Ethyl 2,3-epoxy-3-phenylbutyrate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards, well documented and acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1978

Report date:

1977

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Rats were fed 3 different concentrations of test material (plus control) for 15 wk. Body weights and food consumption were measured periodically. After 15 wk the rats were killed and examined. Rats were also killed and examined at 2 and 6 wks.

GLP compliance:

no

Remarks:

Study predates GLP

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 2,3-epoxy-3-phenylbutyrate

EC Number:

201-061-8

EC Name:

Ethyl 2,3-epoxy-3-phenylbutyrate

Cas Number:

77-83-8

Molecular formula:

C₁₂H₁₄O₃

IUPAC Name:

ethyl 3-methyl-3-phenyloxirane-2-carboxylate

Details on test material:

- Name of test material: Ethyl methylphenylglycidate; EMPG
- Substance type: Flavouring used in food stuffs
- Physical state: No data
- Analytical purity: 99.0 %
- Impurities (identity and concentrations): No data
- Composition of test material, percentage of components:
- Isomers composition: 2 isomers, cis- and trans-
- Purity test date: No data
- Lot/batch No.: No data
- Expiration date of the lot/batch: No data
- Stability under test conditions: No data
- Storage condition of test material: No data
- Source: Givaudan & Co. Ltd, Whyteleafe, Surrey

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent, United Kingdom
- Age at study initiation: No data
- Weight at study initiation: Average per test group, of 79 g to 84 g
- Fasting period before study: No data
- Housing: 5 per cage.
- Diet: Ground Spiller's animal diet
- Water: ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature: No data
- Humidity: No data
- Air changes: No data
- Photoperiod: No data

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

test group 1: 15 wk
test group 2: 2 wk
test group 3: 6 wk

Frequency of treatment:

Continuous - test material included in diet.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.5 other: Percent

Remarks:

500 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

0.1 other: Percent

Remarks:

100 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

0.02 other: Percent

Remarks:

20 mg/kg bw/day

No. of animals per sex per dose:

Test group 1: 15 rats per sex per dose (3 doses + control)
Test group 2: 5 rats per sex per dose (2 doses + control; 0.02 % dose not used)
Test group 3: 5 rats per sex per dose (2 doses + control; 0.02 % dose not used)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Based on previously published papers on the toxicity of the test material.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE-SIDE OBSERVATIONS: Yes, details unclear

BODY WEIGHT
- Time schedule for examinations: day 1, day 2, day 6, day 8, weekly thereafter
- Data are given for day 0, day 22, day 48, day 98

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight
- Time schedule for examinations: day 1, day 2, day 6, day 8, weekly thereafter
- Data are given for day 0, day 22, day 48, day 98

WATER CONSUMPTION
- Time schedule for examinations: 24 hrs preceding weighing; day 1, day 2, day 6, day 8, weekly thereafter.
- Data are given for day 0, day 22, day 48, day 98

URINALYSIS
- Time schedule for collection of urine: Collected during the final week of treatment.
- Animals fasted: No
- Examined for appearance and for the presence of blood, bile salts, protein, ketones, glucose and microscopic constituents.

RENAL FUNCTION
- Time schedule for collection of urine: wk 2, wk 6, wk 13
- Volume and specific gravity of urine produced measured under 3 sets of conditions:
-- 6 hr period of water deprivation
-- 2 hr period following water load of 25 mL/kg
-- 4 hr period following 16 hr water deprivation
- The number of cells was counted in the 2 hr urine sample.

NEUROBEHAVIOURAL EXAMINATION
- Time schedule for examinations: At unspecified intervals.
- Dose groups that were examined: control group, highest dose group (0.5 %).
- Battery of functions tested: motor activity
- A Rotarod was used to assess the co-ordinated motor activity of the animals during the treatment period. The Rotarod consisted of a wooden cylinder, 8 cm in diameter and 12 cm long, bounded at each end by flanges 135 cm in diameter. The cylinder was rotated at 11 revs/min and the animals' ability to remain on the rotating cylinder was assessed. The criteria of adequate performance were that an animal should remain on the rotating rod for a 3 min period of uninterrupted activity and that this should be achieved within ten attempts.

Sacrifice and pathology:

GROSS PATHOLOGY
- A post-mortem examination was carried out and any macroscopic abnormalities noted.

HISTOPATHOLOGY
- At autopsy.
- Brain, heart, liver, spleen, kidneys, stomach, small intestine, caecum, adrenals, gonads, pituitary and thyroids weighed.
- Samples of Brain, heart, liver, spleen, kidneys, stomach, small intestine, caecum, adrenals, gonads, pituitary thyroids, salivary gland, trachea, aorta, lung, lymph nodes, urinary bladder, colon, rectum, pancreas, uterus, skeletal muscle and sciatic and brachial nerves were preserved in 10% buffered formalin.
- Paraffin-wax sections were prepared from all tissues from the control rats and from those fed diet containing 0.5 % test material and from the heart, liver, kidneys and spleen of the animals fed the intermediate dietary levels.
- Tissue sections were stained with haematoxylin and eosin with the exception of the peripheral nerves, which were embedded separately and stained with Luxol fast blue and Cresyl fast violet to demonstrate myelin.

HAEMATOLOGY
- Time schedule for collection of blood: At autopsy
- Animals fasted: Yes, overnight prior to being killed
- Examined for erythrocyte count, leucocyte count.
- Fixed and vitally-stained preparations were prepared from all animals for enumeration of reticulocytes and differential leucocytes, but in the absence of changes in the other haematological parameters, examination of these slides was restricted to those from the control animals and from rats fed diet containing 0.5 % test material at wk 6 and 15.
- Serum separated from the blood collected at autopsy from the rats killed at wk 15 was analysed for urea, glucose, albumin and total protein as well as for the activities of glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase and lactic dehydrogenase.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

with respect to renal function; see below.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

effects observed, treatment-related

Other effects:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
- There were no abnormalities in either condition or general behaviour in animals.
- There were no mortalities during the study.

BODY WEIGHT AND WEIGHT GAIN
- In male 0.02 % level rats, body weights were higher than of the controls to a statistically significant degree (days 29 to 62).

FOOD CONSUMPTION AND COMPOUND INTAKE
- Mean daily food in similar in all groups for a given sex.
- The growth patterns of both male and female rats fed diets containing the test material were similar to those of the control animals. The higher mean body weight values in the male rats fed diet containing 0.02 % test material were not found at any other dietary level or in the female rats at any treatment level.

WATER CONSUMPTION
- Data not discussed by Mason et al.

URINALYSIS
- Data not discussed by Mason et al.

RENAL FUNCTION
- Renal function was unimpaired in the animals fed diets containing 0.5 % EMPG for 13 wk.
- At wk 2 the male rats fed 0.5 % test material induced a smaller volume of urine of a higher specific gravity in the 2 hr period following a water load, although these differences from control values were not statistically significant. The volume of urine collected from this group of animals during the first 6 hr of water deprivation was approximately 50 % of the control value. These data indicate a mild impairment of the ability of the animals to dilute and concentrate urine. There was, however, no increase in the rate of urinary cell excretion.

NEUROBEHAVIOUR
- The mean number of trials required to attain the criterion of 3 min of continuous activity and the number of failures did not differ significantly between the control rats and those fed diet containing 0.5 % test material.
- Data not given in the paper.

HAEMATOLOGY
- There was a lower white blood cell count at wk 6 in the male rats fed 0.5 % test material but there were no other significant findings.
- At wk 15 the level of serum glutamic oxaloacetic transaminase activity was significantly lower in the female rats fed diets containing 0.5% EMPG than in the controls although no similar changes in the male rats.
- No significant increases in the total number of white cells or in the activities of the serum enzymes were observed.
- The isolated lower values of white cells and of glutamic-oxaloacetic transaminase were considered by Mason et al. to be of no toxicological significance.

ORGAN WEIGHTS
- At wk 2, the weights of the empty caecum in the male animals fed 0.1 % or 0.5 % were higher than those of the controls, the differences being statistically significant. However, when expressed relative to body weight the caecum weights did not differ from the control values. Mason et al. conclude that the slight increase in body weights in these animals was the prime factor responsible for these changes.
- After treatment for 6 wk the only statistically significant differences from control values were higher small-intestine weights in female rats fed 0.5 % test material and lower relative stomach weights in male rats fed 0.1 % test material.
- The increased absolute and relative weights of empty caecum, small intestine and stomach found in the female rats fed 0.5 % test material for 15 wk were not found in the male rats fed the same dietary level nor did there appear to be a dose-related effect in the female rats.
- In view of the absence of any histopathology in gastro-intestinal tract tissues, it is difficult to reconcile the increases in weight of the gastro-intestinal tract with any adverse effect of the test material, although the presence of these changes at both 6 and 15 wk in animals fed the same dietary level of the test material suggests that they were related to treatment.

- At autopsy in wk 2 of the male rats fed 0.5 % test material all the kidneys absolute and relative weights were higher than in the corresponding male control rats.

- In the female rats fed 0.1 % test material the pituitary weights were higher than in the controls. Although these increases were statistically significant they were not evident when the weights were expressed relative to body weight.

- The increases in the relative liver weights found in the males and females fed 0.5 % test material at wk 15 were regarded by Mason et al. as being related to treatment, although there was no evidence of histological change.
- The increased liver weight in the male rats fed 0.02 % test material for 15 wk was not evident when expressed relative to body weight and therefore was considered to be unrelated to treatment.

- The increased relative spleen weight in the female rats fed 0.5% EMPG for 15 wk was also probably unrelated
to treatment, since it was not found in any other group of animals.

HISTOPATHOLOGY:
- In the kidneys at wk 2 autopsy of the male rats fed 0.5 % test material, several areas of dense lymphocytic infiltration surrounding both tubules and glomeruli in the cortical area were found. In general the cells in these areas appeared pale and swollen. In the lumen of a few tubules, desquamated cells could be identified, some of which appeared necrotic. Although generally the glomeruli appeared normal, slight thickening of Bowman’s capsule was observed in the areas. These changes to the kidneys, consistent with an early stage of nephrosis, were found in 2/5 animals in the corresponding control group and in 1/5 and 2/5 females in the control group and the group given 0.5% level in their diet respectively for 2 wk. At wk 6, 4/5 male control rats, 2/5 males fed 0.5 % test material and one female rat fed 0.5 % test material had similar lesions. Incidences of early nephrosis in the animals killed at wk 15 are shown in the supporting information.
- In view of the frequent appearance of these lesions, particularly among male rats, and their occurrence in both control and treated animals in the present experiment, it is unlikely that they represent an effect of treatment.
- The relative kidney enlargement found at wk 15 in both male and female rats fed 0.5% test material was not accompanied by either impaired renal function or any histological changes, but the low probability of observing kidney-weight increases of this magnitude by chance suggests that the finding was related to the ingestion of the test material.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

2

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Overall effects

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Histopathology results:

		Incidence of pathological reasons in
		Males at dietary level (%) of		Females at dietary level (%) of
		0	0.5	0	0.5
Tissue and histological finding	No. of animals examined:	12	15	14	15
Lungs
- Severe pneumonitis		2	2	3	1
- Slight to moderate infection		7	12	5	7
Kidney
- Early nephrosis		2	2	1	0
Liver
- Small area of necrosis		1	0	0	1
- Perivascular cuffing		0	1	0	0
- Embolus		0	0	1	0
- Area of old haemorrhage		0	0	0	1
Sciatic nerve
- Vacuolation		4	4	10	13
- Mast cells present		6	8	12	14
- Birefringent deposits in microglial cells		0	0	5	4

The figures indicate the number of animals affected. The incidences in the treated and control animals did not differ significantly (P>0.05 by Pearson's χ² test.

Applicant's summary and conclusion

Conclusions:

Mason et al. found that the NOAEL of the test material was at the 0.5 % level in diet (500 mg/kg bw/day).
Mason et al. found that the NOEL of the test material was at the 0.01 % level in diet (100 mg/kg bw/day).