2,4-di-tert-butylphenyl 3,5-di-tert-butyl-4-hydroxybenzoate

Carcinogenicity

Description of key information

In a two year feeding study in rats no overt reactions to treatment with the test item at dietary levels up to and including 375.6 mg/kg bw/d were seen. No morphological abnormalities were seen in the sections examined which were considered to be related to the administration of the test substance. No effect on the spontaneous tumour incidence was seen.

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:

1975-02-27-1978-05-09

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 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: From Anglia Laboratory Animals (formerly Carworth Europe), Alconbury England, on 20 February 1975
- Age at study initiation: 28 +/- 1 day old
- Weight at study initiation: 70-90 g
- Diet and Water: Free access to tap water and powdered laboratory food, Spratt´s Laboratory Diet 2.
- Housing: Rats were housed 5 to a cage in suspended cages with wire-mesh floors

ENVIRONMENTAL CONDITIONS
- Temperature: 21 +/- 2 °C
- Humidity: 50 +/- 5 %
- Photoperiod: 12 hours light

Route of administration:

oral: feed

Vehicle:

other: diet

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Fresh diets were prepared and fed each week. A pre-mix containing 120,000 ppm was prepared and from this the various dietary concentrations were obtained by direct dilution with further quantities of diet, homogeneity being achieved by mixing for 10 minutes in a rotary double-cone blender. The diets were then stored until use in heat-sealed, opaque polythene bags. At the end of each week, any residual diet was discarded.

DIET PREPARATION
- Rate of preparation of diet (frequency): Each week
- Mixing appropriate amounts with: Spratt´s Laboratory Diet 2

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the diets fed to the rats were taken at 13 weekly intervals and sent to Ciba-Geigy ( U . K . ) Limited, Simonsway, Manchester, M22 5LB, for analysis of the test item content.

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

continously via diet

Post exposure period:

none

Dose / conc.:

2 500 ppm

Remarks:

corresponding to 93.4 mg/kg and 116.3 mg/kg in males and femals, respecitively

Dose / conc.:

5 000 ppm

Remarks:

corresponding to 185.4 mg/kg and 248.2 mg/kg in males and femals, respecitively

Dose / conc.:

10 000 ppm

Remarks:

corresponding to 375.64 mg/kg and 493.6 mg/kg in males and femals, respecitively

No. of animals per sex per dose:

50 females
50 males

Control animals:

yes

Details on study design:

Each animal was weighed and allocated to one of several arbitrary weight ranges, each containing rats with bodyweights differing from one another by no more than +/- 2.5 g. Equal numbers of animals from within each weight range were randomly allocated to each of the 4 treatment groups, and all animals then identified by earmark.

Positive control:

none

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
All sighs of ill-health or reaction to treatment together w i t h any behavioural changes, were recorded. Details of any skin lesions, cataracts and palpable growths, together with the progression or regression of such lesions were recorded. Any rat showing signs of severe debility or intoxication was isolated. Animals in extremis were sacrificed to prevent cannibalism.
All mortalities were recorded as date and nature of death. All rats found dead, or killed for humane reasons, were subjected to detailed macroscopic
examination in an attempt to define the cause of death. A full spectrum of tissue samples was preserved and macroscopically abnormal tissues were subjected to histological examination.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded initially and at weekly intervals for the first eight weeks and at two-weekly intervals thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The efficiency with which food was utilised was assessed by calculation of mean food conversion ratios (FCR values) during the period of fastest growth, i . e . weeks 1 to 26, as weights of food consumed per unit gains in bodyweight.
The quantity of food consumed by each cage of rats was recorded and the mean weekly intake calculated.

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

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- During weeks 6, 12 and 26, water consumption was recorded for a seven-day period for 5 cages of males and 5 cages of females from Groups 1 (Control) and 4 (10,000 ppm).

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced and during weeks 12, 26, 52, 78 and 108
- Dose groups that were examined: Control and Group 4 (10000 ppm)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During weeks 0, 6, 12, 25, 52, 77 and 102
- Anaesthetic used for blood collection: Yes: Light ether anaestesia from the orbital sinus
- Animals fasted: Yes, overnight
- How many animals: 10 males and 10 femals from the Control and Group 4 (10000 ppm)
- Paramters examined: Packed cell volume, Haemoglobin, Red cell count, Absolute indices, total white cell count, Differential count, platelet count, thrombotest.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During weeks 6, 12, 25, 52, 77 and 102
- How many animals: 10 males and 10 femals from the Control and Group 4 (10000 ppm)
- Animals fasted: Yes, overnight
- Parameters examined: Plasma urea, Plasma glucose, total serum proteins, serum protein electrophoresis and AG ratia, serum alkaline phosphatase, serum glutamic pyruvic transaminase, electrolytes

URINALYSIS: Yes
- Time schedule for collection of urine: During weeks 6, 12, 26, 52, 78 and 103
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes, overnight
- Parameters examined: pH, specific gravity, protein, reducing substances, glucose, ketones, bile pigments, urobilinogen, hemoglobin, microscopic examination of spun deposit.

Sacrifice and pathology:

GROSS PATHOLOGY
All superficial tissues, including the urinogenitol orifices and tail, each pinna, orbit and external 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 cervical subcutaneous structures. The nares, buccal cavity, tongue, pharynx and auditory region were then examined, and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection, all subcutaneous tissues were examined, including regional lymph nodes, mammary and salivary glands. 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. Stomach and caecum were incised and the mucosae scrutinised. The condition of the thoracic viscera was noted, with due attention to thymus and lymph nodes; the heart was incised and each chamber examined. The oesophagus, intestines and uterus were incised tp expose the mucosae and subjected to visual appraisal in toto. The lungs were removed and all pleural surfaces examined under suitable illumination, while the liver and kidneys were sectioned at intervals of a few millimeters; any lesion suggestive of neoplasia was noted, including details of location, size and multiplicity. Any abnormalities in the appearance and size of the gonads,adrenals, thyroids, intraabdominal lymph nodes and accessory reproductive organs were recorded., Any evidence of adhesion, invasion or other interaction between presumptive neoplasia and the adjacent structures was noted.

ORGAN WEIGHTS
The following organs from all animals were dissected free of fat and weighed: brain, adrenals, ovaries, liver, testes, uterus and kidneys. For inter-group comparison, relative organ weights were calculated as percentages of bodyweight, x 100.

HISTOPATHOLOGY
Samples of the following tissues from all animals were preserved in buffered 10% formalin (except eyes, which were preserved in Davidson's fixative):
adrenals, aorta*, brain, (for medullary, cerebellar, and cortical sections) caecum, colon, duodenum, eye, femur*, heart, ileum, jejunum*, kidneys, liver, lungs, lymph nodes, mammary gland, oesophagus, ovaries, pancreas, pituitary, prostate, salivary glands, sciatic nerve*, second eye*, seminal vesicles, skeletal muscle*, skin*, spleen, stomach, testes, thymus, thyroids, tongue*, trachea*, urinary bladder, uterus

Tissues marked with * were preserved but not processed further in the first instance. Blood smears and sternum bone marrow sections were prepared. Ali nodules, tissue masses and otherwise macroscopically abnormal tissues were routinely preserved, along with samples of adjacent tissues whereappropriat.

Tissues required for microscopic examination were embedded in paraffin wax and sections cut at 5µ; the latter were stained with haematoxylin and eosin. In addition, frozen sections of liver and kidney were stained for fat with Oil Red O and with periodic acid Schiff reagent for glycogen (liver) or basement membranes kidney. The microscopic examination consisted of the following steps:
(i) Where possible, all tissues indicated in the list above, were examined from ail rats from each group.
(ii) All macroscopically observed lesions suggestive of neoplasia from every rat.
(iii) All macroscopically abnormal tissues from decedents in an attempt to ascertain cause of death.

Statistics:

Students ' t ' test and analysis of variance were employed to assess the significance of inter-group differences.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no visible signs of reaction to treatment with the test item. Areas of alopecia in various parts of the body were reported in a number of animals during the course of the study from both control and treated groups and this finding was not considered related to administration of the test substance. In a proportion of animals from each group, brown staining of the fur, particulariy on the head and neck region, and excrescences on the tail were recorded. This is a normal finding usually seen with gang housed rats and the incidence of these findings showed no treatment related distribution.

Dermal irritation (if dermal study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no marked differences between the distribution of mortalities in control and treated groups during the study that could be attributed to treatment

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no significant differences in bodyweight gain among rats receiving the test item when compared with that of the controls. The fluctuation in mean bodyweight gain during the last 3 months of the study reflected the approaching senescence of the rats associated with the effect on mean bodyweight resulting from mortality in a rapidly decreasing population.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food intakes were similar among control and treated rats throughout the duration of the study.

Food efficiency:

no effects observed

Description (incidence and severity):

Calculation of food conversion ratios during the first 26 weeks of the study showed that the efficiency with with food was utilised was unimpaired in rats receiving the test item.

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

no effects observed

Description (incidence and severity):

Water intakes recorded during weeks 6, 12 and 26 were similar in controls and rats receiving the test substance at 10000 ppm. The marginally higher values recorded at week 6 for males receiving 10000 ppm, although attaining a level of statistical significance when compared with the control values, were considered to be of no toxicological significance as all vaiues were within the 'nonmal' range for the age and strain of rat employed.
In the absence of a treatment-related effect on water intake during the first 26 weeks of the study and in the absence of any change noted by visual appraisal of the water bottles, further measurements were not made.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No abnormalities of the eyes were detected that were considered to be related to treatment with the test item.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant increase in neutrophil count was recorded during week 6 among females receiving the test item at 10,000 ppm and at week 78 in moles receiving 10,000 ppm. At the week 102 investigation a high neutrophil count was recorded for 1 male receiving 10,000 ppm. As no similar findings were recorded in either sex at any of the other investigations, this finding was considered of doubtful toxicological significance. Low values for red cell parameters were recorded at week 78 for 1 rat (10,000 ppm) and at week 102 for another rat (10,000 ppm). These findings were not considered to be related to treatment, as values for one animal were within the 'normal' range at the week 102 investigation. Other small differences between values for control and treated rats, although in some cases attaining a level of statistical significance, were considered not to be related to treatment as ail values were within 'normal' limits for the strain and age of rat employed.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A marginally higher SAP value was recorded in week 12 for males receiving the test item at 10,000 ppm. As no similar increase was recorded for female rats receiving the same treatment and the increase was of a small order of magnitude this finding was considered to be of doubtful toxicological significance. SAP values at the subsequent investigations were similar or marginally lower than controls in rats receiving 10,000 ppm. The higher sodium and lower potassium levels recorded at week 25 for males receiving 10,000 ppm were not recorded at subsequent investigations and the finding was, therefore, considered to have arisen fortuitously. Other small differences between vaiues for control and treated rats were not considered to lae of toxicological significance as oil values were within the accepted range for rats of the age and strain employed.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no differences in urinary parameters among control or rats treated with the test item at 10,000 ppm that were considered to be of toxicological significance. Urinalysis performed at week 102 revealed the presence of haemoglobin in the urine of 3 out of 9 male controls and 9 out of 10 males receiving the test item at 10,000 ppm. Further examination performed during week 104 showed the following incidences of rats with haemoglobin in the urine: 6 out of 9 control males, 8 out of 9 males receiving 2,500 ppm and 8 out of 10 males receiving 5,000 ppm. Hoematuria is a relatively common finding in aged Sprague-Dawley rats of this strain and, therefore, this finding was considered to be unrelated to treatment. Other small differences attaining a level of statistical significance were not seen consistently and were considered to have arisen fortuitously.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no differences in organ weight values of control and treated rots that could be attributed to treatment with the test item.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic findings at autopsy were common to animals from control and treated groups and no marked increases in incidence of any pathology were recorded. In particular, there was no obvious evidence of an increase in the number of lesions suspected of being neoplastic.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Heart: Changes in a proportion of rats from control or treated groups included the following changes: myocardial fibrosis, myocardial degeneration, inflammatory ceil infiltration, thrombus-emboii and mineralisation. These changes were considered to be unrelated to treatment.

Lungs: No treatment-related changes were seen. Changes seen in a proportion of rats from control or treated groups included: perivascular, peribronchial and peribronchiolar lymphoid aggregations, mineralisation, venous congestion, oedema, alveolar emphysema, inflammatory changes, fibrosis, accumulations of foam cells, cholesterol clefts, adenomatosis, foci of osteoid tissue, thrombi and haemorrhage infarction.

Liver: Changes present in a proportion of rats from control and treated groups included: vacuolation of hepatocytes, regressive changes, hyperplasia, inflammatory cell inifltration, bile duct proliferation, fatty change, ectosis, venous congestion, haemorrhage, parasitic cysts and glycogen depletion.
The hepatic changes listed above ore commonly seen in laboratory rats of this age, and they were considered to be of no toxicological significance.

Urinary system: Progressive glomerulonephrosis, often marked in degree, was observed in a proportion of animals from the control and treated groups, being more frequent in moles than in females. This finding was characterised by glomerular abnormalities, atrophic or cysticaliy dilated nephrons, often containing eosinophilic colloid and interstitial fibrosis with foci of mononuclear cell infiltration. No treatment-related effect was detected in the degree and incidence of this change. Additional findings in the kidneys included: mineralisation, basophilic tubules, fatty change, nephrocalinosis, glomerulonephritis, purulent nephritis, purulent pyelitis , other inflammatory changes, hyperplasia of pelvic epithelium, hydronephrosis, cysts, dilated tubules, haemorrhage, infarction, necrosis and pigmentation. Changes present in the urinary bladder included: epithelial hyperplasia, papillary folding of the mucosa, calcul i , inflammatory changes, haemorrhages and a polyp. In the ureter of one rot there was a loss of epithelium due to formation of thrombi associated with inflammatory changes. The additional changes in the kidneys and the changes in the urinary bladder and ureters are commonly seen in rats of this age and, as no treatment-related effect was observed, they were considered to be of no toxicological significance.

Endocrine glands
Adrenals: Changes present in a proportion of rats from control or treated groups included: vacuolated/distended cells, degeneration, hyperplasia, venous congestion and haemorrhage.

Thyroids: Changes present in the thyroids included: parafollicular cell hyperplasia, large follicles and ultimobranchial bodies. Parathyroids: Hyperplasia was present in a small number of rats.

Pituitary: Changes present in the pituitary included: hyperplasia, venous congestion, haemorrhage and vacuolated cells. The changes of the endocrine glands are commonly seen in rats of this age and, OS no group-related effect was observed, they were considered to be of no toxicological significance.

Reproductive system: Changes were seen in a proportion of rats from both control or treated groups. The following changes were observed: Males: Changes present in the testes included: atrophic changes, mineralisation, interstitial cell hyperplasia, peri-arteritis nodosa, necrosis, haemorrhage, venous congestion and spermatocele granulomata. Additional changes included: inflammatory changes in the prostate; atrophy, dilatation , increase in seminal plasma and inflammatory changes in the seminal vesicles; dilatation of ducts and inflammatory changes in the preputial glands; inflammatory changes in the coagulation glands. Females: Cysts were found in the ovaries. In the uterus, hyperplasia, dilatation/distension of lumen, cysts, polyps, metaplasia, necrosis and inflammatory changes were recorded. None of the changes in the male or female reproductive tracts listed above were considered to be of toxicological significance.

Mammary tissue: Mammary hyperplasia and galoctocele formation were present in a proportion of female rats and in a small number of male rots from control and treated groups. Inflammatory changes and fibrosis were seen in a small number of animals. No treatment-related effect was seen in the incidence and distribution of any of these changes.

Gastro-intestinal tract Stomach: The following changes were recorded in a small number of animals: erosions, ulceration, oedema, inflammatory changes, mineralisation and epithelial hyperplasia. Intestine: Changes present in a small number of animals included: oedema, mineralisation, ulceration, inflammatory changes and nematodiasis. In the gastro-intestinal tract, none of the changes listed above were considered to be of toxicological significance.

Other morphological changes seen in a number of rats from control and/or treated groups, also considered to be of no toxicological significance were as follows: blood vessels: peri-arteritis nodosa, mineralisation, aneurysmal dilatation, thrombus/thrombus emboli and bacterial emboli. aorta: mineralisation. spleen: extramedullary haemopoiesis, inflammatory changes, hyperplasia, atrophy and bacterial emboli. pancreas: inflammatory changes, atrophy, peri-arteritis nodosa, aneurysmal dilatation and a nodule undetermined due to post mortem autolytic changes. adipose tissue: necrosis, inflammatory changes and cysts, lymph nodes: venous congestion, inflammatory changes, hyperplasia, haemorrhage and dilatation of sinusoidal spaces. thymus: involution and haemorrhage, salivary gland: inflammatory changes, mesentery: inflammatory changes and necrosis. cutaneous tissue: ulceration, hyperplasia, inflammatory changes, fibrosis, abscesses, cysts, dilatation of sebaceous gland ducts and reduction of hair follicles, subcutaneous tissue: necrosis, inflammatory changes, haemorrhage, epidermoid cysts, cyst-like spaces, fibrosis and abscesses, brain: dilatation of ventricles, haemorrhage, necrosis, mineralisation and ossification, bone marrow: hypoplasia, eyes: inflammatory changes and lenticular degeneration, pinnae: inflammatory changes and epidermal hyperplasia.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There was no evidence to suggest that the administration of the test item was associated with the disturbance of the spontaneous tumour profile in the strain of rat employed. The following comments are made in summary:

Endocrine glands: The largest incidence of tumours in this category occurred in the pituitary gland, and this tumour was seen in rats from both control and treated groups. Pituitary gland tumours are commonly seen in laboratory rats of this age, and there was no evidence of a treatment-related change. The majority of tumours seen were pituitary gland adenomata. The incidence of tumours in other endocrine glands showed no evidence of a treatment-related change.

Cutaneous and subcutaneous tissue: There was no evidence of a treatment-related effect in the incidence of tumours in cutaneous and subcutaneouslocations.

Lymphoreticular system: The low incidence of tumours of lymphoreticular origin showed no evidence to suggest a treatment-related effect.

Liver: One malignant and one benign liver cell tumour were recorded. This was within the expected range for this strain of rat in our laboratory.

Reproductive system: The only neoplasms recorded in male rats were three testicular interstitial cell tumours in control rats. Among female rats, the tumours recorded were five ovarian adenomas, one granulosa ceil tumour, one uterine adenocarcinoma and one uterine fibrosarcoma. These tumours of the reproductive system are not uncommon in the laboratory rat and were considered to be of no toxicological significance.

Mammary tissue: No treatment-related effect was seen in the incidence of the fibromas, fibroadenomas, adenomas and adenocarcinomas recorded in this study.

Miscellaneous tumours: The individual tumour types and the incidence of these neoplasms were within the normal range for the rats used in our laboratories.

Total tumour incidence: The total number of rats bearing neoplasms was similar in treated and control groups.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

375.6 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No morphological abnormalities were seen in the sections examined which were considered to be related to the administration of the test substance. No effect on the spontaneous tumour incidence was seen.

Key result

Dose descriptor:

NOAEL

Effect level:

493.6 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No morphological abnormalities were seen in the sections examined which were considered to be related to the administration of the test substance. No effect on the spontaneous tumour incidence was seen.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

376 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Comparable to guideline requirements and scientifically valid, no effect level measured.

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

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

The available experimental test data are also reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/218. 

Additional information

The objective of the available key chronic carcinogenicity study was to assess the toxicity and tumorigenicity of the test item, when administered in the diet to rats for a period of 104 weeks. The study is similar to the OECD Guideline 451. The animals were exposed to dietary concentrations of 2500, 5000 and 10000 ppm (93.4, 185.4 and 375.6 mg/kg in males and 116.3, 248.2 and 493.6 mg/kg in females). Sprague-Dawley rats with 50 females and 50 males were tested. The route of administration was by admixture with the diet. There were no overt reactions to treatment with the test item at dietary levels up to and including 375.6 mg/kg bw/d for males and 493.6 mg/kg bw/d for females. No morphological abnormalities were seen in the sections examined which were considered to be related to the administration of the test item. No effect on the spontaneous tumour incidence was seen.

In the available supporting study the test substance was administered to one group of 20 Wister 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. Animals which died or were killed during the test were necropsied and, at the conclusion of the test, all surviving animals were killed and necropsied. A total of 134 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 carcinogenic under test conditions. Pathologic symptoms were not found more frequently in the treated groups than in the control group. A NOAEL of ca. 8 mg/kg bw/d was calculated.