpora lutea was recorded.
- Females with Total Litter Loss: Females with total litter were euthanized by carbon dioxide inhalation within 24 hours. The number of former implantation sites were recorded. In addition, the number of corpora lutea were recorded for females with total litter loss between LD 0 and 4. Organ weights were collected and tissues preserved.

GROSS NECROPSY
Animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. The numbers of former implantation sites were recorded for females that delivered. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. Uteri of females without macroscopic evidence of implantation were opened and placed in 10% ammonium sulfide solution for detection of early implantation loss.

HISTOPATHOLOGY / ORGAN WEIGHTS
The organs identified below were weighed at necropsy for all scheduled euthanasia animals. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.
- Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries with oviducts, Pituitary gland, Prostate gland, Seminal vesicle (with coagulating gland and fluid), Spleen, Testes, Thymus gland, Thyroids with parathyroids.
Representative samples of the tissues identified below were collected from all animals and preserved in 10% neutral buffered formalin, unless otherwise indicated.
Brain. Coagulating glands, Kidneys Liver, Mammary glands, Ovaries and oviducts, Pituitary gland, Prostate gland, Seminal vesicles, Testes with epididymidesa, and vas deferens, Uterusb with cervix and vagina, all gross lesions.

Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues identified in Text Table 10 for microscopic examination were evaluated from all animals in the control and high-dose groups. Gross lesions were examined from all groups.

Thyroid Hormone Analysis
Serum samples from adult males and from pups terminated on PND 13 will be analyzed for serum levels of Total T4 by the Charles River Bioanalytical Chemistry using a validated UHPLC/MS/MS assay.
Postmortem examinations (offspring):
A gross necropsy was conducted on 1 pup/sex/litter euthanized at scheduled termination (PND 13) with an emphasis on evaluation of developmental morphology and organs of the reproductive system. All pups were euthanized by an intraperitoneal injection of sodium pentobarbital and examined externally for gross abnormalities. At the time of necropsy, the following tissues from 1 pup/sex/litter were preserved in 10% neutral-buffered formalin for possible microscopic examination. Tissue collection: Thyroid (with parathyroid).
Statistics:
Each mean was presented with the standard deviation and the number of animals or cages used to calculate the mean. Where applicable, the litter was used as the experimental unit. Statistical analyses were not conducted on F0 weekly female body weight data after 1 or more animals had entered the gestation phase. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.
Parental mating, fertility, copulation, and conception indices were analyzed using the Chi-square test with Yates’ correction factor. Parental and offspring body weights and body weight changes, parental food consumption, estrous cycle lengths, precoital intervals, gestation lengths, former implantation sites, unaccounted-for sites, live litter size on PND 0, numbers of pups born, absolute and relative organ weights, thyroid hormone values, anogenital distance (absolute and relative to the cube root of body weight), and number of nipples/areolae were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. Mean litter proportions of postnatal survival and pup sexes at birth (percentage of males per litter) were subjected to the Kruskal-Wallis nonparametric ANOVA.
Reproductive indices:
Male Mating Index (%); Female Mating Index (%); Male Fertility Index (%); Female Fertility Index (%); Male Copulation Index (%); Female Conception Index (%); Mean Estrous Cycle Length (days), Mean Pre-Coital Interval (days).
Offspring viability indices:
The mean number of pups born, live litter size, the percentage of males at birth, postnatal survival, body weight, Anogenital Distance, Nipple Anlagen.
Clinical signs:
no effects observed
Description (incidence and severity):
All F0 males and females in the control, 500, 750, and 2500 ppm groups survived to the scheduled necropsy. No test substance-related clinical observations were noted at the daily examinations at any dosage level. Findings noted in the test substance-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
Mean body weights and body weight gains in the 500, 750, and 2500 ppm group F0 males were unaffected by test substance administration throughout the study. The only statistically significant difference from the control group was a higher mean body weight gain in the 2500 ppm group males during Study Days 7–14.

Females:
Premating:
Mean body weights and body weight gains in the 500, 750, and 2500 ppm group F0 females were unaffected by test substance administration during the premating period. The only statistically significant difference from the control group was a higher mean body weight gain in the 500 ppm group females when the entire premating period (Study Days 0–14) was evaluated.

Gestation
Mean body weights and body weight gains in the 500, 750, and 2500 ppm groups were unaffected by test substance administration during gestation. None of the differences from the control group were statistically significant.

Lactation
Mean body weights and body weight gains in the 500, 750, and 2500 ppm groups were unaffected by test substance administration during lactation. None of the differences from the control group were statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males
Mean food consumption, evaluated as g/animal/day, in the 500, 750, and 2500 ppm group F0 males was similar to that in the control group throughout the study. None of the differences from the control group were statistically significant.

Females
Premating
Mean food consumption, evaluated as g/animal/day, in the 500, 750, and 2500 ppm group F0 females was unaffected by test substance administration during the premating period. None of the differences from the control group were statistically significant.

Gestation
Mean maternal food consumption, evaluated as g/animal/day, in the 500, 750, and 2500 ppm groups was unaffected by test substance administration during gestation. None of the differences from the control group were statistically significant.

Lactation
Mean maternal food consumption, evaluated as g/animal/day, in 500, 750, and 2500 ppm groups was unaffected by test substance administration during lactation. None of the differences from the control group were statistically significant.
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:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis
Lower mean T4 levels were noted in the 500, 750, and 2500 ppm group males; the differences from the control group were statistically significant in the 500 and 2500 ppm groups. However, no dose-response relationship was noted in the value of the thyroid hormone level, and no effects on thyroid macroscopic examination and mean thyroid weights were noted at any dosage level. Therefore, the lower mean T4 levels in the F0 males were not considered test substance-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test substance-related histologic changes. Any histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No test substance-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance-treated groups. The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value. The mean lengths of estrous cycles in these groups were also similar to the control group value. None of these differences were statistically significant.

Parameter Dosage Level (ppm) CRL HC Mean (Range)
0 500 750 2500
Male Mating Index (%) 90.0 100.0 100.0 100.0 98.0 (83.3–100.0)
Female Mating Index (%) 90.0 100.0 100.0 100.0 98.0 (83.3–100.0)
Male Fertility Index (%) 80.0 100.0 100.0 100.0 93.9 (80.0–100.0)
Female Fertility Index (%) 80.0 100.0 100.0 100.0 93.9 (80.0–100.0)
Male Copulation Index (%) 88.9 100.0 100.0 100.0 95.7 (80.0–100.0)
Female Copulation Index (%) 88.9 100.0 100.0 100.0 95.7 (80.0-100.0)
Mean Estrous Cycle Length (days) 4.0 4.1 4.1 4.0 4.2 (3.9–5.2)
Mean Estrous Cycle Length (days) 2.1 2.0 2.6 3.7 2.7 (1.4–4.5)


Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Mean gestation lengths in the 500, 750, and 2500 ppm groups were similar to those in the control group. No statistically significant differences were noted. No signs of dystocia were noted in these groups.
F0 male and female mating and fertility, male copulation and female conception indices, estrous cycle lengths, mean number of days between pairing and coitus, gestation lengths, and the process of parturition were unaffected by test substance administration at all dietary concentrations.
There were no test substance-related effects on gross observations, organ weights, or histologic changes observed in the F0 generation. There were no test substance-related effects on the mean number of former implantation sites or unaccounted-for sites at any exposure level.
Key result
Dose descriptor:
NOAEL
Remarks:
Maternal
Effect level:
158 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test substance-related effects were noted on general toxicity and reproductive performance in P0 males and females
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.
Sex:
female
Basis for effect level:
other: No test substance-related effects were noted on reproduction of males and females
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
No effects observed at any concentration level
Key result
Dose descriptor:
NOAEL
Remarks:
Neonatal
Effect level:
158 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test substance-related effects were noted on F1 generation.
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
No adverse effect observed at any concentration level.
Critical effects observed:
no
Lowest effective dose / conc.:
158 mg/kg bw/day (actual dose received)
System:
other: No adverse effect observed at any concentration level.
Organ:
other: No adverse effect observed at any concentration level.
Clinical signs:
no effects observed
Description (incidence and severity):
The general physical condition (defined as the occurrence and severity of clinical observations) of all F1 pups in this study was unaffected by test substance exposure.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Two (2), 4(3), 2(2), and 4(3) pups (litters) in the control, 500, 750, and 2500 ppm groups, respectively, were found dead. Zero (0), 2(2), 2(2), and 2(1) pups (litters) in the same respective groups were missing and presumed to have been cannibalized.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean absolute F1 birth weights (PND 1) in the 2500 ppm group males and females were 4.9% and 5.2% lower, respectively, than the control group. Mean body weight gain in these pups were comparable to the control group during PND 1–4 and slightly lower than the control group during PND 4–10; the differences were statistically significant only for PND 10–13. The significant lower body weight gains in the 2500 ppm group during PND 10-13 were mainly due to lower body weight gains noted in a single litter (No. 4892). As a result, mean absolute male and female pup body weights that were up to 7.8% and 10.4% lower, respectively, than the control group during PND 4–13. The effects on mean body weights and body weight gains at 2500 ppm were test substance-related but not adverse as with the exception of a single litter, all mean body weights were within the range of values in the Charles River historical control data (version 2019.04).
Mean male and female pup body weights and body weight changes in the 500 and 750 ppm groups were unaffected by parental administration of the test substance. No statistically significant differences from the control group were noted.
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:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid Hormone Analysis (PND13)
Mean T4 levels in the 2500 ppm group F1 males and females were 12.6% and 19.4% lower, respectively, than the control group on PND 13; the difference was statistically significant for females. The effects on mean T4 levels at 2500 ppm were considered to be test substance-related but not adverse as the values were within the range of values in the Charles River historical control data.
Mean T4 levels in the 500 and 750 ppm group F1 males and females were comparable to the control group; no statistically significant differences were noted.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distances (absolute, relative to pup body weight, and relative to the cube root of pup body weight) in the 500, 750, and 2500 ppm groups were similar to the control group values. Differences from the control group were slight and not statistically significant.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Areolae/nipple anlagen in the F1 male pups was unaffected by parental administration of the test substance when evaluated on PND 13. The test substance-treated group values were not statistically different from the control group values
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related effects on thyroid weights in the F1 males and females at any dosage level on PND 13. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Two (2), 4(3), 2(2), and 4(3) pups (litters) in the control, 500, 750, and 2500 ppm groups, respectively, were found dead. No internal findings that could be attributed to parental test substance administration were noted at the necropsies of pups that were found dead. Aside from the presence or absence of milk in the stomach, no other internal findings were noted.
Histopathological findings:
no effects observed
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Mean absolute F1 birth weights (PND 1) in the 2500 ppm group males and females were 4.9% and 5.2% lower, respectively, than the control group. Lower mean body weight gains in these pups during PND 4–13 resulted in mean absolute male and female body weights that were up to 7.8% and 10.4% lower, respectively, than the control group. These differences were not statistically significantly different compared to the control group except for female F1 pups in the 2500 ppm group on PND 13. Also, the lower mean pups body weight in the 2500 ppm group was mainly due to lower body weights in a single litter (No. 4892) and the body weight mean in other litters in the 2500 ppm group were all within Charles River historical control data range; therefore, the effects on mean body weights and body weight gains at 2500 ppm were considered test substance-related and nonadverse. Mean pup body weights and body weight gains in the 500 and 750 ppm groups were unaffected by test substance administration. There were no test substance-related effects on mean number of pups born, pup survival, liver litter size, mean sex ratio, anogenital distance, areolae/nipple anlagen (males only), thyroid hormone levels (total T4) on PND 13, and thyroid weights. There were no clinical observations or necropsy findings that could be attributed to F0 maternal administration of the test substance at any exposure concentration.
Key result
Dose descriptor:
NOAEL
Remarks:
Neonatal
Generation:
F1
Effect level:
158 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test substance-related effects were noted on F1 generation (males and females)
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
No test substance-related effects were noted on F1generation (males and females) at any dose level.
Critical effects observed:
no
Lowest effective dose / conc.:
158 mg/kg bw/day (actual dose received)
System:
other: test substance-related effects were noted at any exposure concentration
Organ:
other: No test substance-related effects were noted at any exposure concentration.
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
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):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Not evaluate in OECD 421 guideline.
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
158 mg/kg bw/day
Treatment related:
no
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:

The objective of this study was to provide preliminary information on the potential adverse effects of the test substance on male and female reproduction within the scope of a screening study. This encompassed gonadal function, mating behavior, conception, parturition, and lactation of the parental generation and the development of offspring from conception through day 13 of postnatal life.

The study design was as follows:

Group Number Test Substance Dose Level (ppm): Basal Diet, Benzyl Salicylate 500 ppm, 750 ppm, 2500 ppm

Number of Animals/sex/group: 10

Mean Calculated Test Substance Consumption (mg/kg/day):

 Theoretical Dietary

Concentration (ppm)

Mean Test Substance Consumption (mg/kg/day)

 

 

  

 Males

Females

 

 

 

 

 

 

 

 

 Prior to Mating

   Prior to Mating

Gestation

 Lactation

 250

 34

 32

 33

 

 67

 

 

 

 750

 49

 48

 51

 

 101

 

 

 

 2500

 166

 158

 170

 

 324

 

 

 

Food consumption generally increases during lactation due to milk production and direct consumption of diet by offspring in the latter portion of the lactation period.

Animals were administered the test substance continuously in the diet. F0 males were dosed for 14 days prior to mating and continuing through the day of euthanasia. F0 females were dosed for 14 days prior to mating and continuing through Lactation Day 13. The following parameters and end points were evaluated in this study: 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.

Mean compound consumption was 34, 49, and 166 mg/kg/day in the 500, 750, and 2500 ppm group F0 males during the premating treatment period (Study Days 0–14). Mean compound consumption was 32, 48, and 158 mg/kg/day during the premating period (Study Days 0–14), 33, 51, and 170 mg/kg/day during gestation (Gestation Days 0–20), and 67, 101, and 324 mg/kg/day during lactation (Lactation Days 1–13) in the 500, 750, and 2500 ppm group females, respectively. All F0 males and females in the control, 500, 750, and 2500 ppm groups survived to the scheduled necropsy. There were no test substance-related clinical observations noted at the daily examinations at any exposure level.

No test substance-related effects on F0 body weight or food consumption parameters were noted for males and throughout the study at any exposure concentration. F0 male and female mating and fertility, male copulation and female conception indices, estrous cycle lengths, mean number of days between pairing and coitus, gestation lengths, and the process of parturition were unaffected by test substance administration at all dietary concentrations. No test substance-related effects on mean T4 levels were noted in the 500, 750, and 2500 ppm group F0 males on Study Day 28. There were no test substance-related effects on gross observations, organ weights, or histologic changes observed in the F0 generation. There were no test substance-related effects on the mean number of former implantation sites or unaccounted-for sites at any exposure level. Mean absolute F1 birth weights (PND 1) in the 2500 ppm group males and females were 4.9% and 5.2% lower, respectively, than the control group. Lower mean body weight gains in these pups during PND 4–13 resulted in mean absolute male and female body weights that were up to 7.8% and 10.4% lower, respectively, than the control group. These differences were not statistically significantly different compared to the control group except for female F1 pups in the 2500 ppm group on PND 13. Also, the lower mean pups body weight in the 2500 ppm group was mainly due to lower body weights in a single litter (No. 4892) and the body weight mean in other litters in the 2500 ppm group were all within Charles River historical control data range; therefore, the effects on mean body weights and body weight gains at 2500 ppm were considered test substance-related and nonadverse. Mean pup body weights and body weight gains in the 500 and 750 ppm groups were unaffected by test substance administration.

There were no test substance-related effects on mean number of pups born, pup survival, liver litter size, mean sex ratio, anogenital distance, areolae/nipple anlagen (males only), thyroid hormone levels (total T4) on PND 13, and thyroid weights. There were no clinical observations or necropsy findings that could be attributed to F0 maternal administration of the test substance at any exposure concentration.

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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
158 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Reproductive Toxicity according to OECD Guideline 421 and GLP.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Short description of key information:
NOAEL for reproductive effects was 158 mg/kg bw/d in male and female rats. The NOAEL for developmental effects in the pups was 158 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

All females survived to the scheduled necropsy on Gestation Day 21; there were no test substance‑related clinical observations during the study.Mean maternal body weight loss in the 4000 ppm group and lower mean body weight gain in the 3000 ppm group were noted following administration of the first dose (Gestation Days 6–7), but mean body weight changes in these groups were comparable to the control group thereafter. Lower mean food consumption was also noted in these groups during Gestation Days 6–9. Mean absolute body weights in the 4000 ppm group were up to 6.4% lower than the control group during Gestation Days 7–21. The effects on mean body weights, body weight gains, and food consumption at 4000 ppm were considered test substance-related and adverse due to the magnitude of the changes. Mean gravid uterine weight in the 4000 ppm group was also lower than the control group. Mean body weights in the 3000 ppm group were ≤ 4.3% lower than the control group. Mean absolute body weights in this group were lower than the control group during Gestation Days 7–15 (statistically significantly on Gestation Days 7, 9, 11, and 15). Mean body weight gain in the 3000 ppm group was statistically significantly lower compared to the control group during Gestation Day 6-7. Thereafter, mean body weight gains in this group were comparable to the control group, including for the overall treatment period (Gestation Days 6–21). The effects on mean body weights, body weight gains, and food consumption at 3000 ppm were considered test substance-related but not adverse due to the overall magnitude of the changes and the transient nature. Mean net body weight, net body weight gain, and gravid uterine weight in this group were comparable to the control group. Mean maternal body weights, body weight gains, and food consumption in the 1000 ppm group, mean gravid uterine weights in the 1000 and 3000 ppm groups, and mean net body weights and net body weight gains in the 1000, 3000, and 4000 ppm groups were comparable to the control group.

There were no test substance-related macroscopic findings noted at the scheduled necropsy. Mean fetal body weights (male, female, and combines sexes) were 7.1% and 10.7% lower in the 4000 ppm group, compared to the concurrent control group. The effects on fetal body weights at 4000 ppm were considered test substance‑related and adverse. Intrauterine growth in the 1000 ppm group and intrauterine survival in the 1000, 3000, and 4000 ppm groups were unaffected by test substance administration.

There were no test substance-related fetal malformations noted in any group. A higher mean litter proportion of 14th rudimentary rib(s) was noted in the 4000 ppm group and higher mean litter proportions of bent rib(s) were noted in the 3000 and 4000 ppm groups compared to the concurrent control group. These findings corresponded to the test substance-related lower mean fetal body weights observed at 3000 and 4000 ppm but were not considered adverse because both have been noted to resolve postnatally. No test substance-related developmental variations were noted at 1000 ppm.

Based on adverse mean body weight loss, lower mean body weights and food consumption at 4000 ppm, a dosage level of 3000 ppm, equivalent to 214 mg/kg/day, was considered to be the no observed adverse effect level (NOAEL) for maternal toxicity. Based on lower mean fetal body weights in the 4000 ppm group, a dosage level of 3000 ppm, equivalent to 214 mg/kg/day, was considered to be the NOAEL for embryo/fetal development when benzyl salicylate was administered orally (via the diet) to time-mated Crl:CD(SD) rats. 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
214 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
An Oral (Dietary) Prenatal Developmental Toxicity Study of Benzyl Salicylate in Sprague Dawley Rats. OECD Guideline 414, GLP study.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Justification for selection of Effect on developmental toxicity: via oral route:

NOAEL for developmental effects in the pups was 214 mg/kg bw/day.

Toxicity to reproduction: other studies

Description of key information

F0 male and female mating and fertility, male copulation and female conception indices, estrous cycle lengths, mean number of days between pairing and coitus, gestation lengths, and the

process of parturition were unaffected by test substance administration at all dietary concentrations. No test substance-related effects on mean T4 levels were noted in the 500, 750, and 2500 ppm

group F0 males on Study Day 28. There were no test substance-related effects on gross observations, organ weights, or histologic changes observed in the F0 generation. There were no test substance-related effects on the mean number of former implantation sites or unaccounted-for sites at any exposure level. 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.

Additional information

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 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.

Justification for classification or non-classification

Based on data generated on Benzyl Salicylate, for classification and labelling purpose this substance would not be classified in accordance with Regulation (EC) No 1272/2008 (CLP).