Hexamethylene bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate]

Carcinogenicity

Description of key information

In a two 2-year carcinogenicity study in rats no evidence for an elevated incidence of tumorgenicity was found throughout the study period. (BASF 1978)

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1978-03-02 to 1980-05-13

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Scientifically acceptable and guideline compliant study report

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

no

GLP compliance:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, USA
- Age at study initiation: 28 days
- Weight at study initiation: 65 - 85 g
- Housing: in groups of 5/sex in suspended cages with wire-mesh floors
- Diet: ad libitum, Spratt`s Laboratory diet No. 2
- Water: ad libitum, tap water
- Acclimation period: two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with: Spratt's Laboratory Diet No. 2
- Storage temperature of food: at room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before treatment commenced, and during weeks 1, 7, 13, 26, 53, 78 and 104 samples of all dietary levels were sent to the Sponsor for analysis. The samples were shipped in heat-sealed polythene bags and included all dietary levels (200 g) and a sample of the test substance (5 g). The test substance was extracted with distilled chloroform and then determined by liquid chromatography.
Dietary analysis performed by the Sponsor on samples of diet prepared for weeks 1, 7, 13, 26, 53, 78 and 104 of the study showed the levels to be in good agreement with the intended dietary levels of the test substance. Levels achieved were within 10 % of the intended concentration.

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

daily

Post exposure period:

no

Remarks:

Doses / Concentrations:
50, 150, 450 ppm (eq. 1.8-2.3, 5.2-6.9, 15.4-20.0 mg/kg bw /d)
Basis:
nominal in diet

No. of animals per sex per dose:

60

Control animals:

yes, plain diet

Details on study design:

The withdrawal period, during which all rats were fed control diet began at the end of the 104-week treatment period and continued until any one group within a sex had reached approximately the 20 % survival point. Using this criterion, all surviving male rats were killed after 112 weeks of the study, and all surviving female rats were killed after 114 weeks.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during the first 4 weeks and afterwards once per week.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each group (5 animals) determined and mean weekly diet consumption calculated as g food/kg body weight: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All superficial tissues, including the urogenital orifices and toil, each pinna, eye and extemal auditory meatus, were examined visually and by palpation for distortion, swelling or other evidence of tumour formation; similar attention was given to the mammary tracts and the subcutaneous structures. The extemal nares, buccal cavity and tongue were then examined, and the cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. The nasal cavity was exposed and inspected. After ventral midline incision and skin reflection, all subcutaneous tissues were examined, including regional lymph nodes, mammary and thyroic porathyroid glands. The condition of the thoracic viscera was noted, with due attention to the thymus, lymph nodes and heart. The abdominal viscera were examined before and after removal; the urinary bladder was briefly distended with fixative in situ, then removed, opened and examined under low-power magnification. The oesophagus, stomach and intestines including caecum were incised and examined. The lungs were removed and all pleural surfaces were examined under suitable illumination. The liver was sectioned at intervals of a few millimetres; the kidneys were incised and examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded. Any lesion suggestive of neoplasia was noted, including details of location, size and multiplicity. Any evidence of adhesion or possible invasion to adjacent structures was noted.

HISTOPATHOLOGY: Yes
The following tissues were preserved at autopsy and further examined:
adrenals, aorta, bone, brain (medullary, cerebellar, thalamic and cortical sections), caecum, duodenum, eye, harderian gland, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (cervical and mesenteric), mammary gland, mid-colon, middle ear, nosal cavity, oesophagus, optic nerve, ovaries, pancreas, pituitary, prostate, salivary gland, sciatic nerve, skeletal muscle, skin, spinal cord (cervical), spleen, sternum (for bone marrow), stomach (glandular and non-glondular), testes, thymus (where present), thyroids (with porothyroids), urinary bladder, uterus

Other examinations:

ORGAN WEIGHTS: Yes
The following organs were weighted: heart, kidney, liver, thyroids, brain, gonads, adrenals, thymus, spleen

Statistics:

Analysis of variance followed by Student's 't' test was used to assess the significance of intergroup differences. Group mean total food consumption over specified time periods (weeks 1-52; 53-104; 1-104; 105-112; 105-114) was calculated as the mean of all the cage food intakes in the group over that time period. The cage totals are a sum of the weekly cage mean food intakes. The weekly cage means are calculated using the amounts of food given to and left by each cage in each week and the number of animals surviving in the cage for the majority of days in the week. The cage mean food consumption figures are rounded to the nearest whole number, but the cage totals were calculated using the exact cage mean weekly figures. Group mean bodyweight change over specified time periods (weeks 0-52; 52-104; 104-112; 104-114) was calculated as the mean of all the individual weight changes of rats surviving throughout the time period. Analysis of organ weights was performed using analysis of covariance. Organ weights were adjusted for final bodyweight as covariate, where this was the more efficient method of analysis. Where appropriate, organ weights were log transformed to stabilise variance. Group means were compared using both Student's 't' test and Williams' test. Analysis by Kruskal-Wallis and distribution free Williams' test was performed where appropriate.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
One week before dosing commenced a number of male and female rats from all groups exhibited clinical signs of sialo-dacryo-adenitis. This is a viral disease which causes swollen salivary and Harderian glands, and it is not uncommon among young rots. After veterinary examination performed 2 days prior to the scheduled start of the study it was considered that the rats were in a suitable state of health to commence treatment, and by week 2, no rats in any group exhibited the clinical signs of sialo-dacryo-adenitis. Throughout the treatment period, no signs of ill, health or behavioural changes that could be related to treatment were noted. There was no indication of a treatment-related effect on the number of rats exhibiting palpable masses.
The distribution of deaths gave no indication of a treatment-related effect on survival either in the treatment or the withdrawal groups. The data show that the group receiving the highest dietary level of 450 ppm of the test substance had marginally fewer deaths than any other group. During the withdrawal period, mortality was similar in all groups. The pathological changes seen in decedents during either the treatment of withdrawal periods were those usually encountered in rats of this age and strain.

BODY WEIGHT AND WEIGHT GAIN
During the treatment period, bodyweight gains of treated rats were similar to those of the controls. During the withdrawal period there was one overall group mean loss of bodyweight which was essentially similar for treated and control rats. A bodyweight loss at this stage of a long term study is commonly recorded for Sprague-Dawley rats and reflects the approaching senescence of the surviving rats.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food intake for females receiving 450 ppm was 4 to 5 % lower than that of the controls during the study although the difference only attained a level of significance (P<0.05) during the first year of treatment. Food intake of other treated groups was essentially similar to that of the controls throughout the treatment period. During the withdrawal period, food intake of previously treated males was similar to control values, whereas previously treated females ate marginally more than their corresponding controls.
At weekly intervals the intake of the test compound was calculated from the group mean estimated mid-week bodyweight and food consumption. The mean group intakes during the treatment were as follows:

50 ppm: 1.8 (male) and 2.3 (female) mg/kg bw/day
150 ppm: 5.2 (male) and 6.9 (female) mg/kg bw/day
450 ppm: 15.4 (male) and 20.0 (female) mg/kg bw/day

FOOD EFFICIENCY
Efficiency of food utilisation was unimpaired by treatment.

ORGAN WEIGHTS
Although differences from control values in some organ weights attained statistical significance, these were not considered large enough to be of any biological significance.

GROSS PATHOLOGY
In both control and treated groups there was a higher incidence in female rats, in comparison to males, of subcutaneous masses and enlarged and haemorrhagic pituitaries. In both control and treated male groups, there was a higher incidence of cortical scarring of kidneys, in comparison to that of the female rats. These are normal findings related to sexually dimorphic patterns of senescence in the Sprague-Dawley rat. The incidence and distribution of macroscopic lesions suggestive of neoplasia were considered to fall within the expected background range of such lesions and therefore were considered unrelated to treatment with the test substance.

HISTOPATHOLOGY
No treatment-related changes were seen during histopathology. Spontaneous, usually age-related changes were recorded in many organs and tissues. Such changes were most frequently seen in the heart, lungs, thymus, liver, kidneys, gonads and endocrine glands. However, they showed no treatment-related trend on their incidence. All neoplastic changes observed in this study were neither treatment-related effects on the incidence of any tumour type nor on the total number of rats with tumours (benign or malignant) per group and there was no deviation from the expected tumour profile. Mammary tumours among females and pituitary tumours among males and females were the most commonly encountered tumours. The greater frequency of these two tumour types among female as compared to male rats is an expected sex-related feature. Although the incidence of pancreatic islet cell adenoma and adrenal phaeachromocytoma among male rats appears to be high, they are within the normal background range for males of this strain of laboratory maintained rats. For rats dying or killed in a moribund state, during this study the pathologist attempted to ascertain the major factor or factors in the cause of death or moribund state for each individual rat. In some instances more than one factor per animal was recorded. No factor or group of factors contributory to death were found to be associated with treatment. Subcutaneous tumours, mammary tumours, pituitary tumours and renal disease were among the most common cause of death. However, none of these factors showed any relationship to the treatment.

Dose descriptor:

NOAEL

Effect level:

ca. 15.4 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: highest tested dose

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Dose descriptor:

NOAEL

Effect level:

ca. 20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: highest tested dose

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

15.4 mg/kg bw/day

Study duration:

chronic

Species:

rat

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available key study is considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance does not need to be classified and labelled for carcinogenic toxicity under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EC.

 

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

The available key study is reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance does not need to be classified and labelled for carcinogenic toxicity under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation EC No 605/2014.

Additional information

Key study

The potential tumorigenicity of the test substance was examined in a 2-year carcinogenicity study with rats equivalent or similar to OECD guideline 451 (Huntingdon, 1982). After acclimatisation, four groups of 60 male and 60 female CD Sprague-Dawley rats were exposed to the test substance for 104 weeks at concentrations of 0 (control), 50, 150 and 450 ppm in diet. The ppm concentrations expressed mg/kg bw/d are as follows:

Group	Dietary level	Intake of test substance
		male	female
1	-	-	-
2	50	1.8	2.3
3	150	5.2	6.9
4	450	15.4	20.0

The stability of the test substance in diet was analytically verified. After 104 weeks the rats were maintained on untreated diet. The study was terminated when any one group (sexes were reviewed separately) reached the 20 % survival level. All male survivors were killed in week 113 and all female survivors in week 115. No treatment related effects were observed for clinical signs, body weight, organ weight, pathology and histopathology. Furthermore the distribution of deaths did not indicate any treatment related effect on survival. Efficiency of food conversion was unimpaired by the test substance. Overall food intake by females receiving 450 ppm of the test substance was slightly lower than that of the controls. Females receiving lower dietary levels and all treated male groups had food intakes similar to those of the control group. During the withdrawal period all female treated groups had a higher recorded food consumption than the controls. Among rats fed the test substance at a dietary level of 450 ppm, a slight lowering of food intake was recorded (5% lower than controls) among females only during the first year of the study. However this apparent difference in food intake did not affect the rate of bodyweight gain. None of the parameters assessed were disturbed by treatment. Therefore, it may be concluded that the test substance at a dietary level of 450 ppm equivalent to an average daily intake of 15.4 mg/kg bodyweight for males and 20.0 mg/kg bodyweight for females was not tumorigenic to rats.

Supporting study

Aim of the supporting study was to provide information on the possible health hazard likely to arise from repeated exposure to the test item (Centre d'explorations et de recherches medicales, 1977). The test substance was administered to one group of 20 Wistar rats/sex for 2 years via the diet at 100 ppm. The animals were observed closely for signs of toxicity and for the development of neoplastic lesions. All animals that died during the test or survived until the end were necropsied. A total of 110 animals (80 males and 40 females necropsied at the age of 2 years; 14 rats (sex not specified) received the same diet but no test item and served as control group. On the day of their scheduled necropsy, all survivors were killed, weighed, examined macroscopically and organs/tissues were processed for histopathological examinations. Information on haematological analyses was provided without further details. The test item was found not to be cancerogenic under the test conditions. Pathologic symptoms were not found more frequently in the treated group than in the control group.

Justification for selection of carcinogenicity via oral route endpoint:
Scientifically acceptable study report.