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Description of key information

Five oral repeated dose toxicity studies are available. The studies were conducted using rats and mice as the test species. The studies were conducted for a period of 14 days or 13 weeks. There is no indication that the studies conformed to guidelines or were GLP compliant. The NOAEL derived for subchronic exposure to rats was 250 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
Male and female F344/N rats obtained from Harlan Industries and observed for 12 days. Animals were approximately 6 weeks old when placed on study. Groups of five rats of each sex were administered 0, 250, 500, 1,000, 2,000, or 4,000 mg/ kg benzyl acetate in corn oil by gavage for 14 consecutive days. Animals were housed five per cage and received water and feed ad libitum. Rats were observed daily for mortality and were weighed weekly. On day 16, surviving animals were killed and necropsies were performed on all animals.
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Benzyl Acetate
- Physical state: water-white liquid with a pear-like odour

Melting Point: -51.3°C
Boiling Point: 213°C
Vapor Pressure: 1.99 mm Hg at 60'C
Density: 1.05
Refractive Index: 1.4998
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Harlan Industries Indianapolis. I N
- Age at study initiation: 6 weeks
- Weight at study initiation: 103.0-127.4g
- Housing: Bedding: Beta-Chips heat treated hardwood chips
Cages: Polycarbonate
Cage filters: Spun-bonded polyester filters
- Diet (e.g. ad libitum): Wayne Lab-Blox Allied Mills. ad libitum
- Water (e.g. ad libitum): Tap water supplied through automatic watering system. ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 40-60
- Air changes (per hr): 15

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Doses were prepared on a weight-to-volume basis by pipetting the appropriate amount of benzyl acetate into a vessel and adding enough corn oil to give the desired concentration. Solutions were mixed until they were visually homogeneous. Rats received 5 ml/kg. Benzyl acetate/corn oil mixtures were analyzed at Midwest Research Institute and found to be stable at room temperature for at least 7 days. Once prepared, benzyl acetate/corn oil mixtures were stored at 5O°C for no longer than 11 days.


VEHICLE
- Justification for use and choice of vehicle (if other than water): No data
- Concentration in vehicle: Male and female rats: 0, 250, 500, 1,000, 2,000 or 4,000 mg/kg body weight in corn oil
- Amount of vehicle (if gavage): 5 ml/kg for rat
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of benzyl acetate in corn oil were selected at random and analyzed periodically at Southern Research Institute. Results of these analyses and of referee analyses at Midwest Research Institute indicated that benzyl acetate/ corn oil mixtures were properly formulated.
Duration of treatment / exposure:
14 consecutive days
Frequency of treatment:
Daily
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Dose / conc.:
4 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 animals
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on an acute study
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Obserted daily for mortality and clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Observed daily for mortality and clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: at the start and end of dosing

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: Necropsies performed on all animals
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: No
Other examinations:
No further data
Statistics:
No data
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
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
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY All rats that received 4,000 mg/kg body weight were dead by the afternoon of day 2, and all rats that received 2,000 mg/ kg were dead by the afternoon of day 5. No other rats died.

BODY WEIGHT AND WEIGHT GAIN Final mean body weights of both sexes of dosed rats were comparable to control animals

GROSS PATHOLOGY The cecum was redder than normal in 3/5 males and 3/ 5 females that received 4,000 mg/ kg.

Dose descriptor:
NOAEL
Effect level:
ca. 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects clinical signs; mortality; body weight
Critical effects observed:
no

Male/Female

Dose

Survival

Final weight relative to control %

Male

0

5/5

-

250

5/5

99.2

500

5/5

96.1

1000

5/5

95.4

2000

0/5

-

4000

0/5

-

Female

0

5/5

-

250

5/5

97.8

500

5/5

99.5

1000

5/5

97.7

2000

0/5

-

4000

0/5

-

Conclusions:
The NOAEL was found to be 500 mg/kg body weight after dosing rats with benzyl acetate in corn oil for 14 days.
Executive summary:

Male and female F344/N rats were obtained from Harlan Industries and observed for 12 days. Animals were approximately 6 weeks old when placed on study. Groups of five rats of each sex were administered 0, 250, 500, 1,000, 2,000, or 4,000 mg/ kg

benzyl acetate in corn oil by gavage for 14 consecutive days. Animals were housed five per cage and received water and feed ad libitum. Rats were observed daily for mortality and were weighed weekly. On day 16, surviving animals were killed and necropsies were performed on all animals. The NOAEL was found to be 500 mg/kg body weight after dosing rats with benzyl acetate in corn oil for 14 days.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Guideline-comparable and GLP studies are available with older studies of acceptable methodology

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeat Dose Toxicity: Gavage

In a study conducted by Abdo (1986), male and female F344/N rats were obtained from Harlan Industries and observed for 12 days. Animals were approximately 6 weeks old when placed on study. Groups of five rats of each sex were administered 0, 250, 500, 1,000, 2,000, or 4,000 mg/ kg

benzyl acetate in corn oil by gavage for 14 consecutive days. Animals were housed five per cage and received water and feed ad libitum. Rats were observed daily for mortality and were weighed weekly. On day 16, surviving animals were killed and necropsies were performed on all animals. The NOAEL was found to be 500 mg/kg body weight after dosing rats with benzyl acetate in corn oil for 14 days.

In a study conducted by Abdo (1986), male and female B6C3F1 mice were obtained from Harlan Industries and observed for 12 days. Animals were approximately 6 weeks old when placed on study. Groups of five mice of each sex were administered 0, 125, 250, 500, 1,000, or 2,000 mg/ kg benzyl acetate in corn oil by gavage for 14 consecutive days. Animals were housed five per cage and received water and feed ad libitum. Mice were observed daily for mortality and were weighed weekly. On day 16, surviving animals were killed and necropsies were performed on all animals. The NOAEL was found to be 1000 mg/kg body weight after dosing mice with benzyl acetate in corn oil for 14 days.

Thirteen-week studies were conducted by Abdo (1986) to evaluate the cumulative toxicity of benzyl acetate and to determine the doses to be used in the 2-year studies. Four-week-old male and female F344/ N rats were obtained from Harlan Industries, observed for 13 days, and assigned by species and sex to cages according to a table of random numbers. The cages were then assigned to dosed and control groups according to another table of random numbers. Rats were housed five per cage in polycarbonate cages covered with spun-bonded polyester filters. Racks and filters were replaced every 2 weeks. Cages and bedding were replaced twice per week. Water, via an automatic watering system, and Wayne Lab Blox were available ad libitum. Groups of 10 rats of each sex were administered 0, 62.5, 125, 250, 500, or 1,000 mg/ kg benzyl acetate in corn oil by gavage, 5 days per week for 13 weeks. Animals were checked for mortality and signs of morbidity twice daily. Those animals that were judged moribund were killed and necropsied. Each animal was given a clinical examination weekly, including palpation for tissue masses or swelling. Individual body weight data were collected weekly. On days 92-96, survivors were killed with carbon dioxide. Necropsies were performed on animals that survived to the end of the study and on all animals found dead, unless precluded in whole or in part by autolysis or cannibalization. The following specimens were examined microscopically for controls, for the highest dosage group with at least 60% survivors at the time of the group kill, and for all animals that died before the survivors of the group were killed: gross lesions, tissue masses, abnormal lymph nodes, skin, mandibular lymph nodes, mammary gland, salivary gland, thigh muscle, bone, bone marrow, thymus, trachea, lungs and bronchi, heart,thyroid, parathyroid, esophagus, stomach, duodenum, jejunum, ileum, colon, mesenteric lymph nodes, liver, gallbladder (mice), pancreas, spleen, kidneys, adrenals, urinary bladder, seminal vesicles/ prostate/ testes or ovaries/ uterus, nasal cavity, brain, pituitary, and spinal cord, Tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The NOAEL was found to be 500 mg/kg body weight after dosing male rats with benzyl acetate in corn oil for 13 weeks. The NOAEL was found to be 250 mg/kg body weight after dosing female rats with benzyl acetate in corn oil for 13 weeks.

Thirteen-week studies were conducted by Abdo (1986) to evaluate the cumulative toxicity of benzyl acetate and to determine the doses to be used in the 2-year studies. Four-week-old male and female B6C3F1 mice were obtained from Harlan Industries, observed for 13 days, and assigned by species and sex to cages according to a table of random numbers. The cages were then assigned to dosed and control groups according to another table of random numbers. Mice were housed five per cage in polycarbonate cages covered with spun-bonded polyester filters. Racks and filters were replaced every 2 weeks. Cages and bedding were replaced twice per week. Water, via an automatic watering system, and Wayne Lab Blox were available ad libitum. Groups or 10 male mice were administered 0, 62.5, 125, 250, 500, or 1,000 mg/ kg benzyl acetate in corn oil by gavage, 5 days per week for 13 weeks. Groups of 10 female mice were administered 0, 125, 250, 500, 1,000, or 2,000 mg/ kg on the same schedule. Animals were checked for mortality and signs of morbidity twice daily. Those animals that were judged moribund were killed and necropsied. Each animal was given a clinical examination weekly, including palpation for tissue masses or swelling. Individual body weight data were collected weekly. On days 92-96, survivors were killed with carbon dioxide. Necropsies were performed on animals that survived to the end of the study and on all animals found dead, unless precluded in whole or in part by autolysis or cannibalization. The following specimens were examined microscopically for controls, for the highest dosage group with at least 60% survivors at the time of the group kill, and for all animals that died before the survivors of the group were killed: gross lesions, tissue masses, abnormal lymph nodes, skin, mandibular lymph nodes, mammary gland, salivary gland, thigh muscle, bone, bone marrow, thymus, trachea, lungs and bronchi, heart, thyroid, parathyroid, esophagus, stomach, duodenum, jejunum, ileum, colon, mesenteric lymph nodes, liver, gallbladder, pancreas, spleen, kidneys, adrenals, urinary bladder, seminal vesicles/ prostate/ testes or ovaries/ uterus, nasal cavity, brain, pituitary, and spinal cord, Tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The NOAEL was found to be 1000 mg/kg body weight after dosing mice with benzyl acetate in corn oil for 13 weeks.

In a study conducted by Abdo et al (1998), the effects of Glycine on benzyl acetate was examined in male F344 rats. The rats were fed a diet containing the test substance at 0, 2000, 3500 or 5000mg/kg bw/day Benzyl Acetate for up to 28 days. Two additional groups were fed NIH-07 diet with 5000 mg/kg bw/day benzyl acetate and 2700 mg/kg bw/day glycine or 5000 mg/kg bw/day benzyl acetate and 3200 mg/kg bw/day L-alanine; supplemental glycine and L-alanine were equimolar. The L-alanine group served as an amino nitrogen control. A third group was fed NIH-07 diet with 3200 mg/kg bw/day L-alanine and served as an untreated isonitrogenous control. Benzyl Acetate caused increase in mortality, body weight loss. The incidence of abnormal neurobehavioral signs such as ataxia and convulsions, along with astrocyte hypertrophy and neuronal necrosis in the cerebellum, hippocampus and pyriform cortex of the brain. These effects were reduced significantly by supplementation with glycine but not with L-alanine. These results suggest that the neurodegeneration induced by SA is mediated by a depletion of the glycine pool and the subsequent excitotoxicity.

The key study was determined to be the study conducted by Abdo in 1986, including information on repeated dose toxicity over an exposure period of 14 days and 13 weeks in rats and mice.

Repeated dose dietary toxicity

The toxicity of benzyl acetate has also been investigated in high quality range-finding studies in teh rat and mouse (NTP, 1993).

Groups of 10 male and 10 female F344/N rats were fed diets containing 0, 3130, 6250, 1250, 25000 or 50000 ppm (0, 230, 460, 900, 1750 or 3900 mg/kg bw in males and 0, 240, 480, 930, 1870 or 4500 mg/kg for females) benzyl acetate for 13 weeks. Nine male and nine female 50000 ppm rats died or were killed moribund between weeks 2 and 8 of the study. The mean body weight gain and the final mean body weight of 25000 ppm males were significantly lower (p ≤0.01) than those of the controls. Feed consumption by exposed rats, except the 25000 and 50000 ppm males and 50000 ppm females, was similar to that of the controls. The reduced feed consumption by 25000 ppm and 50000 ppm males may have been due to toxicity or decreased palatability. Tremors and ataxia occurred only in the 50000 ppm group; these findings were first observed on day 15 in nine males and six females and continued until the end of the study. Cholesterol levels in the 12500 and 25000 ppm females were lower than the controls. Chemical-related lesions occurred in the brain, kidney, tongue, and skeletal muscles of the thigh. Necrosis of the brain involving the cerebellum and/or hippocampus, degeneration and regeneration of the renal tubule epithelium and degeneration and sarcolemma nuclear hyperplasia of the tongue and skeletal muscles occurred in most male and female 50000 ppm rats.

No chemical related changes in sperm morphology were observed. The NOAEL in female rats was 6250 ppm (equivalent to 480 mg/kg bw) based on reduced cholesterol levels in the 12500 ppm females. The NOAEL in male rats was 12500 ppm (equivalent to 900 mg/kg bw) based on reductions in body weight and food consumption in the 25000 ppm males.

Groups of 10 male and 10 female B6C3F1 mice were fed diets containing 0, 3130, 6250, 1250, 25000 or 50000 ppm (0, 425, 1000, 2000, 3700 or 7900 mg/kg bw in males and 0, 650, 1280, 2980, 4300 or 9400 mg/kg for females) benzyl acetate for 13 weeks. One 50000 ppm male died and one 50000 ppm female was killed moribund before the end of the study. Mean body weight gains and final mean body weights of all exposed mice were significantly lower than those of the controls, and the mean body weight gains decreased with increasing exposure level. Feed consumption by 3130 ppm males and all exposed females was lower than that of the controls. Tremors occurred only in females and were first observed on day 16 in three 50000 ppm females, day 94 in one 25000 ppm female and day 93 in one 12500 ppm female. The tremors continued until the end of the study. Necrosis of the brain involving the hippocampus occurred in four 50000 ppm mice (1 male and 3 females). Hepatocellular necrosis also occurred in the male with brain lesions. No chemical related changes in sperm morphology were observed. A NOAEL could not be identified for this study, on the basis that reduced body weights were observed in all treated groups.

The DNEL was derived from the gavage study conducted by Abdo in 1986, specifically from an NOAEL of 250 mg/kg bw/day.

Justification for classification or non-classification

Based on the results of the key study, the test substance does not require classification according to Regulation EC No. 1272/2008.