Registration Dossier

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:
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

Reference
Name:
Unnamed
Type:
Constituent
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 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 / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 20,000, 35,000, or 50,000 ppm benzyl acetate
Basis:
nominal in diet
Groups I, II, III and IV
Remarks:
Doses / Concentrations:
50,000 ppm benzyl acetate supplemented with 27,000 ppm glycine
Basis:
nominal in diet
Group V
Remarks:
Doses / Concentrations:
50,000 ppm benzyl acetate supplemented with 32,000 ppm L-alanine
Basis:
nominal in diet
Group VI
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.