Registration Dossier

Administrative data

Description of key information

ORAL
A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test is available in which the NOAEL of the substance was determined to be 3 mg/kg in male and female rats.
Furthermore a 2 year chronic study is available in which the NOAEL of the substance was determined to be 1.5 mg/kg in male and female rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
1.5 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Three studies are available to address this endpoint. Matsumoto et al. (1991) and Takahashi & Hiraga (1978) have both been awarded a reliability score of 2; van Otterdijk (2015) has been awarded a reliability score of 1. The quality of the database is therefore considered to be high.

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

Oral

In the first key study, the repeated dose toxicity of the test material was investigated in a combined repeated dose toxicity study with the reproduction / developmental toxicity screening test conducted in accordance with the standardised guidelines OECD 422 and US EPA OPPTS 870.3650 under GLP conditions. The study was awarded a reliability score of 1 in line with the principles for assessing data quality as defined by Klimisch et al. (1997).

Based on the results of a 10-day dose range finding study, the dose levels for this study were selected to be 3, 10 and 30 mg/kg.

The test material, formulated in corn oil, was administered daily by oral gavage to SPF-bred Wistar Han rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41 to 56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), clinical pathology (end of treatment), macroscopy at termination, organ weights and histopathology on a selection of tissues. Reproduction/developmental parameters were also evaluated.

Formulations were analysed once during the study to assess accuracy, homogeneity and stability. Analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

Histopathological examination showed hepatocellular hypertrophy up to moderate degree in both sexes at 10 and 30 mg/kg. This was supported at necropsy by enlargement and/or accentuated lobular pattern of the liver for some animals at 30 mg/kg. At 30 mg/kg, hepatocellular necrosis was present in a single male and a single female at minimal degree. Additionally, higher liver weights (absolute and/or relative to body weights) were recorded at 10 and 30 mg/kg; relative liver weights were increased 39 and 63 % in males and females at 30 mg/kg, respectively, and 21 % in females at 10 mg/kg. The higher liver weights in females at 10 and 30 mg/kg along with the combined occurrence of hepatocellular hypertrophy with necrosis at 30 mg/kg were considered to be adverse in nature.

Mucosal hypertrophy of the cecum in males treated at 10 and 30 mg/kg was noted up to a slight degree only and occurred in absence of any other indicators of toxicity in this organ. This histopathological lesion was therefore not considered adverse in nature.

Decreased splenic haematopoiesis was observed in females treated at 10 and 30 mg/kg, which occurred along with lower reticulocyte counts. However, red blood cell counts showed an increase rather than a decrease at this dose level. Also, since there were no other indicators of toxicity in the spleen, these changes were not considered to represent an adverse effect on red blood cell turn over.

At 30 mg/kg, other changes in blood of females that were considered to be related to treatment consisted of lower relative neutrophil counts, higher relative lymphocyte counts, lower mean corpuscular volume and mean corpuscular haemoglobin, higher total protein, albumin, calcium, cholesterol, potassium and glucose and lower urea and total bilirubin. Higher potassium and lower total bilirubin were also noted for males at 30 mg/kg.

At 10 mg/kg, changes in blood consisted of lower mean corpuscular volume and mean corpuscular haemoglobin in females, lower total bilirubin in males and females, and higher cholesterol in females. In the absence of any concurrent morphological lesions, and given the slight nature of these changes (i.e. within or just outside the range considered normal for rats of this age and strain), these were not considered adverse in nature.

Females at 10 and 30 mg/kg showed lower food consumption during the lactation period, as well as notable weight loss during lactation ranging from 4 to 9 % of Day 1 lactation values. The lower maternal food intake and body weight gain and mean pup body weight gain appeared unrelated on an individual animal basis. Also, these changes were not accompanied by supportive clinical signs or inadequate maternal care. As such, these changes in food intake and body weight gain during lactation were considered not adverse in nature.

No toxicologically relevant clinical signs or changes in functional observation parameters were noted.

No toxicologically relevant changes in reproductive parameters were noted.

With regard to developmental effects, at 30 mg/kg, the mean number of living pups at first litter check appeared slightly lower than controls. Also, an increased postnatal loss and lower viability index was noted at 10 and 30 mg/kg. At 10 mg/kg, a total of 3 dams showed postnatal loss. At 30 mg/kg, a total of 5 dams showed postnatal loss. The significance of these findings is not clear at this stage and are proposed to be further investigated.

Mean pup body weights for both sexes combined on Day 4 of lactation were approximately 16 and 20 % lower than the control mean at 10 and 30 mg/kg, respectively. Although there were no other developmental changes noted for these pups (macroscopy and clinical signs), the magnitude of changes in pup body weight was considered to represent an adverse effect on pup development. No apparent relationship could be found between maternal food intake and mean pup body weight gain on an individual animal basis.

Under the conditions of the study, the repeated dose NOAEL was determined to be 3 mg/kg (based on higher liver weights at 10 and 30 mg/kg, with hepatocellular hypertrophy and necrosis at 30 mg/kg).

In the second key study (Matsumoto et al., 1991), the potential of the test material to cause repeated dose toxicity was investigated in a study conducted using methodology similar to that outlined in the standardised guideline OECD 452. The study was awarded a reliability score of 2 in line with the principles for assessing data quality as defined by Klimisch et al. (1997).

Groups of 40 Slc:Wistar rats of both sexes were fed diet containing 0, 30, 100, 300 and 1000 ppm of the test material for up to 24 months, with interim examinations at 6, 12 and 18 months.

The general condition of the animals was observed and body weights were recorded throughout the study. At 6, 12, 18 and 24 months after the start of test material administration, haematological and serum biochemical examinations were conducted for all dose groups. Haemoglobin concentration, mean corpuscular volume, platelet count, blood urea nitrogen, phospholipids, total cholesterol, glutamate oxaloacetate transaminase and gamma-glutamyl transpeptidase were all analysed. Also following 6, 12, 18 and 24 months of test material administration, histopathological examinations were performed for all groups.

Mortality in treated rats was comparable to that of controls. No remarkable general findings were observed in the control and treated groups throughout the experimental period. Significant reduction of body weight gain was found in the female 1000 ppm group from 12 months onward. No remarkable changes in food consumption were observed in the control and treated groups throughout the experimental period.

The haematological, biochemical and histopathological examinations revealed slight microcytic anaemia, changes in some biochemical parameters relating to liver function and focal necrosis of liver cells following test material administration. The changes observed in females were more severe than those in males. No neoplastic response following test material administration was noted.

It was concluded that the test material causes liver injury characterised by focal necrosis with microcytic anaemia and elevations of serum phospholipids and cholesterol levels presumably occurring as secondary effects following the liver injury.

Under the conditions of this study, the no observed adverse effect level (NOAEL) was determined to be 30 ppm (ca. 1.5 mg/kg bw/day). The lowest observed adverse effect level (LOAEL) was determined to be 100 ppm (ca. 5 mg/kg bw/day).

 

In the supporting study (Takahashi & Hiraga 1978), the relationship between haemorrhage induced by butylated hydroxytoluene and its antioxidant properties or structural characteristics was investigated. The study was awarded a reliability score of 2 in line with the principles for assessing data quality as defined by Klimisch et al. (1997).

Rats were fed one of five antioxidants or six substituted phenols at concentrations which were equal to the haemorrhagic LC50(40 days) of BHT to determine the mechanism of BHT-induced haemorrhage.

The test material was one of the substituted phenols. Ten male Sprague-Dawley rats were administered the test material in the diet at a concentration of 5.46 mmol/100 g diet for up to 3 weeks.

The number of dead animals, the presence of haemorrhage in dead and surviving animals, outward signs of intoxication, food intake and body weight were recorded. Intake of test material was calculated from food intake to be 1.98 mmol/kg/day (mean chemical intake).

All 10 animals administered the test material died during the dosing period. Lethal times were Days 5 (1 animal), 6 (1 animal), 8 (2 animals), 9 (3 animals), 10 (2 animals) and 11 (1 animal).

In all of the dead animals, haemothorax, haematocoelia, intracranial haematoma, intranasal haemorrhage, intramuscular haematoma, intratesticular haematoma and intraepididymis haemorrhage were observed.

Under the conditions of this study, the LT50 of male rats which received a mean chemical intake of TBP of 1.98 mmol/kg/day was 7.4 days (95 % confidence limit 6.3 to 8.7 days).

The results of the overall study suggest that the haemorrhagic effect of BHT appears to be related to its structure.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Two studies are considered to be key; a modern, guideline compliant OECD 422 study conducted under GLP conditions and a study conducted using methodology similar to that outlined in the standardised guideline OECD 452. The value obtained in the 2 year study was used for risk assessment purposes, due to both the longer duration and lower value.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance requires classification with respect to Specific Target Organ Toxicity (STOT) (repeated exposure) as Category 1 (H372: Causes damage to organs (liver) through prolonged or repeated exposure).