Benzyl acetate

Neurotoxicity

Administrative data

Endpoint:

neurotoxicity: short-term oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1998

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Glycine Modulates the Toxicity of Benzyl Acetate in F344 Rats

Author:

Abdo, K.M., Wenk, M.L., Harry, G.J., Mahler, J., Coehl, T.J., Irwin, R.D.

Year:

1998

Bibliographic source:

Toxicologic Pathology, vol. 26. no. 3. pp. 395-402. 1998

Materials and methods

Test guideline

Qualifier:

no guideline required

Principles of method if other than guideline:

Groups of male F344 rats were fed NIH-07 diet containing 0 20,000, 35,000, or 50,000 ppm benzyl acetate for up to 28 days. Two additional groups were fed NIH-07 diet with 50,000 ppm benzyl acetate and 27,000 ppm glycine or 50,000 ppm benzyl acetate and 32,000 ppm 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 32,000 ppm L-alanine and served as an untreated isonitrogenous control.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Benzyl acetate

EC Number:

205-399-7

EC Name:

Benzyl acetate

Cas Number:

140-11-4

Molecular formula:

C9H10O2

IUPAC Name:

benzyl acetate

Details on test material:

- Name of test material (as cited in study report): Benzyl Acetate
- Analytical purity: >99.0%
- Other: The identity was confirmed by infrared spectrophotometry. The purity of benzyl acetate was determined by gas liquid chromatography.

Specific details on test material used for the study:

- Name of test material (as cited in study report): Benzyl Acetate
- Analytical purity: >99.0%
- Other: The identity was confirmed by infrared spectrophotometry. The purity of benzyl acetate was determined by gas liquid chromatography.

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Germantown, NY
- Age at study initiation: 35 days
- Weight at study initiation: No data
- Fasting period before study: No
- Housing: individually housed in polycarbonate cages
- Diet (e.g. ad libitum): Ground NIH-07 diet (Zeigler Bros., Gardners, PA), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50% ± 5%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 hr/12 hr

IN-LIFE DATES: No data

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency):All diets were provided fresh daily
- Storage temperature of food: All diets were stored at -20°C

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

29 days

Frequency of treatment:

Daily through the diet

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 20,000, 35,000, or 50,000 ppm benzyl acetate
Basis:
nominal in diet
Groups I, II, III and IV

Doses / concentrations 2

Remarks:

Doses / Concentrations:
50,000 ppm benzyl acetate supplemented with 27,000 ppm glycine
Basis:
nominal in diet
Group V

Doses / concentrations 3

Remarks:

Doses / Concentrations:
50,000 ppm benzyl acetate supplemented with 32,000 ppm L-alanine
Basis:
nominal in diet
Group VI

Doses / concentrations 4

Remarks:

Doses / Concentrations:
32,000 ppm L-alanine
Basis:
nominal in diet
Group VII isonitrogenous control

No. of animals per sex per dose:

Groups I, II, III: 30 animals/group
Group IV: 50 animals
Group V, VI, VII: 10 animals/group

Control animals:

yes, plain diet

other: Group VII was an isonitrogenous control for groups V and VI

Details on study design:

Groups I, II, III and IV received diet containing 0, 20,000, 35,000, or 50,000 ppm benzyl acetate, respectively. Group V received diet containing 50,000 ppm benzyl acetate supplemented with 27,000 ppm glycine. To determine whether glycine effects were due to added amino nitrogen, group VI received a diet containing 50,000 ppm benzyl acetate supplemented with 32,000 ppm L-alanine. Group VII received a diet containing only 32,000 ppm L-alanine and served as an isonitrogenous control for groups V and VI.

Examinations

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at initiation of the study and at weekly intervals including time of terminal euthanasia
- Cage side observations checked: Cage-side observations of coat condition, color of eyes and ears, secretions from eyes, nose, or mouth, oral and perianal excretions, lethargy, body position, muscle tone, ataxia, tremor, convulsions, and laboured respiration were made for individual animals

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: at initiation of the study and at weekly intervals including time of terminal euthanasia

OPHTHALMOSCOPIC EXAMINATION: No

Specific biochemical examinations:

No

Neurobehavioural examinations performed and frequency:

Screening by the Functional Observational Battery (FOB) was conducted as a systematic evaluation of nervous system effects. 10 animals from each exposure group were randomly selected for screening of behavioural and neurological effects at days 5, 12, 20, and 26 by the FOB. In addition, moribund animals in each dose group were also screened for behavioural and neurological effects. The FOB was conducted outside of the home cage and consists of tests for assessment of various aspects of neurological functioning such as autonomic nervous system functions (lacrimation, salivation, respiration, pupil response, palpebral closure), convulsive measures (tremors, convulsions), excitability measures (arousal, handling reactivity, ease of removal from a cage), neuromuscular measures (gait score, righting reflex), and sensorimotor measures (approach and touch response).

Sacrifice and (histo)pathology:

Whole brains (including cerebellum and brain stem) from all animals at euthanasia on day 29 and from all animals that died or were euthanatized in moribund condition prior to study termination were collected, weighed, and immersion fixed in 10% neutral buffered formalin. Following 48 hr of fixation, brains were cut by hand to obtain 3 coronal sections: the first at the level of the anterior cerebral cortex and basal ganglia, the second at the level of the posterior cortex, hippocampus, and thalamus, and a third to include the medulla and cerebellum. Trimmed samples were dehydrated in graded ethanols, embedded in paraffin, and cut to 6 um thickness. Brain sections were stained with hematoxylin and eosin (H&E) for light microscopic examination of neuronal necrosis.
For all brain samples, 6 um sections were cut serial to the sections stained by H&E, and astrocyte reactivity was evaluated by immunohistochemistry using a polyclonal antibody specific to the structural protein of astrocytes, glial fibrillary acidic protein (GFAP). Rehydrated sections were treated with 3% H20 2 for 10 min to remove endogenous peroxidase activity, rinsed in phosphate-buffered saline (PBS) for 20 min, and incubated with nonimmune goat serum for 20 min, followed by a 60 min incubation with anti-rabbit GFAP (1 :850). Sections were then incubated with a biotinylated secondary anti rabbit IgG antibody for 30 min, washed in PBS, incubated in avidin-biotin-peroxidase complex reagent for 30 min, rinsed with PBS, and stained with a diaminobenzoic acid substrate containing CO2+ ions. Areas of neuronal necrosis previously identified were examined by light microscopy for increased level of GFAP immunostaining and astrocyte morphology.

Other examinations:

No

Positive control:

No

Statistics:

Two approaches were employed to assess the significance of pairwise comparisons between dosed and control groups in the analysis of continuous variables. Brain and body weight data were analyzed using the parametric multiple comparison procedures of Williams and Dunnett. Lesion data were analyzed using nonparametric multiple comparison methods of Dunn.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Clinical biochemistry findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

effects observed, treatment-related

Other effects:

not examined

Description (incidence and severity):

Migrated information from 'Further observations for developmental neurotoxicity study'



Details on results (for developmental neurotoxicity):Not applicable (migrated information)

Details on results:

CLINICAL SIGNS AND MORTALITY All control rats, all rats receiving L-alanine alone or 20,000 ppm benzyl acetate, and 90% of rats receiving 35,000 ppm benzyl acetate or 50,000 ppm benzyl acetate with glycine survived until the end of the study. Rats receiving 50,000 ppm benzyl acetate survived a maximum of 12 days, and rats receiving 50,000 ppm benzyl acetate supplemented with L-alanine did not survive beyond day 6.

BODY WEIGHT AND WEIGHT GAIN Body weight gain of rats exposed to either 35,000 or 50,000 ppm BA was significantly decreased.

FOOD CONSUMPTION AND COMPOUND INTAKE Benzyl acetate administration caused a dose-related decrease in food consumption that, at the 50,000 ppm dose, was partially reversed by the co-administration of glycine.

NEUROBEHAVIOUR In the 35,000 ppm Benzyl acetate dose group, 37% of rats showed ataxia and 60% showed tremors. Because of generalized systemic toxicity, the distinct signs of ataxia were evident in 8% and tremors in 26% of rats in the 50,000 ppm Benzyl acetate dose group. No neurobehavioral signs were observed in rats given 50,000 ppm Benzyl acetate supplemented with glycine. Tremors were observed in 40% of rats receiving 50,000 ppm Benzyl acetate supplemented with 32,000 ppm L-alanine. The functional observations conducted prior to death or at termination of the study showed that Benzyl acetate induced alterations in gait, increased incidence of tremors, and signs of increased respiration at dose levels of 35,000 ppm or 50,000 ppm. For each end point, supplementation with L-alanine did not moderate the Benzyl acetate effect, but dietary supplementation with glycine ameliorated all behavioural signs of neurotoxicity. Control rats and rats receiving 20,000 ppm Benzyl acetate or L-alanine also did not show any signs of neurotoxicity.

GROSS PATHOLOGY Absolute brain weights were significantly lower in these groups. Relative to body weight, brain weights of rats receiving 35,000 ppm BA were significantly greater than those of the controls. Compared with controls, rats exposed to L-alanine alone showed significant increases brain weights and a significant decrease in its relative weight. Supplementation with glycine resulted in body weight and brain weight similar to those of control animals.

NEUROPATHOLOGY Gross lesions that could be attributed to compound administration or correlated to microscopic effects in target organs were not evident at necropsy. Neuronal necrosis at various brain sites was present in rats receiving 35,000 or 50,000 Benzyl acetate. Necrosis occurred at specific sites in the hippocampus and cerebral cortex and more diffusely in the cerebellum. Necrosis was present in all early death animals and in those surviving to scheduled euthanasia. In the hippocampus, necrosis primarily affected the large neurons of the dentate gyrus. In mild lesions, necrotic cells were confined to the dentate gyrus and were intermingled with viable neurons. In more severe lesions, all cells of the dentate gyrus were affected, and there was also viable involvement of CAlCA4 pyramidal cells of the hippocampus. In the majority of brains displaying hippocampal necrosis, neuronal death of similar severity was typically present in the piriform cortex of the ventrolateral cerebral cortex. Necrosis followed the contours of the neuronal layers in this area, resulting in a laminar pattern.
Neuronal necrosis in the cerebellum was confined for the most part to the small neurons of the granular layer. In minimal to mild cases, pyknotic nuclei were scattered among viable cells. In more severe lesions, there was diffuse necrosis of the granular layer, and occasional necrotic Purkinje cells were present.
In rats dying early or euthanatized at day 8, neuronal necrosis was typically more severe in the hippocampus and piriform cortex than in the cerebellum. One rat that died early and that had received benzyl acetate supplemented with glycine had marked hippocampal necrosis but sparse necrosis at other identified brain sites. At day 29, the severity of neuronal necrosis in rats supplemented with glycine was attenuated.
In areas of neuronal necrosis, there was concomitant astrocyte hypertrophy indicated by an increase in immunoreactivity for GFAP. Cellular processes of hypertrophied cells were thickened and intensely inununostained. In general, regional GFAP staining intensity was proportional to the degree of necrosis and to the duration of the treatment. Hypertrophy and increased reactivity were most apparent in rats treated with 35,000 ppm Benzyl acetate and surviving to day 29. Dietary supplementation with glycine resulted in decrease in the degree of astrocyte hypertrophy and the severity of necrosis.


OTHER FINDINGS

Effect levels

Dose descriptor:

NOAEL

Effect level:

20 000 ppm

Based on:

test mat.

Basis for effect level:

behaviour (functional findings)

Any other information on results incl. tables

Group	Survival (%)	Food intake (g/rat/day)	Body weight (g)	Absolute brain weight (g)	Relative brain weight (g)
I	100	16.0	223.0	1.63	9.9
II	100	15.2	199.0	1.6	10.7
III	90	8.7	87.4	1.43	17.1
IV	0	NA	NA	NA	NA
V	90	11.9	166.7	1.47	9.9
VI	0	NA	NA	NA	NA
VII	100	16.3	216.9	1.71	7.9

 

 

Group	Abnormal gait	Laboured/ increased respiration	Convulsions	Tremors
I	0/10	0/10	0/10	0/10
II	0/10	0/10	0/10	0/10
III	9/9	49/	0/9	5/9
IV	7/11	5/11	1/11	3/11
V	0/8	0/8	0/8	0/8
VI	7/10	4/10	0/10	2/10
VII	0/10	0/10	0/10	0/10

 

Group	Ataxia	Tremors
I	0/30	0/30
II	0/30	0/30
III	11/30	18/30
IV	4/50	13/50
V	0/10	0/10
VI	0/10	4/10
VII	0/10	0/10

 

Group	Rats examined	Neuronal necrosis hippocampus	Neuronal necrosis pyriform cortex	Neuronal necrosis cerebellum
I	10	0	0	0
II	10	0	0	0
III (early deaths)	3	3	3	3
III (terminal)	9	9	9	9
IV	50	50	50	50
V (early deaths)	1	1	1	0
V (terminal)	9	8	7	6
VI	10	10	9	10
VII	10	0	0	0

Applicant's summary and conclusion

Conclusions:

Glycine supplementation of the diet protected rats from the toxic effects of benzyl acetate.

Executive summary:

Groups of male F344 rats were fed NIH-07 diet containing 0 20,000, 35,000, or 50,000 ppm benzyl acetate for up to 28 days. Two additional groups were fed NIH-07 diet with 50,000 ppm benzyl acetate and 27,000 ppm glycine or 50,000 ppm benzyl acetate and 32,000 ppm 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 32,000 ppm L-alanine and served as an untreated isonitrogenous control. Glycine supplementation of the diet protected rats from the toxic effects of benzyl acetate.