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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Dec 2018 to 27 Mar 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study OECD 408 (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:
Sub-chronic toxicity study (90-day), oral route (Annex IX, Section 8.6.2.; test method: EU B.26./OECD TG 408) in rats with the registered substance.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
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
Details on species / strain selection:
The Crl:CD(SD) rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- 10 animals/group/sex
- Age at study initiation: 7 weeks:
- Weight at study initiation:176 and 272 g
- Fasting period before study: Animals were fasted overnight prior to blood collection for clinical pathology evaluations and prior to necropsy.
- Housing: 2 to 4 animals of the same sex until randomization. Following randomization, animals were group housed (2 animals of the same sex and same dosing group together) in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve. 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: at least one week

DETAILS OF FOOD AND WATER QUALITY: The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are on file at the Testing
Facility. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
Water: Municipal tap water after treatment by reverse osmosis and ultraviolet irradiation was freely available to each animal via an automatic watering system. Water bottles were provided, if required.
Periodic analysis of the water was performed, and results of these analyses are on file at the Testing Facility. It was considered that there were no known contaminants in the water that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26°C
- Humidity (%): 30-70%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle

IN-LIFE DATES: From:02 Jan 2019 To: 04 Apr 2019

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
The route of administration was oral (dietary) because the oral route is the most likely route of human exposure.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For administration to Group 1 control animals, an appropriate amount of PMI Nutrition International, LLC Certified Rodent LabDiet 5002 (meal) was weighed out weekly and was placed in a labeled bag.

DIET PREPARATION
Test substance dietary formulations were prepared at appropriate concentrations to meet dose level requirements. The test substance was added to PMI Nutrition International, LLC Certified Rodent LabDiet 5002 (meal) on a weight/weight basis (no adjustment for purity) and mixed in a Hobart mixer for 5 minutes. The resulting premix was then mixed thoroughly with the remaining amount of feed in a Hobart mixer for 10 minutes. The dietary formulations were prepared weekly and stored at room temperature (18°C to 24°C) until use.

VEHICLE
Not need (dietary study).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical Method:
Analyses were performed by a high performance liquid chromatography (HPLC) method with ultraviolet light absorbance using a validated analytical procedure.

Concentration Analysis:
Duplicate sets of samples (50 g) for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.

Homogeneity Analyses:
Duplicate sets of samples (50 g) for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Homogeneity results were considered acceptable if the relative standard deviation of the mean concentration value at each sampling location was ≤ 10% and if mean sample concentration results were within or equal to ± 15% of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.

Stability Analyses:
Test substance diet formulations have been previously shown to be stable over the range of concentrations used on this study for at least 11 days at room temperature. Therefore, stability of test substance formulations was not assessed on this study.
Duration of treatment / exposure:
Basal diet and dietary admixed containing test substance were administered ad libitum for at least 90 consecutive days.
Doses / concentrationsopen allclose all
Dose / conc.:
1 500 ppm
Remarks:
Equivalent to 86 mg/kg bw/d for males and 106 mg/ kg bw/d for females
Dose / conc.:
3 000 ppm
Remarks:
Equivalent to 177 mg/kg bw/d for males and 204 mg/kg bw/d for females
Dose / conc.:
6 000 ppm
Remarks:
Equivalent to 357 mg/kg bw/d for males and 429 mg/kg bw/d for females
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
- Dose selection rationale:
Dosage levels were based on the findings of a 14-day range-finding study, in which nonadverse test substance-related effects on body weights (decreased body weights, lower body weight gains or body weight losses) and decreased food consumption were noted in 3000 ppm females (decreased body weight) and 6000 ppm males and females.
3 It was anticipated that the high-exposure level would show compound-specific effects but not produce an incidence of fatalities that would prevent a meaningful evaluation. The lower exposure levels were selected at intervals that were predicted to be narrow enough to reveal any dose-related trends. Though priority was given to detecting a dose-related trend, it was expected that the low-exposure level would be the no-observed-adverse-effect level (NOAEL).

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
Cage side observations were performed daily, beginning on Day 1 and lasting throughout the study period (see Appendix 1 – Study Protocol and Deviations). Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
During social housing, some observations (e.g., fecal observations) may not have been
attributable to an individual animal.

Throughout the study, animals were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. Animals were not removed from the cage during observations, unless necessary for identification or confirmation of possible findings.


DETAILED CLINICAL OBSERVATIONS:
The animals were removed from the cage, and a detailed clinical observation was performed within 4 days of receipt, 1 week (± 2 days) prior to randomization, on the day of randomization, on Day 1 (prior to dosing), weekly (± 2 days) during the study period, and on the day of the scheduled necropsy.

BODY WEIGHT:
Animals were weighed individually 1 week (± 2 days) prior to randomization, on the day of randomization, on Day 1 (prior to dosing), weekly (± 2 days) during the study period, on the day prior to the scheduled necropsy, and on the day of the scheduled necropsy. A fasted weight was recorded on the day of the scheduled necropsies.

FOOD CONSUMPTION AND COMPOUND INTAKE :
Food consumption was quantitatively measured weekly (± 2 days) starting on Day 1 and
continued throughout the study period.
The mean amounts of test substance consumed (mg/kg/day) by each sex per dose group were calculated from the mean food consumed (g/kg/day) and the appropriate target concentration of test substance in the food (mg/kg). Food efficiency (body weight gained as a percentage of food consumed) was calculated and reported.


OPHTHALMOSCOPIC EXAMINATION:
Ocular examinations were conducted by a board certified veterinary ophthalmologist during the pretreatment period (Day -2) and near the end of the dosing period (Day 91). An indirect ophthalmoscope and slit lamp biomicroscope were used to examine the ocular structures. Prior to examination, an appropriate mydriatic agent was used for pupillary dilation.

HAEMATOLOGY:
Animals were fasted overnight prior to blood collection. Blood samples for hematology and serum chemistry were collected from the jugular vein. Blood samples for coagulation parameters were collected by necropsy personnel from the inferior vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation. K2EDTA was used for the anticoagulant on samples collected for hematology. Sodium citrate was used for samples collected for clotting determinations. Samples for serum chemistry were collected without anticoagulants.
Parameters checked: Differential leukocyte count (percent and absolute), Erythrocyte count
Total hemoglobin, Hematocrit, Mean corpuscular hemoglobin, Mean corpuscular volume, Mean corpuscular hemoglobin concentration, Platelet count, red cell distribution width, reticulocyte count, Total leukocyte count, Activated partial thromboplastin time, Fibrinogen, Prothrombin time, Sample qualitya.


CLINICAL CHEMISTRY
Blood samples were processed for serum, and the serum was analyzed for the parameters: Alanine aminotransferase, Albumin, A/G ratio (calculated), Alkaline phosphatase, Aspartate aminotransferase, Calcium, Chloride, Creatinine, Gamma glutamyltransferase, Globulin (calculated), Glucose, Phosphorus, Potassium, Sodium
Sorbitol dehydrogenase, Total bilirubin, Total cholesterol, Total protein, Triglycerides,
Urea nitrogen,Appearance.


URINALYSIS
Urine was collected overnight using metabolism cages. Urine samples were processed and analyzed for the parameters: Bilirubin, Color and clarity, Glucose, Ketones, Occult blood, pH, Protein, Specific gravity, Volum.


Thyroid Hormone Analysis:Triiodothyronine (T3), Thyroxine (Total T4), Thyroid Stimulating Hormone (TSH).
Blood samples (approximately 1.2 mL) were collected from a jugular vein for thyroid hormone analyses into tubes without anticoagulants.
Sacrifice and pathology:
Animals were subjected to a complete necropsy examination, which included evaluation of all external surfaces and orifices; the cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. A veterinary pathologist, or other suitably qualified person, was available

GROSS PATHOLOGY:
The organs were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.
Organs weighted at necropsy: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries with oviducts, Pituitary, Prostate with seminal vesicles, Spleen, Testes, Thymus
Thyroid with parathyroids, Uterus.

HISTOPATHOLOGY:
Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues were evaluated from all animals in the control and high-dose groups at the terminal necropsy. Gross lesions were examined from all animals in the low- and mid-dose groups at the terminal necropsy.
Tissues evaluated: Adrenal glands, Aorta, Bone with marrow, Femur (with joint), Sternum, Bone marrow smear (from femur), Brain, Cervix, Epididymides, Eyes with optic nerves, Gastrointestinal tract, Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Harderian glands, Heart, Kidneys, Larynx, Liver (sections of 2 lobes), Lungs (including bronchi, fixed by inflation with, fixative), Lymph nodes, Axillary, Mandibular, Mesenteric, Nasal cavityf, Ovaries (2) with oviductsd, Pancreas, Peripheral nerve (sciatic)g
Peyer’s patches, Pharynx, Pituitary, Prostate, Salivary glands, Seminal vesicles, Skeletal muscle (quadriceps), Skin with mammary glande, Spinal cord (cervical, thoracic, lumbar), Spleen, Testes (2)b, Thymus, Thyroid (with parathyroids [2]), Tongue, Trachea, Urinary bladder, Uterus, Vagina, Gross lesions (when possible).
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and are reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible. Values may also be expressed as a percentage of predose or control values or fold change of control values when deemed appropriate. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. Calculated values on Provantis tables may not be reproducible from the individual values presented because all calculations were conducted using non-rounded values.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no test substance-related clinical observations. All clinical observations in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in an exposure-related manner, and/or were common findings for laboratory rats of this age and strain.
Mortality:
no mortality observed
Description (incidence):
All animals survived until scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related lower mean body weight gains were noted in the 6000 ppm group males and females generally throughout the exposure period; differences from the control group were occasionally statistically significant. As a result, statistically significantly lower mean body weight gains were noted for males and females in this group compared to the control group when the entire exposure period (Days 1–91) was evaluated. In addition, mean body weights in the 6000 ppm group males and females were lower (up to 9.9% and 13.2%, respectively) compared to the control group during Days 15–91. The aforementioned body weight effects noted in the 6000 ppm group males and females were considered adverse due to the magnitude of the body weight losses, the extended period during the study in which the body weight effects occurred and the lack of any trend for body weights to return to concurrent control values. These body weight effects correlated with lower mean food efficiency in both males and females at 6000 ppm.
Body weights in the 1500 and 3000 ppm group males and females were unaffected by test substance administration. Differences from the control group were not statistically significant and/or did not occur in an exposure-related manner.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test substance-related statistically significantly lower mean food consumption was noted in the 6000 ppm group males compared to the control group during Days 1–8; due to the small magnitude of change (2 g lower compared to the control group) and a lack of an effect during the remainder of the dosing period, this difference was considered nonadverse. Statistically significantly lower mean food efficiency was observed for males in the 6000 ppm group compared to the control group sporadically throughout the dosing period (Days 1–15, 22–29, and 43–50). Lower mean food efficiency (2 to 6 g lower compared to the control group) was also noted for females in the 6000 ppm group during 7 of the 13 weekly intervals during the study. The effects on food efficiency were considered test substance-related and adverse as the effects were a contributing factor to the adverse effects on body weights in males and females at 6000 ppm. Food consumption in the 1500 and 3000 ppm group males and females and the 6000 ppm group females was unaffected by test substance administration. Differences from the control group were not statistically significant and/or did not occur in an exposure-related manner.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmic lesions indicative of toxicity were observed in any of the test substance-treated groups. All findings observed were typical in prevalence and appearance for laboratory rats of this age and strain.
Haematological findings:
no effects observed
Description (incidence and severity):
Hematology parameters were unaffected by test substance administration. Some parameters such as mean corpuscular hemoglobin (MCH) and red cell distribution width (RDW) were statistically significantly different from the concurrent control group; however, the magnitude of the changes were small and mean values were within the reference ranges in the Charles River Ashland historical control data (version 3.6).

Coagulation parameters were unaffected by test substance administration. There were no statistically significant differences when the control and test substance-treated groups were compared.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related changes in serum chemistry parameters. Some parameters such as sorbitol dehydrogenase (SDH), total bilirubin (TBILI), total protein (TPROT), globulin (GLOB), and albumin (ALB) were different from the concurrent control group. Some of these differences were statistically significant, however, the magnitude of the changes were small and mean values were within the reference ranges in the Charles River Ashland historical control data (version 3.6). The mean differences were also attributable to individual animal values in both control and treatment groups, a normal biological variation. Changes noted in other parameters, including those which may be have been statistically significant, were also not considered test substance-related and were attributed to biologic variation because they were of a magnitude of change commonly observed in Sprague Dawley rats under similar study conditions or the change was in a direction not of toxicologic importance.

There were no direct test substance-related effects on thyroid hormone (T3, T4, and TSH) levels in males or females at any dietary concentration tested. There was a decrease (not statistically significant) in mean T3 levels for males in the treated groups. These changes were not observed in an exposure-related manner. There was also an exposure-responsive decrease in mean T4 levels for males in all treated groups. These effects were not considered to be a direct result of test substance administration, and were considered secondary effects resulting from decreased final body weight.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis parameters were unaffected by test substance administration. There were no test substance-related changes in urinalysis parameters. Changes noted in other parameters, including those which may be have been statistically significant, were also not considered test substance-related and were attributed to biologic variation because they did not occur in a dose-related manner and were within the range of historical control values (version 3.6).
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related alterations in organ weights. Some organ weight differences were statistically significant when compared to the control group but were considered to be a result of a test substance-related effect on final body weight. Statistically significantly higher brain, heart, kidney, liver, and spleen weights relative to terminal body weights in the 3000 and/or 6000 ppm group females were all considered to be due to a test substance-related effect on terminal body weight and not directly test substance-related. Statistically significantly lower absolute pituitary gland weights were noted in the 6000 ppm group males. There were no correlating histologic findings and the mean pituitary gland weight remained within the mean ± 2 standard deviations in the Charles River Ashland historical control database (version 3.6). Thus, this finding was considered to be due to biologic variation and not test substance-related.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histologic examination of the tissues revealed no observations that were considered to be associated with administration of the test substance. Histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. Of note, marked, regionally extensive hepatocellular necrosis was noted in a 6000 ppm group male (No. 4004) at the terminal euthanasia. This inding correlated with the gross observation of mass in the caudate lobe of the liver. The location and histologic appearance of this finding was consistent with a liver lobe torsion which is considered an occasional spontaneous lesion in rats11 and was thus not considered test substance-related. Additionally, minimal retinal dysplasia and minimal mononuclear infiltrate in the skeletal (quadriceps) muscle noted in 6000 ppm group males and/or females were considered background lesions and are reported in the Charles River Ashland historical control database (historical control finding for mononuclear infiltrate reported under skeletal muscle; version 3.9). 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

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
177 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food efficiency

Target system / organ toxicity

Critical effects observed:
no
Lowest effective dose / conc.:
177 mg/kg bw/day (nominal)
System:
other: body weight
Organ:
other: body weight and weight gain

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, dietary administration of benzyl salicylate to Crl:CD(SD) rats at concentrations of 1500, 3000, and 6000 ppm for a minimum of 90 days resulted in adverse lower body weights, lower body weight gains, and decreased food efficiency for males and females at 6000 ppm. Therefore, the no-observed-effect level (NOEL) was considered to be 3000 ppm (equivalent to 177 and 204 mg/kg/day for males and females, respectively).
Executive summary:

The objective of this study was to evaluate the potential toxicity of the Benzyl Salicylate when administered via the diet to Sprague Dawley rats for at least 90 consecutive days. The study design was as follows:

Group Number

 Treatment  Dietary Concentration (ppm)*

 Equivalent dosage in mg/kg bw/d

(males and females respectively)

 Number of Animals/sex/group
 1  Basal Diet  0  10
 2  Benzyl Salicylate  1500  86 - 106 mg/kg bw/d  10
 3  Benzyl Salicylate  3000  177 - 204 mg/kg bw/d  10
 4  Benzyl Salicylate  6000 357 - 429 mg/kg/d   10

* Test substance was presented as a fixed dietary admix concentration without adjustment for body weight. There was no adjustment for purity when preparing the test or control diets.

Animals were administered the test substance continuously in the diet for at least 90 consecutive days. The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, ophthalmology, clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis), thyroid hormone concentration (T3, T4, and TSH), gross necropsy findings, organ weights, and histopathologic examinations. Average compound consumption for the 1500, 3000, and 6000 ppm groups was 86, 177, and 357 mg/kg/day for males and 106, 204, and 429 mg/kg/day for females, respectively. All animals survived to the scheduled necropsy. There were no direct test substance-related clinical, ophthalmic, macroscopic, or microscopic observations or effects on serum chemistry, hematology, coagulation, urinalysis, thyroid hormone concentration, and organ weights. Adverse test substance-related lower body weights and body weight gains were noted in the 6000 ppm group males and females generally throughout the dosing period. In the 6000 ppm group males, lower food consumption was noted during the first week of dosing and lower food efficiency was noted throughout the first month of the dosing period. Lower mean food efficiency was also noted for females in the 6000 ppm group during 7 of the 13 weekly intervals during the study. Effects on mean food efficiency were considered adverse as they were a contributing factor to the adverse effects on body weights in males and females at 6000 ppm. Based on the results of this study, dietary administration of benzyl salicylate to Crl:CD(SD) rats at concentrations of 1500, 3000, and 6000 ppm for a minimum of 90 days resulted in adverse lower body weights, lower body weight gains, and decreased food efficiency for males and females at 6000 ppm. Therefore, the no-observed-effect level (NOEL) was considered to be 3000 ppm (equivalent to 177 and 204 mg/kg/day for males and females, respectively).