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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
repeated dose toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
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:
publication
Title:
Unnamed
Year:
1981
Report Date:
1981

Materials and methods

Principles of method if other than guideline:
Male and female rats were fed test substance in their diet for 2 years and observed.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Ethyl methylphenylglycidate; strawberry aldehyde; Aldehyde C16; ethyl ester of 2,3-epoxy-3-phenylbutanoic acid; EMPG)
- Substance type: No data
- Physical state: No data
- Analytical purity: 99 %
- Impurities (identity and concentrations): No data
- Composition of test material, percentage of components:
- Isomers composition: No data
- 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
- Other: Supplied by Givaudan & Co. Ltd, Whyteleafe, Surrey, United Kingdom
- Other: Relative density at 20 °C: 1.085 to 1.097
- Other: Refractive index at 20 °C: 1.505 to 1.509
- Other: Acid value: Max 1.0

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: A. Tuck & Son, Battlesbridge, Essex, United Kingdom
- Age at study initiation: No data
- Weight at study initiation: males mean weight 81 g; females mean weight 77 g.
- Fasting period before study: No data
- Housing: Grid floor metal cages, 4 animals per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 °C ± 2 °C
- Humidity (%): No data
- Air changes (per hr): Not quantified, but described as "ventilated".
- Photoperiod (hrs dark / hrs light): No data

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with: Reground Spratt's Laboratory Animal Diet No. 1
- Storage temperature of food: No data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
104 wks
Frequency of treatment:
Continuous - test substance included in ad libitum diet.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.02 %
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.01 %
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.5 %
Basis:
nominal in diet
No. of animals per sex per dose:
48 per sex per dose.
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Based on previously published academic papers.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
OBSERVATIONS
- Extent of observations not specified
- Time schedule: Daily
- Rats showing signs of ill health were isolated, to be returned to their group if their condition improved, but otherwise to be killed and autopsied.

BODY WEIGHT
- Time schedule for examinations: Observations at 0 wks, 4 wks, 9 wks, 12 wks, 20 wks, 27 wks, 40 wks, 53 wks, 67 wks, 79 wks, 91 wks, 103 wks.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY
- Time schedule for collection of blood: Observations at 16 wks, 24 wks, 54 wks, 2 yrs
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 per sex (control, 0.1 %, and 0.5 % groups only)
- Parameters examined:
-- Haemoglobin, packed cell volume, erythrocyte count, total leucocyte count (control, 0.1 %, and 0.5 % groups)
-- Reticulocyte, differential white counts (control and 0.5 % groups only)
-- Blood serum analysed for urea, glucose, total albumin and activities of glutamic-pyruvic transaminase and lactic dehydrogenase (female control, female 0.5 %, male control, male 0.5% , male 0.02% groups at 2 yr only)

URINALYSIS
- Time schedule for collection of urine: 12 wks, 24 wks, 54 wks, 79 wks
- Collection
-- 6 hr period of water deprivation.
-- 2 hr period following water load of 25 mg/kg bw.
-- 4 hr period following water deprivation of 16 hr.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- No. of animals: 10 per sex per dose (12 wks, 24 wks); 5 per sex for doses of 0, 0.1 %, 0.5 % (54 wks, 79 wks).
- Parameters examined: specific gravity, volume.
-- 6 hr samples examined semi-quantitatively for protein, glucose, ketone bodies, bile salts, blood.
-- 2 hr samples examined for No. of cells

NEUROBEHAVIOURAL EXAMINATION
- Time schedule for examinations: 2 wks, 15 wks, 21 wks, 62 wks, 84 wks
- Dose groups that were examined: 15 male control group, 15 male 0.5 % group, 15 female control group, 15 female 0.5 % group.
- Battery of functions tested: motor activity
- Details: Tested for ability to remain on rotarod at 11 rpm for 3 mins. No. of attempts required recorded, maximum attempts allowed 10.
Sacrifice and pathology:
GROSS PATHOLOGY: No data

HISTOPATHOLOGY
- Survivors at wk 104
- Macroscopic abnormalities noted.
- Weighing of brain, heart, liver, spleen, kidneys, stomach, small intestine, caecum (with and without contents), adrenal glands, gonads, pituitary and thyroid.
- Samples of brain, heart, liver, spleen, kidneys, stomach, small intestine, caecum, adrenal glands, gonads, pituitary, thyroid, salivary gland, trachea, lungs, aorta, thymus, lymph nodes, urinary bladder, colon, rectum, pancreas, uterus, mammary gland, prostate, seminal vesicles, skeletal muscle, eye, Harderian gland, spinal cord, sciatic nerve, brachial nerve and any other tissues of abnormal appearance fixed, in a 10 % buffered formalin. Microscopic examinations of these carried out on 5 µm paraffin wax sections stained with haematoxylin and eosin.
Statistics:
No data

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):
effects observed, treatment-related
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
not examined
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- No. of deaths amongst 0.5 % group was less than control group, but not statistically significant.

BODY WEIGHT AND WEIGHT GAIN
- Body weights were significantly lower in females fed 0.5 % test material, although differences were small (less than 10 % of the control).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- Food intake was slightly lower in females fed 0.5 % test material. In this group, there was no difference in water intake.

HAEMATOLOGY
- Statistical difference (P < 0.05) between treated and control rats - a high leucocyte count in wk 54 in males fed 0.5 % test substance. There was no parallel finding in the females. At wk 104, haematological values were similar in all groups.

URINALYSIS
- No consistent effects
- Semi-quantitative tests for glucose, bile salts, blood and ketones negative. Protein and microscopic constituents similar in control and test groups.

NEUROBEHAVIOUR
- No evidence of neuropathy

ORGAN WEIGHTS
- Isolated values for absolute or relative organ weights in the treated animals differed from the control groups, but none in the 0.5 % treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
- In general, the frequency of the histological findings was similar in the control and treated groups but male animals fed 0.5 % showed a greater incidence of fatty vacuolation of the liver.
- In males fed 0.1 % (but not 0.5 %), there were significantly more animals with liver necrosis. However, the extent of the lesions in affected rats was similar in treated and control animals, and there was no statistically significant difference in females.
- Dilation of the pancreatic duct occurred with great frequency in males fed 0.5 % test substance, and in females fed 0.02 %.
- A marginal treatment-related increase in dilation of lymph node sinusoids occurred in male but not female rats, the difference being statistically significant in rats fed 0.5 % test substance.

HISTOPATHOLOGY: NEOPLASTIC
- See table 9 in the attached supporting materials

Most of the tumours were encountered as isolated findings or in very low incidence in any one group with no statistically-valid differences between the treated and control groups. The exceptions to this were interstitial cell tumours of the testis, which occurred more frequently in the males given 0.02 % or 0.5 % test material, and pituitary adenomata, which were more common amongst females of the same groups that in the controls. In both cases, the incidence in the intermediate group did not differ significantly from the control group.

A number of malignant tumours of vascular origin were seen. Two haemangiosarcomata were present in female animals, one in the 0.1 % group and the other in the 0.5 % group. In males, similar tumours were found in single animals from the control and two lowest dose groups. In addition, haemangioendotheliomata were identified in one female in the 0.5 % group (in the liver) and in one in the 0.02 % group (in the uterus). Benign tumours of vascular origin (haemangiomata) were confined to one control male and 0.1 % dose female.

Lymphosarcomata were encountered in occasional animals in most groups. Affected were the spleen of a control male and multiple organs, including the lymph nodes, of a single male in each of the groups fed 0.02 % and 0.1 % test material. In females, lymphosarcomata were identified in the thyrus of two rats from the 0.02 % dose group and in the bone of one in the 0.5 % dose group. Two male animals, one from 0.02 % dose and one from the 0.1 % dose group had reticulum cell sarcomata.

Some malignant tumours of common origin were found in more than one treated animal. An osteosarcoma occurred in one male from the 0.02 % group and in one from the 0.5 % group, the tumour in the latter case being identified from a metastatic deposit in the lung. Spindle cell sarcoma was found in the large intestine of one male fed 0.5 % test material and in the bladder of a male fed 0.02 % and adrenal-gland cortical carcinomata occurred in 2 males (in the 0.02 % and 0.5 % groups). In females, mammary adenocarcinoma was found in 1 animal from each of the 2 higher dosage groups.

Single occurrences of malignant tumours in treated animals without a similar control finding included a uterine endometrial sarcoma and neurofibrosarcoma of the ovary in animals fed 0.5 % and 0.1 % test material respectively, while an astrocytoma was present in the brain of a male animal fed 0.1 % test material. An anaplastic carcinoma of the pancreas and basal-cell carcinoma of the skin were found in males from the high dose group.

There were more pituitary adenomata in the treated female rats than in the controls but Dunnington et al. considered such tumours to be particularly common in old rats and cite incidences of 50-70 % that have frequently been reported. They also reported that similar incidences 50-70 % have been reported in their laboratory in control animals. They thus considered the number seen in the treated animals as not unusual, and the difference in incidence between control and treated groups seems to have been due to a low incidence (42 %) in the control group rather than to treatment with the test material.

There were no statistically significant differences in the overall incidence of tumours, either benign or malignant, between the treated and control male rats. In the females there was a higher incidence of benign tumours, but this was no longer the case when the pituitary tumours were excluded and incidences of pituitary tumours do not seem to have been due to tumours.

Interstitial cell tumour of the testis was the only other individual tumour that occurred in treated rats with a frequency of statistical significance. Dunnington cite reports that such tumours are the most common testicular neoplasmin the rat. Control data from Dunnington et al.'s laboratory indicate an incidence in some studies as high as 10 % in Wistar rats, with much variation between groups. In this study there was an incidence of 5 % in the control animals, while in those fed 0.02 % test material there was an incidence of 23 % and in the group fed 0.5 % test material an incidence of 22 %. The incidence among the rats fed 0.1 % test material however was 3 % so there was no dose-response relationship.

All of the tumours were present in animals killed at the end of the study; none was found at earlier stages. The lack of any relationship between dosage and the number of tumours found or of any evidence for a shortening of the latent period suggested to Dunnington et al. that the distribution of these testicular tumours was more likely to be a reflection of variations in the normal incidence in rats than an effect of the test material, although the latter possibility cannot be ruled out entirely.

There was no dose-association pattern in the occurrence of other benign tumours in treated rats and all the types encountered were well documented in previous literature.

A number of malignant tumours occurred in treated animals but not in controls. However, reticulum-cell sarcoma, haemangiosarcoma, adrenal carcinoma, basel-cell carcinoma, endometrial sarcoma, spindle-cell sarcoma of the bladder and of the large intestine, an mammary adenocarcinoma have all been reported to arise spontaneously in the rat. Two of these types of tumour of particular interest to Dunnington et al: Lymphosacroma and reticulum cell sarcoma form a group of tumours that occur spontaneously in the lymphoretuclar system of ageing rats, and cite an incidence of 20 % that had been reported in the Furth strain of Wistar rat. In Dunnington's laboratory, there was an overall incidence of 3.3 % for males and 1.2 % for females among control Wistar animals. The overall incidences in the various groups were similar to this background figure and there was no evidence of any dose-relationship, particularly since none of these sarcomata were present in animals on the highest dose level. It seems likely therefore to Dunnington et al. that the incidence of lymphoreticular tumours recorded in the investigation was affected by the administration of the test material.

Vascular neoplasms formed the second group of tumours that occurred with relatively high frequency in the study. Malignancies of vascular origin were present in the lymph nodes, liver, kidney and uterus.

The overall incidence of tumours of vascular origin classified as malignant (haemangiosarcoma and haemangioendothelioma) was 1, 1, 1,0 in the control, 0.02 %, 0.1 % and 0.5 % dose males respectively, and 0, 1, 1, 2 in the females. These incidences were not statistically-significant and showed no overall dose relationship. Although these tumours were classified as malignant, doubt remains as to their exact nature because of a combination of local invasive properties and benign cytological features. This problem had been encountered with vascular tumours at other sites.

Although some of the benign tumours occurred with increased frequency in the treated animals, Dunnington et al. found that there was no conclusive evidence to link their development with treatment with the test material.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOAEL
Effect level:
ca. 35 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: overall effects
Dose descriptor:
NOAEL
Effect level:
ca. 60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: overall effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Dunnington et al. found that the no observed effect level of the test material to be 0.1% in the diet of Wistar Rats. This is equivalent to 35mg/kg bw/d for Males and 60mg/kg bw/d for Females.