Registration Dossier

With reference to the effects observed in several repeated dose studies (low body weight and body weight gain, increased bilirubin level, histopathological changes in adrenals and liver) a 14 day short term oral toxicity study with special focus on the reported effects was performed in rats (non-pregnant and pregnant). This study followed in principle the design as lined out in OECD 407. The test item, Hostanox O 3 P, was administered to Wistar rats orally (by gavage) at the doses of 100 (low dose), 300 (mid dose) and 600 mg/kg/day (to 3 cohorts of rats: the repeated dose phase (14 days), the satellite group (3 and 10 days treatment) and the Embryo Fetal Developmental phase (gestation day 5 to 19 treatment). The repeated dose phase and the satellite group consisted of 6 rats/sex/group, while the Embryo Fetal Developmental phase consisted of 6 presumed pregnant rats (gestation day 0).
In this study, assessments included clinical signs, body weights, food consumption, clinical laboratory investigations of blood (for hematology, acute phase proteins, clinical chemistry and hormone analysis), gross pathology, and histopathological evaluation (repeat dose phase and satellite group). The microscopic examination was initially performed for the control and high dose groups of males and females, and extended to the following organs of lower dose groups as higher incidence of lesions was present in high dose groups

The following observations were reported:

1. Immunological response
- increased number of neutrophils, leukocytes and monocytes from day 10 on at 300 and 600 mg/kg bw/d (highest dose tested)
- increase in inflammatory markers at day 10-group in all animals at doses ≥ 300 mg/kg bw/d, at day 14-group at 600 mg/kg bw/d dose group and for the developmental phase rats (Gestation Day 20) at all dose groups.

- histopathological changes of thymus and spleen at 600 mg/kg bw/d and
- lymphangiectasis in the gastro intestinal tract from day 3 onwards ≥ 300 mg/kg bw/d

2. Adrenals

Histopathological changes were reported from day 3 onwards. However, it has to be emphasized that the hormonal status remains unchanged and comparable to that of the control animals. Therefore, an endocrine activity is excluded.

3. Liver:
- the bilirubin concentration in blood is increased at 300 and 600 mg/kg bw/d.
- furthermore, the AST and ALT level as well as the triglyceride level are slightly elevated at 600 mg/kg bw/d
- vacuolization of hepatocytes from day 3 on at 300 and 600 mg/kg bw/d.

4. Stomach
- hyperplasia and ulcers in non-glandular and glandular stomach.

Based on the above mentioned effects the NOAEL was set at 100 mg/kg bw/d.

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable without restriction

System:

immune system

Organ:

adrenal glands

duodenum

ileum

jejunum

liver

stomach

Reference

Endpoint:: repeated dose toxicity: inhalation, other
Data waiving:: study scientifically not necessary / other information available
Justification for data waiving:: other:
Critical effects observed:: not specified

Endpoint conclusion:

no study available

Reference

Endpoint:: repeated dose toxicity: inhalation, other
Data waiving:: study scientifically not necessary / other information available
Justification for data waiving:: other:
Critical effects observed:: not specified

Endpoint conclusion:

no study available

Reference

Endpoint:: repeated dose toxicity: dermal, other
Data waiving:: study scientifically not necessary / other information available
Justification for data waiving:: other:
Critical effects observed:: not specified

Endpoint conclusion:

no study available

Reference

Endpoint:: repeated dose toxicity: dermal, other
Data waiving:: study scientifically not necessary / other information available
Justification for data waiving:: other:
Critical effects observed:: not specified

Endpoint conclusion:

no study available

A combined chronic toxicity and carcinogenicity study (130 weeks) was performed with Bis-(3,3-di-(4-oxy-3-tert. butyl-phenyl)butane acid) glycol ester fed to rats at dietary levels of 0, 0.16, 0.4 or 1.0%.
The study was conducted with weanling rats obtained from mothers which had been fed the same diets during 30 days before mating and during gestation and lactation. From the results of the reproduction phase of the study it was concluded that the test substance failed to induce any deleterious effect other than a slight growth depression in parent males at 0.4 and 1.0% and in pups at 1.0% (please refer to chapter 7.8.1). This study deals with the results obtained with F1 rats during a life-time period (130 weeks).
The data of mortality was not affected. Body weight was relatively low at the 1.0% dose level from week 12 onwards in both sexes. The differences with the controls were generally smaller than 10%. There were no outstanding differences in food intake and food efficiency amongst the groups.

There were no changes in haematological characteristics that could be ascribed to the feeding of DTB-glycolester. The only effect considered to be treatment-related occurred in the blood biochemistry and consisted of an increase in total Bilirubin in the groups treated with 0.4 and 1.0% DTB-glycolester in both sexes. The direct Bilirubin level was not affected. Since histologically no abnormality was found in the liver, bile ducts or spleen and the complete absence of signs of haemolytic anemia, the elevated total Bilirubin level in the mid- and high-dose group is considered to be of minor toxicological relevance. Kidney function tests did not show any dose-related changes. Organ to body weight ratios of 8 different organs were not affected. Gross examination did not reveal any treatment-related effects. Histopathological examination did not reveal any change. No indications were found for carcinogenic properties of the test item.

Based on the absence of toxicological relevant effects the NOAEL is considered to be 1.0% in diet corresponding to 500 mg/kg bw/d (considering a dietary conversion factor (ppm to mg/kg bw/d) of 20 for older rats).

The toxicity of DTB-glycolester was examined in a sub-chronic (16 week) study in which the test substance was fed to groups of 50 male and 50 female weanling rats each at levels of 0 (control), 0.1, 0.4 or 1.6% in stock diet. The rats were derived from parents which had been fed the diets for 31 days before mating and during gestation and lactation. Observations were made on general appearance and behavior, growth, food intake and food efficiency, hematology, blood biochemistry and urine composition. At week 17 all rats were killed and examined grossly for pathological changes. The major organs were weighed and extensive histopathological examinations were carried out on 15 males and 15 females of each group.
General condition and behavior were not adversely affected at any level of the test substance.
Gain in body weight was relatively low in males of the high-dose group. Food intake was decreased at 1.6% dose level in both sexes during the first two weeks. Food efficiencies were not adversely affected.
Haematological examinations did not reveal any treatment related differences amongst various groups.
There were no toxicologically significant differences amongst the groups with respect to enzyme activities and protein values of blood serum.
Kidney-function tests and urine composition did not exhibit any harmful effects of the test substance.
There were slight increases in the relative weights of the kidneys, liver and ovaries at the high-dose level. The weights of ovaries were also increased in the medium-dose group.
Gross and microscopic pathology failed to reveal changes that could be attributed to ingestion of the test substance.
It was concluded that the NOAEL of DTB-glycolester in the present study was 1.6% (1600 mg/kg bw/d, considering a diet conversion factor (ppm to mg/kg bw/d) of 10) in the diet of rats for 16 weeks.

Bis-(3,3 -Di-(4 -oxy-3 -tert. butyl-phenyl)-butanic acid)-glycol ester has been tested in a 90 day feeding study with SPF Wistar K rats in a control group and 3 dosage groups of 5%, 1% and 0.1% in food. All animals within the testing groups 1% and 0.1% showed normal behaviour and no indication of toxicity. All animals of the dosing group 5% died within day 10 and 39 after the start of the study. Food consumption of all animals in the two lower dose groups was comparable to the control group. Animals of the top dose group (5%), however, did not take up sufficient food. These animals showed significant body weight loss until they died. Histological findings of the animals in the lower dosed groups were not different from control. Histology examination of the top dose animals were diagnosed as significant tissue changes of lung, liver, spleen, adrenals, pancreas and testes which might be related to the malnutrition. The diagnosis was partially disturbed by autolyis.
Based on the results of this study, the no-observed-effect-level (NOEL) for the test item, Hostanox O 3, was considered to be 1% in diet (corresponding to 1000 mg/kg bw/d considering a diet conversion factor (ppm to mg/kg bw/d) of 10 for young rats)).

After dosing dogs (6 animals/sex/dose level) with 100, 500 or 600-1100 ppm DTB-Glykolester (Hostanox O 3) in diet for 2 years no treatment-related clinical/neurological changes as well macroscopical/microscopical effects were observed. Food consumption and body weight gain were slightly reduced in males at 1100 ppm compared to the control animals.The NOAEL is set at 1100 ppm substance in diet corresponding to 84.6 mg/kg bw/d (males, highest dose tested) and 75.2 mg/kg bw/d (females, highest dose tested), respectively.

After dosing dogs (4 animals/sex/dose level) with 100, 500 or 750 ppm DTB-Glykolester (Hostanox O 3) in diet for 90 days no clinical/neurological changes as well macroscopical/microscopical effects were observed. The NOEL is set at 750 ppm substance in diet corresponding to 47.6 mg/kg bw/d (males, highest dose tested) and 39.6 mg/kg bw/d (females, highest dose tested), respectively.

With reference to the effects observed in several repeated dose studies (low body weight and body weight gain, increased bilirubin level, histopathological changes in adrenals and liver) a 14 day short term oral toxicity study with special focus on the reported effects was performed in rats (non-pregnant and pregnant). This study followed in principle the design as lined out in OECD 407. The test item, Hostanox O 3 P, was administered to Wistar rats orally (by gavage) at the doses of 100 (low dose), 300 (mid dose) and 600 mg/kg/day (to 3 cohorts of rats: the repeated dose phase (14 days), the satellite group (3 and 10 days treatment) and the Embryo Fetal Developmental phase (gestation day 5 to 19 treatment). The repeated dose phase and the satellite group consisted of 6 rats/sex/group, while the Embryo Fetal Developmental phase consisted of 6 presumed pregnant rats (gestation day 0).
In this study, assessments included clinical signs, body weights, food consumption, clinical laboratory investigations of blood (for hematology, acute phase proteins, clinical chemistry and hormone analysis), gross pathology, and histopathological evaluation (repeat dose phase and satellite group). The microscopic examination was initially performed for the control and high dose groups of males and females, and extended to the following organs of lower dose groups as higher incidence of lesions was present in high dose groups

The following observations were reported:

1. Immunological response
- increased number of neutrophils, leukocytes and monocytes from day 10 on at 300 and 600 mg/kg bw/d (highest dose tested)
-increase in inflammatory markers at day 10-group in all animals at doses≥ 300 mg/kg bw/d, at day 14-group at 600 mg/kg bw/d dose group and for the developmental phase rats (Gestation Day 20) at all dose groups.

- histopathological changes of thymus and spleen at 600 mg/kg bw/d and
- lymphangiectasis in the gastro intestinal tract from day 3 onwards≥ 300 mg/kg bw/d

2. Adrenals

Adrenals were identified as target organ. Histopathological changes were reported from day 3 onwards. However, it has to be emphasized that the hormonal status remains unchanged and comparable to that of the control animals. Therefore, an endocrine activity is excluded.

3. Liver:
- the bilirubin concentration in blood is increased at 300 and 600 mg/kg bw/d.
- furthermore, the AST and ALT level as well as the triglyceride level are slightly elevated at 600 mg/kg bw/d
- vacuolization of hepatocytes from day 3 on at 300 and 600 mg/kg bw/d.

4. Stomach
- hyperplasia and ulcers in non-glandular and glandular stomach.

Based on the above mentioned effects the NOEAL was set at 100 mg/kg bw/d.

According to the criteria laid down in the EU Classification Labelling and Packaging Regulation (1272/2008/EC) and based on available data no classification is warranted for the registration substance.