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Description of key information

As evident from all available repeated dose toxicity studies, the analogous substances possess very low toxicity potential by prolonged exposures both by oral and dermal routes of exposure. They did not induce mortality and no toxicologically significant clinical observations were detected in treated animals. Behavioural and functional performance parameters and sensory reactivity were generally without changes except some incidental transient findings. In the sub-chronic studies, animals showed statistically significant reduced body weight, while in sub-acute studies bodyweight development was not meaningful changed. Haematological and blood chemical parameters were slightly altered, probably as consequence of the adaptive change in liver function and had minimal physiological effect on the overall health of the animals. At necropsy, no toxicologically significant macroscopic abnormalities were detected. Significantly increased liver weight and centrilobular hepatocyte enlargement are common observations, which are often observed in rodents following the administration of xenobiotics and are usually the result of detoxification mechanism. The histopathological changes observed in liver and in other organs collectively represent adaptive response and considered non-adverse effects of treatment. The NOAEL established was the highest dose level tested in four repeated dose toxicity studies. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
186 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study is a GLP compliant and has Klimish score 2 (due to read-across).

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study is a GLP compliant and has Klimish score 2 (due to read-across).

Additional information

To assess toxicity potential at prolonged exposures to reaction mass of isomers of: C7 -C9 -alkyl 3 -(3,5 -di-tert-butyl-4hydroxyphenyl) propionate the following repeated dose toxicity studies on the related substances were used:

90 -day repeated dose oral (dietary) toxicity study in rats (Butyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate (CAS No. 52449 -44 -2))

The study was conducted according to the OECD guideline 408 (Dunster, 2009; Report No. 0525/0900).The test material was administered continuously by dietary admixture to three groups, each of ten male and ten female Wistar rats for ninety consecutive days, at dietary concentrations of 500, 1200 and 2500 ppm (equivalent to a mean achieved dosage of 39, 90 and 186 mg/kg/day respectively). A further group of ten males and ten females was exposed to basal laboratory diet to serve as a control. Clinical signs, functional observations, bodyweight development, dietary intake and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on control group and high dose animals before the start of treatment and during week 12 of the study. All animals were subjected to gross necropsy examination and a comprehensive histopathological evaluation of tissues was performed.

There were no mortalities and no clinically observable signs of toxicity in test or control animals throughout the study period. Females treated with 2500 ppm showed a reduction in bodyweight gain during week 11 whilst males from this treatment group showed a reduction in bodyweight gain during the final week of treatment. No toxicological significant effects were detected in animals of either sex treated with 1200 or 500 ppm. Males from all treatment groups showed a statistically significant reduction in bodyweight gain during Week 4. Males treated with 1200 ppm also showed a statistically significant reduction in bodyweight gain during week 12. In the absence of a true dose-related response the intergroup differences were considered of no toxicological importance.

There were no toxicologically significant changes in the behavioural parameters, in the functional performance parameters and in sensory reactivity.

No adverse effect on dietary intake was detected. There were no treatment-related ocular effects. Haematological parameters were without changes. Females treated with 2500 and 1200 ppm showed a statistically significant increase in plasma bilirubin. No such effects were detected in males treated with 2500 or 1200 ppm or animals of either sex treated with 500 ppm.

At necropsy, no toxicologically significant macroscopic abnormalities were detected. Males from all treatment groups and females treated with 2500 and 1200 ppm showed a statistically significant increase in liver weight in both absolute and relative to terminal bodyweight. No such effects were detected in females treated with 500 ppm. Histopathological findings were detected at all dietary concentrations. Centrilobular hepatocyte enlargement was seen in relation to treatment for animals of either sex treated with 2500 ppm (P <0.001), 1200 ppm (P <0.001), or at 500 ppm (P <0.05-0.001). Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, this is generally considered to be adaptive in nature. In spleen, lower grades of severity of extramedullary haemopoiesis were seen for females only treated with 2500 ppm or at 1200 ppm. Follicular cell hypertrophy was seen as a consequence of treatment for animals of either sex treated with 2500ppm (statistically significant for males only P <0.001), and for males only treated with 1200 ppm (P <0.01) or at 500 ppm (P <0.05). In lungs, a higher incidence of groups of alveolar macrophages was seen for males treated with 2500 ppm (P <0.01) or at 1200 ppm (P <0.01) compared with the control group. Females were not similarly affected. Groups of alveolar macrophages are seen occasionally as a spontaneous background change among laboratory maintained rats and the group distribution can be variable. A generally lower cellularity of the bone marrow, as evidenced by higher grades of severity of adipose infiltration, was observed for animals of either sex at all treatment levels, attaining statistical significance for animals of either sex treated with 2500 ppm (P <0.05) and for females only treated with 500 ppm (P <0.05). The response was not dose related and there were no indications of changes in any haematological parameters.

The oral administration of test material to rats for a period of ninety consecutive days at dietary concentrations of 500, 1200 and 2500 ppm resulted in treatment-related effects in animals of either sex at all dose levels. A No Observed Effect Level (NOEL) has, therefore not been established.

The microscopic effects detected at all dietary concentration were not considered to be an adverse effect of treatment. The “No Observed Adverse Effect Level” (NOAEL) was therefore considered to be 2500 ppm.

 

Oral (Dietary) One Generation Reproduction Toxicity Study in the Rat (OECD 415)(Butyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate (CAS No. 52449 -44 -2))

The administration of test material (by dietary admixture to rats for either a period of up to one hundred and twenty seven consecutive days for males or at least fifteen consecutive days prior to mating and throughout the gestation and lactation phases of the reproductive cycle for females, resulted in adult systemic effects at dose levels of up to 2500 ppm (Marr, 2010; Report No. 0525/0901). There were no clinical observations attributed to test material toxicity. Reductions in bodyweight development and dietary intake were observed together with increased liver and thyroid organ weight measurements. In addition, histopathological changes in the liver, thyroid, lung and bone marrow were observed. Microscopically examination of liver revealed centrilobular hepatocyte enlargement, which is, however, commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, is generally considered to be adaptive in nature. An increase in thyroid weights both absolute and relative to terminal bodyweight was also detected throughout the female treatment animals, in comparison to controls. Histopathological examination of the thyroid gland showed a greater incidence and severity of follicular cell hypertrophy. This may be associated with the changes seen in the liver and probably connected with increased thyroxine excretion which in turn is the consequence of induced hepatic enzymes in the liver. In the lungs a higher incidence of groups of alveolar macrophages was seen for the 2500 ppm males, compared with the control group. In addition, the changes observed in the bone marrow displayed by generally lower cellularity of the bone marrow, as evidenced by higher grades of severity of adipose infiltration are considered to represent functional adaptations to treatment and are not indicative of systemic toxicity. In summary, the changes observed for adults collectively represent adaptive responses that can be considered non-adverse effects of treatment. There were no treatment related effects upon fertility and reproductive performance at dose levels up to 2500 ppm. The lack of any significant differences in numbers of successful matings and pregnancies clearly indicate that there was no effect upon reproduction.

The effects detected at dietary concentrations up to 2500 ppm would not be categorised as an adverse effect of treatment according to criteria outlined in ECETOC Technical Report 85 (2002). The “No Observed Adverse Effect Level” (NOAEL) was therefore considered to be 2500 ppm. There were no effects upon prenatal offspring viability and development. The reductions in offspring postnatal bodyweight development during lactation correlate with adult adaptive responses and therefore reflect a further manifestation of the same treatment effects. The change is therefore adaptive in nature. The ‘No Observed Adverse Effect Level’ (NOAEL) for reproduction and offspring development is 2500 ppm, equivalent to 205 mg/kg/d.

 

Twenty-eight Day Repeated Dose Oral (Gavage) Toxicity Study in the Rat (Butyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate (CAS No. 52449 -44 -2))

The study was performed according to the OECD guideline 407 and fulfilled the criteria of GLP (Jones et al., 2000; Report No. 525/207). The test material was administered by gavage to four groups, each of five male and five female Sprague-Dawley Crl:CD@BR strain rats, for twenty-eight consecutive days, at nominal dose levels of 15, 50, 150 and 500 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). Three recovery groups, each of five males and five females, were treated with the high or intermediate II dose (500 or 150 mg/kg/day respectively) or the vehicle alone for up to twenty-eight consecutive days and then maintained without treatment for a further fourteen days.

Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Oral administration of the test material to rats resulted in treatment-related effects at 500, 150, 50 and 15 mg/kg/day. These included clinically observable signs, reduced body weight development and dietary intake, increased water consumption, increased urine volume of reduced specific gravity, increased clotting (prothrombin) and activated partial thromboplastin time, reduced haemoglobin, haematocrit and erythrocyte count, increased alkaline phosphatase and total plasma protein, reduced albumin/globulin ratio, increased absolute and relative liver weight, increased spleen weight, macroscopic liver changes, microscopic liver and thyroid changes identified as centrilobular hepatocyte enlargement and follicular cell hypertrophy with associated colloid depletion respectively at 500 mg/kg/day. Effects at 150 mg/kg/day included increased urine volume of reduced specific gravity, increased clotting (prothrombin) and activated partial thromboplastin time, reduced haemoglobin, haematocrit and erythrocyte count, increased absolute and relative liver weight and microscopic liver and thyroid changes identified as centrilobular hepatocyte enlargement and follicular cell hypertrophy with associated colloid depletion respectively.

The liver and thyroid changes detected were entirely adaptive in nature and were considered not to represent an adverse health effect. Since these were the only changes seen at dose levels of 50 and 15 mg/kg/day, for the purposes of hazard evaluation, the "No Observed Effect Level" (NOEL) should be regarded as 50 mg/kg/day.

 

Twenty-eight Day Repeated Dose Oral (Gavage) Toxicity Study in the Rat (Benzenepropanoic acid, 3,5 -bis(1,1 -dimethylethyl)-4 -hydroxy-,2 -ethylhexylester (CAS No. 144429 -84 -5)

The GLP 28-day oral study in rats was designed to investigate the systemic toxicity of the Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-,2-ethylhexylester (related substance to the target chemical) and complies with the OECD guideline 407 (Dunster et al., 2007; Report No. 0525/0757). The test material was administered by gavage to three groups of Sprague-Dawley rats for twenty-eight consecutive days, at dose levels of 25, 150 and 750 mg/kg/day. A control group was dosed with vehicle alone (Arachis oil BP). Two recovery groups were treated with the high dose (750 mg/kg/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Clinical signs, bodyweight development, food and water consumption and functional observations were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from non-recovery high dose and control animals was performed.

There were no unscheduled deaths during the study. No toxicologically significant clinical observations were detected in the treated animals. There were no treatment-related changes in their behaviour, in the functional performance parameters and in sensory reactivity. Bodyweight development was unchanged and no effects on food consumption and on food efficiency were detected.

Treatment-related findings were confined to the liver and thyroid. Microscopic examination revealed centrilobular hepatocyte enlargement for animals of either sex treated with 750, 150 and 25 mg/kg/day. Organ weight data supported this finding with increased absolute and relative liver weights detected in 750 and 150 mg/kg/day dose groups. Increased hepatic activity of this type is often observed in the rodent liver following the administration of xenobiotics and is usually the result of detoxification mechanisms involving hepatic enzyme induction. Haematological and blood chemical investigations revealed a possible altered metabolism, probably as a consequence of the adaptive change in liver function. Increases were evident in clotting time, platelet count and activated partial thromboplastin time along with changes in cholesterol, triglycerides, total protein and alanine aminotransferase, all of which can be associated with altered liver function. Animals of either sex from all treatment groups showed an increase in reducing substances present in the urine. Non-recovery 750 mg/kg/day males also showed an increase in urine volume together with a reduction in urine specific gravity and an increase in water consumption; thereby again suggesting the possibility of altered metabolism.

Microscopic changes were also identified in the thyroid gland. Follicular cell hypertrophy was observed for animals of either sex treated with 750, 150 and 25 (males only) mg/kg/day. Thyroxine is ultimately excreted via the bile, having first been conjugated in the liver. It is conceivable that conjugating hepatic enzymes may have been induced as a response to the test material therefore increasing thyroxine excretion and stimulating compensatory TSH and thyroxine production, resulting in the microscopic change identified and, as such, is generally regarded as adaptive in nature.

Effects of treatment were apparently reversible since only increases in cholesterol and liver weights were evident in recovery 750 mg/kg/day females and there was also evidence of regression in the microscopic conditions seen in the liver and thyroid among recovery 750 mg/kg/day dose groups following fourteen days without treatment.

A “No Observed Effect Level” (NOEL) has not been achieved because treatment related effects were detected at all dose levels. However, the changes detected in all treatment groups were confined to adaptive microscopic liver and thyroid changes and together with associated increased liver weights and a slightly altered metabolism were considered not to represent “serious damage” to health as defined by the criteria given in the EC labelling guide of Commission Directives 2001/59/EC. For this reason 750 mg/kg/day may be regarded as a “No Observed Adverse Effect Level” (NOAEL).

 

A 4 -Week Dermal Toxicity Study in Rats with a 2 -weeks recovery phase (Butyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate (CAS No. 52449 -44 -2))

A 4-week dermal toxicity study in rats with a 2-week recovery phase was conducted in accordance with US-EPA Health Effects Test Guidelines OPPTS 870.3200 (August 1998) and was focusing more specifically on the potential target organ effects (Rusty and Rusch, 2002; Report No. SLI 3263.268). The test material was administered to rats by the dermal route at dosage levels of 100, 250, 500 and 1000 mg/kg/day. Control animals received the control material (mineral oil) at the same dose volume as the high-dose level under the same experimental conditions. Detailed clinical observations, dermal irritation examinations, food consumption, individual body weights and clinical pathology parameters including thyroid hormone analyses were performed during the study. All animals were subjected to a complete gross necropsy examination at scheduled euthanasia (day 26 or 39 for main and recovery animals respectively). Fresh organ weights were obtained for all animals. Selected tissues, including treated skin, were preserved from all rats. All tissues and organs collected at necropsy from animals in the control and high-dose groups were examined microscopically, as well as the adrenal glands, brain, heart, kidneys, liver, ovaries, prostate, spleen, testes with epididymides, thymus, thyroids, uterus and vagina from the low- and mid-dose groups.
No mortality, test article-related clinical abnormalities or notable dermal findings were observed during the main and recovery phases of this study. In addition, there were no toxicologically meaningful changes in body weights, food consumption or gross necropsy observations during this project. In the 1000 mg/kg/day males, a slight increase in haematocrit, mean corpuscular volume (MCV) and bilirubin and a slight decrease in Mean corpuscular haemoglobin concentration (MCHC) were observed on day 26. These changes could be interpreted as slight haemolytic anaemia, however, the absence of microscopic changes in the bone marrow, spleen or liver along with the lack of increases in the number of reticulocytes or polychromatic cells suggests that, if anaemia was present, it was minimal and had little impact on the overall health of the animal. In the 1000 mg/kg/day females, statistically elevated reverse triiodothyronine (RT3) was observed at the end of the treatment period on day 26. In addition, statistically increased absolute/relative thyroid weights were noted in these animals on day 26 and follicular cell hypertrophy was observed microscopically at both the day 26 and 39 intervals. These findings appear to be associated with test article treatment. However, the absence of correlative changes in the other key thyroid hormones (ex., increases in serum thyroid stimulating hormone (TSH) or decreases in triiodothyronine (T3) or thyroxine (T4) suggests that the above changes, if real, had minimal physiological effect on the thyroid and on the overall health of the animal.
Based on the results of this study, dermal administration of test material for four consecutive weeks (5 days/week) at up to 1000 mg/kg/day did not produce any mortality, significant clinical abnormalities or notable dermal findings. Minimal thyroid follicular cell hypertrophy was observed in the 1000 mg/kg/day females at the end of the treatment and recovery periods. However, the above change did not have a notable effect on the thyroid serum hormones levels and had no apparent effect on the overall health on the animals. Therefore, the no-observed-adverse-effect level (NOAEL) for this study was considered to be 1000 mg/kg/day in the males and 500 mg/kg /day in females.

 


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The study with the longest duration (90-days) and the lowest NOAEL was chosen.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Dermal study and dermal route of exposure in humans.

Justification for classification or non-classification

The related substances Butyl 3,5 -bis(1,1 -dimethylethyl)-4 -hydroxybenzenepropanoate (CAS No. 52449 -44 -2) and Benzenepropanoic acid, 3,5 -bis(1,1 -dimethyl)-4 -hydroxy-, 2 -ethylhexyl ester (CAS No. 144429 -84 -5) do not produce adverse systemic effects in test animals. The changes observed in the repeated dose toxicity studies are considered to be adaptive in nature. According to the Directive 67/548/EEC and (Regulation (EC) No. 1272/2008) classification and labelling of reaction mass of isomers of: C7-C9- alkyl 3 -(3,5-di-tert-butyl-4hydroxyphenyl)propionate is not required for prolonged exposure.

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