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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 25 july 1991 to 07 july 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was GLP, standardized guidelines compliant and well described.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: US EPA Food guideline 1982
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Manston Road, Margate, Kent, England
- Age at study initiation: approximately 28 days old
- Weight at study initiation: 12 g (females) to 15 g (males)
- Fasting period before study: no data
- Housing: individually
- Diet (e.g. ad libitum): free access to SDS rat and mouse N°1 modified maintenance diet.
- Water (e.g. ad libitum): free access to tap water
- Acclimation period: 9 days. A further period of acclimatisation of 7 days was allowed between allocation of animals and beginning of treatment.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2°C
- Humidity (%): 55 +/- 10 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12h / 12h


IN-LIFE DATES: from 16 aug 1991 to 15-20 nov 1991

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
DIET PREPARATION
- Rate of preparation of diet (frequency): each week
- Mixing appropriate amounts with (Type of food): SDS rat and mouse N°1 modified maintenance diet. A premix of suitable strength was prepared each week by grinding the required quantity of Ethyl vanillin with untreated diet. Blending of the premix was achieved by mixing in a turbula mixer for a minimum period of 2 minutes.
The required concentrations were prepared by direct dilution of the prepared premix. Blending of the inclusion levels for feeding was achieved by mixing in a double-cone blender for a minimum period of 7 minutes.
The concentrations of Ethyl vanillin were changes as necessary.
- Storage temperature of food: -20°C in airtight containers.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Only samples of diet prepared for week 1, 6 and 13 were analysed for inclusion levels. Following issue of the final report, the stored samples will be discarded with no further analyses performed. Chemical analysis was carried out by HRC Department of Analytical chemistry. The results of these analyses are available in addendum in the study report.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0; 500; 1000; 2000 mg/kg/day
Basis:
nominal in diet
No. of animals per sex per dose:
20 males and 20 females
Control animals:
yes, plain diet
Details on study design:
Post-exposure period: none
- Dose selection rationale: issue from the palatability study (Huntingdon, 1992).
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: at time of allocation, on the day of the start of treatment, and once a week thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal: The quantity of food consumed by each animal was recorded on a weekly basis.

FOOD EFFICIENCY: Food conversion ratios were calculated, where appropriate, from bodyweight and food consumption data as weight of food consumed per unit gain in bodyweight.


WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily monitoring by visual appraisal of the water bottles was maintained throughout the study. Water consumption was measured accurately, by weight, over daily periods during Week 12 for all rats in all groups.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the start of the treatment, then during Week 13, the eyes of all surviving animals in the control and high dosage level groups were examined. Prior to examination the pupils were dilated using a Tropicamide ophtalmic solution.
- Dose groups that were examined: all surviving animals in the control and high dosage level groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: twice
- Anaesthetic used for blood collection: Yes (no data on identity)
- Animals fasted: Yes
- How many animals: 20 (10 males and 10 females)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: twice
- Animals fasted: Yes
- How many animals: 20 (10 males and 10 females)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Adrenals, brain, heart, kidneys, liver, lungs, ovaries, pituitary, prostate, spleen, testes (with epididymes), thyroids, uterus.

HISTOPATHOLOGY: Yes
Adrenals, alimentary tract, aorta, brain, eyes, femur, Harderian gland, heart, kidneys, larynx and pharynx, liver, lungs, lymph nodes, mammary gland, ovaries, pancreas, pituitary, prostate, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal column, spleen, sternum, testes (with epididymes), thymus (where present), thyroid (with parathyroid), tongue, trachea, urinary bladder, uterus, vagina.
Statistics:
The following sequence of statistical tests was used for food conumption, water consumption, bodyweight, organ weight and clinical pathology data:
- If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%), the proportion of animals with values different form the mode was analysed by Fisher's exact test or Mantel's test. Otherwise:
- Bartlett's test was applied to test heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
- Where no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out. If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks was used.

Analyses of variance were followed by Student's "t" test and William's test for a dose-related response, although only the one thought most appropriate for the response pattern observed was reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the "t" test and William's test.

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:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY: A control female died during the Week 6 routine blood sampling procedures, as a result of suspected ether anesthesic overdose. There were no clinical signs noted during the 13-week treatment period considered to be of toxicological importance.

BODY WEIGHT AND WEIGHT GAIN: The group mean bodyweight gain of male receiving 2000 mg/kg/day was inferior to that of controls troughout the whole study. For males receiving 500 or 1000 mg/kg/day the mean bodyweight gain was inferior to that of controls during the first 4 weeks. However, although the differences from control were statistically different they were not dosage-level in degree. It was noted that for these groups the food intake was lower than control.
During the remaining weeks of the study, the weekly mean bodyweight gains of males receiving 500 or 1000 mg/kg/day were considered to be essentially comparable with those of controls. However, this improvment in bodyweight gain was not sufficient for either the overall weight gain for period Weeks 4 to 13, or the absolute bodyweights for these male rats to regain parity with concurrent controls.
For females, overall, inferior bodyweight gain was recorded in the 2000 mg/kg/day group, while weight gain of females receiving 500 or 1000 mg/kg/day was considered to be unaffected by treatment with Ethylvanillin.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): during the first 4 weeks of the study the cumulative mean food intake by all the male treated groups was stastistically lower than that of controls.
During the subsequent period the food intake by all the treated groups of males was without any notable differences from control.
During the first week of the study, the mean intake by females receiving 2000 mg/kg/day was marginally inferior to that controls. Subsequently, during the remainder of the study, the food intake by the same rats was comparable to that of controls.
The food intake by females receiving 500 or 1000 mg/kg/day was considered to be similar to that of controls throughout the study.

FOOD EFFICIENCY: The efficiency of food utilisation was assessed by calculation of food conversion ratios. The food conversion ratios for rats of either sex treated with 2000 mg/kg/day during 13 weeks were inferior to those of their respective control. The marginally inferior food conversion ratios noted for rats of both sexes treated with 500 or 1000 mg/kg/day were considered to reflect the marginally lower bodyweight gains reported earlier for these groups.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Measurement of water intake during Week 12 of treatment revealed no notable differences between the water intake of treated rats and their respective controls.

OPHTHALMOSCOPIC EXAMINATION: no treatment-related changes were recorded during the Week 13 examination.

HAEMATOLOGY: Investigation of haematological parameters during Week 6 and 13 did not reveal any differences from control that were considered to be attributable to treatment, despite the occasional parameters that achieved levels of statistical significance.

CLINICAL CHEMISTRY: biochemical investigations showed slightly higher than control values for glutamic-pyruvic transaminase, alkaline
phosphatase, cholesterol and total plasma protein at the high dosage level, only occasionally involving the intermediate dosage level. These
differences from control may be related to treatment in view of the histopathological changes in liver. The remaining differences from the control
were considered unlikely to be of toxicological importance.

ORGAN WEIGHTS : for rats receiving 1000 or 2000 mg/kg/day, liver, spleen and kidneys weights were higher than the control. The absolute liver
weights of these animals were only marginally higher than the respective controls. Spleen and kidney weights were not associated with
histopathological change.
The remaining statistical differences from control were considered to have arisen as a result of the lower than control terminal bodyweights for the
treated male groups or were considered to reflect normal biological variability for rats of this age and strain and were therefore considered to be
of no toxicological importance.


GROSS PATHOLOGY : Fur: yellow discolouration of some or all of the fur was observed in 1/20 male and 1/20 female rats treated with
500 mg/kg/day, 6/20 male and 16/20 females rats treated with 1000 mg/kg/day, and all male and female rats treated with 2000 mg/kg/day,
compared with 0/20 male and 0/19 female control rats. This finding was also noted clinically and considered to be of no toxicological importance.
Cervical lymph nodes: enlargement was noted in a greater number of male rats treated with 1000 mg/kg/day or 2000 mg/kg/day compared with male and female control rats.
Deep cervical lymph nodes: enlargement was observed in 7/20 female rats treated with 2000 mg/kg/day compared with 0/19 female control rats.
Adipose tissue: a reduction in adipose tissue was observed in 7/20 male and 1/20 female rats treated with 2000 mg/kg/day compared with 0/20 male and 0/19 female control rats.
Spleen: enlargement was seen in 2/20 male treated with 500 mg/kg/day, 3/20 male treated with 1000 mg/kg/day and in 9/20 male and 4/20 female rats treated with 2000 mg/kg/day compared with 0/20 male and 0/19 female control rats.


HISTOPATHOLOGY: NON-NEOPLASTIC : Hepatic peribiliary inflammatory change and minor bile duct hyperplasia attributable to dietary administration of ethylvanillin were seen at dosages of 2000 and 1000 mg/kg/day. No changes were seen in the liver parenchyma and no degenerative or inflammatory changes were seen in the bile duct epithelium.
Associated reactive changes of lymphoid tissues were also seen.
No treatment-related changes were observed at the 500 mg/kg/day dosage level.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Spleen weight higher than control group (relative) related with histopathological changes.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEL is 1000 mg/kg bw/day based on spleen weight which is higher than control group (relative) related with histopathological changes.
Executive summary:

In a subchronic toxicity study (Hooks, 1992), ethylvanillin was administered to CD(SD) BR rats (males and females, 20 per sex per dose), in diet at dose levels of 0; 500; 1000; 2000 mg/kg/day for 13 weeks. Yellow discolouration of some or all of the fur of rats treated with 500 mg/kg/day, 1000 mg/kg/day, or 2000 mg/kg/day, were not considered to be of toxicological importance.

For males and females, inferior bodyweight gain was recorded in the 2000 mg/kg/day group. For males receiving 500 or 1000 mg/kg/day, the mean bodyweight gain was inferior to that of controls during the first 4 weeks and the cumulative mean food intake by all the male treated groups was stastistically lower than that of controls. The food intake by females receiving 500 or 1000 mg/kg/day was considered to be similar to that of controls throughout the study. The decrease of bodyweight gain is observed in the fourth first weeks, related with a decrease in food consumption. This variation is due to the unpathability of the substance, and was not considered to related with toxicological property of the substance.

Biochemical investigations showed slightly higher than control values for glutamic-pyruvic transaminase, alkaline phosphatase, cholesterol and total plasma protein at the high dosage level, only occasionally involving the intermediate dosage level. These differences from control may be related to treatment in view of the histopathological changes in liver. The remaining differences from the control were considered unlikely to be of toxicological importance.

Concerning observations done on the increase of liver weight (relative), it appears to be not correlated with hepathological changes, because these effects were observed in the control group too, that would be due to the strain of rat used in this study. Therefore the increase of the liver weight is not considered as adverse effect.

The NOAEL is 1000 mg/kg bw/day based on spleen weight which is higher than control group (relative) related with histopathological changes.