Ethylenebis(oxyethylene) bis[3-(5-tert-butyl-4-hydroxy-m-tolyl)propionate]

Endpoint summary

Description of key information

The substance caused an adaptive enlargement of liver and thyroid upon repeated oral exposure to rats as observed in a subchronic feeding study and a subchronic gavage study. The NOELs were 50 mg/kg bw and 11 mg/kg bw for gavage and feed application, respectively. No adverse effects were observed in a 90-day feeding study in dogs at the highest tested dose of 300 mg/kg bw as applied in a capsule. In a follow up 28-day study in cynomolgus monkey which was designed to investigate liver and thyroid effects, no adverse effects were observed  at the highest tested dose of 1000 mg/kg bw. Based on the absence of effects in dog and monkey, the liver and thyroid effects are rat specific findings and the substance is not expected to pose a hazard for human health.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20.1.1982 to 28.10.1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Deviations:

yes

Remarks:

10 hour light cycle, morbidity and mortality checked once a day, and urinalysis was not performed.

GLP compliance:

yes

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal production stein (Tierfarm) CIBA-GEIGY Ltd.
- Age at study initiation: Males 26-30 weeks; Females 26 -32 weeks
- Weight at study initiation: Males 7.0 - 9.9 kg; Females 5.7 - 9.2 kg
- Housing: 2 dogs to a kennel, each equipped with underfloor heating
- Diet (e.g. ad libitum): dogs were fed 350 g/day of a pelleted standard diet
- Water (e.g. ad libitum): tap water was provided ad libitum
- Acclimation period: not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 20 to 70
- Photoperiod (hrs dark / hrs light): 14/10

IN-LIFE DATES: From: February 17, 1982 To: May 25, 1982

Route of administration:

oral: capsule

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
Batches of diet were assayed for composition and contaminant levels by the manufacturer. The test article was filled in gelatine capsules by the galenical department of the Pharmaceutical Division of CIBA-Geigy Ltd.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

3 months

Frequency of treatment:

once/day

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Details on study design:

Animals were randomized into groups and given 0, 10, 30, 100, or 300 mg/kg bw/day

Positive control:

None used.

Observations and examinations performed and frequency:

MORTALITY: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: mortality and signs of local and/or symtemic toxicity checked daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Calculated using the following formula - food consumption/group and week (g) / number of animals per group

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretest and week 13
- Dose groups that were examined: all animals

HEARING TEST
Auditory perception test performed at pretest and week 13

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week -1; week 7; week 13
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all animals
- Parameters checked: ERYTOEIOCYTES (RBC), HAEMATOCRIT (PCV), HAEMOGLOBIN (Hb), THROMBOCYTES (PLT), LEUCOCYTE COUNT (WBC): TOTAL COUNT, DIFFERENTIAL COUNT, RETICULOCYTES, INCLUSION BODIES, THROMBINE TIME (TT), PROTHROMBINE TIME (PT), PARTIAL THROMBOPLASTIN TIME (PIT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:week -1; Feb 17, 1982, week 7; April 6, 1982; week 13; May 17, 1982 (between 7:30 and 9:00 am)
- Animals fasted: No data
- How many animals: all animals
- Parameters checked: glucose, Urea (Urea-N), Total Bilirubin, Total Protein, Protein Electrophresis, Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Phosphorus Inorganic, Asp. Aminotransferase (GOT), Ala. Aminotransferase (GPT), Alkaline Phosphatase (AP), Gamma-Glutamyl transpeptidase (G-GPT).

URINALYSIS: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At 3 months all dogs were anaesthetized with intravenous injection of T61 (Hoechst) and bled. The total weight of each animal was determined, complete autopsy of all dogs was performed and the following organs were weighed: liver, kidneys, adrenals, thyroid, gonads, brain

HISTOPATHOLOGY: Yes
Small tissue portions of brain (cerebrum, cerebellum, brainstem), spinal cord, eye, pituitary, lacrymal gland, salivary gland, heart, thymus, thyroid, parathroid, lungs (including bronchi), trachea, spleen, bone (with marrow), lymph nodes, aorta, urinary bladder, oesophagus, stomach, small and large intestine, adrenal glands, pancreas, liver, kidneys, ovaries, testes, epididymis, prostate, uterus, skin, sternum, skeletal muscle, sciatic nerve, gall bladder, mammary gland, were fixed in buffered 10% neutral formalin. The fixed tissue samples were embedded in paraplast and sectioned at 3-5 um. The routine stain was haematoxylin and eosin.

Statistics:

For each time point and parameter an uni-variate statistical analysis was conducted. Each treated group was compared to the control group in respect of dispersion and displacement.

Clinical signs:

no effects observed

Description (incidence and severity):

In both, control and dose groups, diarrhoea was observed. Diarrhoea is often observed in the breed of beagles used and is not considered to be due to the administration of the test article. No animal died.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No difference in body weight development was observed between controls and treatment groups throughout the test.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No difference in food consumption was observed between controls and treatment groups throughout the test.

Food efficiency:

no effects observed

Description (incidence and severity):

No difference in food conversion was observed between controls and treatment groups throughout the test.

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmoscopic examination of conjunctiva, sclera, cornea, lens and fundus was performed before and at the end of the administration period, revealed no substance related findings.

Haematological findings:

no effects observed

Description (incidence and severity):

In the assessment of hematology values, the findings of the treated dogs were unremarkable and comparable to those of the controls.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In the assessment of blood chemistry values, the findings of the treated dogs were unremarkable and comparable to those of the controls.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Description (incidence and severity):

No impairment of the auditory perception was observed in the control and treatment groups before and at the end of the treatment period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The mean testicular weights in the dogs of the 10, 100 and 300 mg/kg dose groups were slightly, but statistically significantly increased when compared with those of the controls. With exception of tubular atrophy of spermatogenic epithelium noted in one male control dog (animal No. 1), there were no consistent differences in the testicular histopathology between treated and control animals. In addition, the statistical significant increase is mainly triggered by control animal 1 which showed a very low testicular weight therefore reducing the average testicular weight of the control group. Therefore no experimental relevance is attached to the increased mean weights of the testes in treated animals. There were some intra- and intergroup variations in the weights of some organs among the treated and control animals as well. Apart from higher weights of the testes, the analysis of the organ weights and organ to body weight ratios however, revealed no consistent compound related trends or alterations.

Gross pathological findings:

no effects observed

Description (incidence and severity):

All macroscopical appearances of the treated animals were comparable to those of the controls.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

In one male dog (No. 1) of the control group, it was observed that both testes displayed moderate tubular atrophy of the spermatogenic epithelium; moderate hyperplasia of the interstitial cells of Leydig, and absence of spermatozoa within the epididymes. However, all microscopical changes noted in some control and test animals were only incidental in nature and not due to the application of the tested compound.

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at the highest dose level

Critical effects observed:

not specified

Mean Testicular Weights

Dose group (mg/kg bw)	0	10	30	100	300
absolute testicular weight (g)	13.944	21.534*	19.084	19.598*	18.798*
relative testicular weight	0.134	0.194	0.168	0.179	0.176

* p ≤ 0.05

Individual Testicular Weights

control group		10 mg/kg bw		30 mg/kg bw		100 mg/kg bw		300 mg/kg bw
Animal No	Weight (g)	Animal No	Weight (g)	Animal No	Weight (g)	Animal No	Weight (g)	Animal No	Weight (g)
1	5.15	6	24.18	11	15.25	16	18.58	21	20.65
2	13.4	7	21.09	12	20.05	17	20.83	22	19.3
3	17.16	8	18.63	13	18.02	18	21.72	23	17.72
4	17.02	9	21.71	14	17.89	19	19.48	24	17.63
5	16.99	10	22.06	15	19.21	20	17.38	25	18.69
Mean weight	13.944	Mean weight	21.534	Mean weight	18.084	Mean weight	19.598	Mean weight	18.798

Conclusions:

Daily oral doses of up to 300 mg/kg body weight/day over a period of 3 months did not elicit measureable toxic side effects in male and female beagle dogs.

Executive summary:

In the present study a total of 50 pedigree Beagle dogs (5 males and 5 females per dose group) were used. The test article was administered orally in capsules for 3 months at doses of 0, 10, 30, 100 and 300 mg/kg/day. The results of the study are summarized as follows: In both, control and dose groups, diarrhoea was observed. Diarrhoea is often observed in the breed of beagles used and is not considered to be due to the administration of the test item. No animal died. No differences in body weight development, food consumption and food conversion were observed between controls and treatment groups throughout the test. The ophthalmoscopic examination of conjunctiva, sclera, cornea, lens and fundus was performed before and at the end of the administration period, revealed no substance related findings. No impairment of the auditory perception was observed in the control and treatment groups at the end of the treatment period. In the assessment of haematology and blood chemistry values, the findings of the treated dogs were unremarkable and comparable to those of the controls. Organ weights, organ to body weight ratios and organ to brain weight ratios revealed some intra-and intergroup variations. No toxicological relevance is attributed to these findings. Neither gross nor microscopical changes in the organs and tissues related to the treatment with the test article were noted. All morphological lesions seen in some control and test animals were only incidental in nature and not due to the treatment with the test article. It can be inferred from the above, that daily oral doses of 10, 30, 100 and 300 mg/kg/day of test material, administered over a period of 3 months do not elict measurable toxic side-effects in male and female beagle dogs.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

dog

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The substance exerts a species-specific toxicity profile. In rats, but not in dogs or monkeys, the substance is a strong inducer of xenobiotic metabolism in liver and interferes with thyroid hormone homeostasis. For details, it is referred to the section on specific investigations. The secondary effects on thyroid were observed at doses of 200 mg/kg bw upon 90 day exposure and at doses of 50 mg/kg bw upon two-year exposure.

In the subchronic rat gavage study (OECD 408), a total of 160 RAI rats (20 males and 20 females per dose group) were dosed by gavage with 0, 50, 250 and 1000 mg/kg body weight daily for three months (Ciba-Geigy, 1982). Doses were based on a 28-day range-finder study (Ciba-Geigy, 1981). High dose males and females consumed slightly less food and water during the study, and, as a consequence, showed slightly lower body weights at the end of the treatment period. Intermediate and high dose animals showed a dose-dependent increase of the enzyme activity of alanine aminotransferase and of the blood cholesterol concentration, and in line with these findings, a dose-dependent increase of the absolute and relative liver and kidney weights. Histopathologically, minimal to moderate hepatocyte hypertrophy was found in the high dose group animals and in the intermediate dose group males. In addition, minimal hypertrophy of thyroid follicles was observed in the intermediate and high dose group animals in a dose-dependent manner. A single small thyroid adenoma found was considered unlikely to be substance related. No pathological changes were observed in the kidneys. From the observed findings, and not considering the marginally increased kidney weights at the low dose male animals, it can be deduced that the dose of 50 mg/kg body weight is the NOEL.

In the subchronic feeding study with rats (OECD 408), a total of 200 RAI rats (20 males and 20 females per dose group) were fed with a diet containing 0, 50, 150, 500 and 1500 ppm, corresponding to 3.6, 11.1, 37.4, 112 and 3.9, 11.5, 38.1, 117 mg/kg body weight for males and females, respectively (Ciba-Geigy, 1984). Doses were chosen based on a palatability study (Ciba-Geigy, 1983). Food consumption and body weight gain were comparable in all dose groups except for the low dose females which showed a significant increase of both parameters. In the absence of any dose-relationship, this increase was considered not to be treatment-related. No death and no clinical symptoms showing systemic toxicity occurred. Higher plasma alkaline phosphatase and alanine amino transferase activities, blood and urea nitrogen and cholesterol levels in males of the high dose group and higher cholesterol levels in females of the intermediate and high dose group were observed. Increased liver weight in all animals of the high dose group and slightly increased kidney weight of the high dose males only were not accompanied by macroscopical and microscopical changes. The dose of 11.1 (males) and 11.5 (females) mg/kg body weight per day is considered to be the NOEL.

In subchronic feeding study with dogs (OECD 409), 50 beagles (5 males and 5 females per dose group) were dosed with 0, 10, 30, 100 and 300 mg/kg body weight per day for 3 months (Ciba-Geigy, 1982). The test article was applied in gelatine capsules. No substance-related effects were observed with respect to mortality, body weight, food consumption, clinical symptoms, hematology, clinical chemistry, organ weights (liver, kidneys, adrenals, thyroid, gonads and brain) and pathology (lever, thyroid, spleen, kidney and many others). The overall NOEL was equal to or above 300 mg/kg body weight per day.

In a 28 days repated dose study three groups of three male monkeys were dose with 0, 200 and 1000 mg/kg body weight daily by gavage (CIT, 1997). Apart from the general parameters measured according to OECD guideline 407, blood pressure was measured. Tissue and blood samples were taken for further biochemical analysis. One animal of the high dose group was killed in extremis, but its death was not related to the treatment. Body weight, food consumption, hematological and biochemical parameters of the blood showed no difference as compared to the controls. Slightly increased liver weights with hepatocyte hypertrophy in both dose groups were the only treatment-related finding in this study. In the absence of adverse hepatocyte effects this was attributed to a physiological adaptation process rather than to a toxic effect to the liver. The NOAEL was 1000 mg/kg bw per day.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No serious or irreversible effects were observed at a dose of 300 mg/kg bw or less upon repeated oral exposure of dogs and monkeys. As a result the substance is not considered to be classified for repeated dose oral toxicity under Regulation (EC) No. 1272/2008.