Pentaerythritol tetrakis(3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate)

Carcinogenicity

Description of key information

The substance was found to be non-carcinogenic in a valid carcinogenicity feeding study in mice and in a valid combined chronic toxicity/carcinogenicity feeding study in rats (Huntingdon, 1974). Both studies were performed prior to GLP and OECD testing guideline introduction but are adequate in design and reporting detail. The highest tested doses were 1000 and 10000 ppm for mice and rats, respectively.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1978-06-06, 1980-06-09

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

yes

Remarks:

see below

Principles of method if other than guideline:

Deviations: No careful clinical examination. Missing peripheral nerve and sternum with bone marrow although bone marrow alone was analyzed. No summary table containing non-neoplastic lesions, Analysis procedures/methods not reported. No analysis for homogeneity. Accuracy of preparation deviates more than 20% (7 times in total at all dose levels (4 times at 300 ppm)).

GLP compliance:

no

Species:

mouse

Strain:

other: Tif:MAGf, SPF-bred

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Production Stein, CIBA-GEIGY Ltd.
- Age at study initiation: Approx. 4 weeks
- Weight at study initiation: 24.1 - 24.5 g (males), 21.4 - 22.0 g (females)
- Housing: Groups of 5 in Macrolon cages type 3 with standardised granulated soft wood bedding (societe Parisienne des sciures Pantin)
- Diet (e.g. ad libitum): Pelleted, certified standard diet Nafaq No. 890 Tox, ad libitum (except as noted under Laboratory Investigations).
- Water (e.g. ad libitum): Ad libitum, tap water quality controlled
- Acclimation period: 7 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 10
- Air changes (per hr): 15 - 17
- Photoperiod (hrs dark / hrs light): 14/10

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: admixed to pelleted food

DIET PREPARATION
- Rate of preparation of diet (frequency): Appr. monthly
- Mixing appropriate amounts with (Type of food): Pulverised food (pelleted, certified standard diet Nafag No. 890 Tox) homogeneously mixed with the appropriate concentrations of the compound with 30 % water. The pellets were subsequently airdried.
- Storage temperature of food: Room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance is extracted with distilled chloroform 20 g of each powered sample and control sample for 3 hours in a Soxhlet apparatus. The additive is then determined by liquid chromatography
Sampling times: pretreatment samples (actual concentrations and stability) and regularly during the study (actual concentrations) in duplicate

Duration of treatment / exposure:

24 months / continuous

Frequency of treatment:

Daily

Post exposure period:

1 month

Dose / conc.:

100 ppm

Dose / conc.:

300 ppm

Dose / conc.:

1 000 ppm

No. of animals per sex per dose:

50

Control animals:

yes, plain diet

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily (a.m. and p.m. on working days)

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly, after 3 months monthly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Weekly, after 3 months monthly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

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: No
- Calculated according to: Weekly food consumption (g)/midweek body weight (g) X 1000/7. Unit: g food/kg body weight/day

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Brain (cerebrum, cerebellum, brainstem), spinal cord, eyes, pituitary, salivary gland, heart, thymus, thyroid, aorta, lung, trachea, spleen, bone marrow, lymph nodes (axillary and mesenteric), sciatic nerve, oesophagus, stomach, small and large intestine, adrenal glands, pancreas, liver, gall bladder, kidneys, urinary bladder, ovaries, testes, prostate, epididymis, seminal vesicle, uterus, skin (mammary area), skeletal muscle, organs and tissues showing macroscopical changes

HISTOPATHOLOGY: Yes. Samples from the organs above (control and test mice) were embedded in paraplast, sectioned at 3-5 μm, and stained with haematoxylin and eosin.

Other examinations:

Organ weight: Liver, adrenals, brain, heart, kidneys and testes (for animals surviving until scheduled sacrifice)

Statistics:

Wilcoxon test, Savage test

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality:

not specified

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The analysis of the body weight data revealed a slight but significant depression of the body weight in female groups 3 and 4 (300 and 1000 mg/kg feed). Though statistically significant, this depression is of minor biological importance since it is within a range of about 10 %. In male groups, the analysis of the body weight data revealed no statistically significant intergroup differences. The mean body-weight gain of all treated male groups was similar to that of the control.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The mean food consumption values of all treated male and female groups were generally similar to those of the controls. Statistical analysis of food consumption data revealed transient depressions in food consumption of female groups 3 and 4 (300 and 1000 mg/kg feed) most prominent during week 53, 57, and 84. These erratic values could not be interpreted.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Statistical analysis of the organ weight data and ratios revealed the following significant differences to the respective control group:
- Increase of the adrenal weight in males of groups 2, 3 and 4 (100, 300, 1000 mg/kq feed) and in females of group 3 (300 mg/kg feed).
- Increase of brainweight in female groups 3 and 4 (300 and 1000 mg/kg feed).
Such slight variations are common in aged animals and are considered to be unrelated to the treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Organ weights, organ to body weight and organ to brain weight ratios revealed marked intra and intergroup variations. These variations are common in aged animals. No experimental significance whatsoever is attributed to the slightly increased mean weights and ratios of adrenals in treated male mice and of brain in female mice from the top and intermediate concentration groups. Also the histopathological findings in the adrenals and brain between treated and control animals were similar.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Lesions and changes found in some control and test animals and described as congenital, degenerative or inflammatory in nature are attributed to naturally occurring diseases which are common in aged mice of this breeding colony

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Numerous benign and malignant tumours were observed in both control and treated mice. Frequency and type of the neoplasms occurring in these animals were not influenced by the treatment.

Other effects:

not examined

Dose descriptor:

NOEC

Effect level:

>= 1 000 ppm (analytical)

Sex:

male/female

Basis for effect level:

other: no effects at the highest dose level

Remarks on result:

other: Corresponds to 126 and 107 mg/kg bw per day for males and females, respectively.

Dose descriptor:

NOEC

Effect level:

>= 1 000 ppm (analytical)

Sex:

male/female

Basis for effect level:

other: no effects at the highest dose level

Remarks on result:

other: Corresponds to 126 and 107 mg/kg bw per day for males and females, respectively.

Dosage Levels: the amount of test material in the diet was determined periodically during the course of the study. The results of these analyses revealed a concentration of 68-190 % of the nominal value.

Sample	Day of test	Analytical results (mg/kg feed)
		Nominal 0 (control)	Nominal 100	Nominal 300	Nominal 1000
1	38-72		66/69	223/235	666/659/796
2	71-113		105/106	302/312	1080/1072
3	114-154		91/98	271/258	961/925
4	182-213		97/86	320/302	966/1054
5	250	10	87/94	272/282	924/906
6	310	5	90/84	277/268	963/983
7	400	5	95/94	303/289	936/881
8	484	10	95/89	367/370	655/627/604/628
9	532	10	84/82	588/551	882/830
10	613	10	84/97	439/427	1080/1006
11	704	15	123/132	392/409	1134/1074

Conclusions:

The substance is not carcinogenic in mice upon life-long feeding a diet containing 100, 500 and 1000 ppm of the test item.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1972 - 1974

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Feed analysis was performed, but no details for accuracy of preparation, homogeneity and stability were reported. Limited biochemical parameters measured. Non-GLP. Histopathological examinations as described in the summary were performed 10 years after the finalisation of the study. Lymphoid aggregates in the lungs reported in the first histopathology report were no longer reported in the second report.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

Urinalysis: missing volume and density. Blood chemistry: missing total protein; albumin, glutamic oxalacetic transaminase, g-glutamyl transpeptidase, and ornithine decarbossilase. Pathology: missing salivary glands, oesophagus, peripheral nerve, and s

GLP compliance:

no

Species:

rat

Strain:

other: CFY, a hysterectomy-derived strain of Sprague-Dawley origin

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Carworth Europe, England
- Weight at study initiation: 100 - 150 g (male); 73 - 132 g (female)
- Housing: 5 in suspended metal cages fitted with wire-mesh floors
- Diet (e.g. ad libitum): Ad libitum, Spratt's Laboratory Animals Diet No. 2.
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 7 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 5
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
A premix containing 60000ppm test material was prepared each week, and from this the different dietary concentrations were obtained by direct dilution with further quantities of diet. Homogeneity was achieved by mixing for 10 m in a rotary double-cone blender; all diets were stored until use in heat-sealed opaque polythene bags.

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Powdered Spratt's Laboratory Animals Diet No. 2
- Storage temperature of food: Room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the diet fed to the rats have been analysed for their content of test substance by a thin layer chromatographic method. The estimated relative reproducibility of this method is ± 10 % or better. Within the limits of error of the sampling technique and of the analytical method, there was good correspondence between the concentrations found in the diets and the nominal values.

Duration of treatment / exposure:

two years

Frequency of treatment:

continuous

Post exposure period:

none

Dose / conc.:

1 000 ppm

Remarks:

corresponding to 45-55 mg/kg bw

Dose / conc.:

3 000 ppm

Remarks:

corresponding to 135-166 mg/kg bw

Dose / conc.:

10 000 ppm

Remarks:

corresponding to 446-547 mg/kg bw

No. of animals per sex per dose:

50

Control animals:

yes, plain diet

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked: Mortality and all signs of ill-health and toxicity, and behavioural changes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All signs of ill-health and toxicity, together with any behavioural changes; skin lesions; cataracts or palpable growths; together with the progression or regression of such lesions. Any rat showing signs of severe debility or intoxication was isolated.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly for the first 12 weeks and at 4-week intervals thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Weekly
-The quantity of food consumed by each cage of rats was recorded and the mean weekly intake calculated

FOOD EFFICIENCY:
- Calculation of mean food conversion ratios (FCR values) during the period of fastest growth, a s weight of food consumed per unit gain in bodyweight.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 13, 26, 52, 78, and 103 w
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 males and 10 females from Groups 1 (Control) and 4 (I0000 ppm)
- Parameters checked: Packed cell volume (PCV); Haemoglobin (Hb); Red cell count (RBC); Mean corpuscular haemoglobin concentration (MCHC) and mean cell volume (MCV); Total white cell count (WBC); Differential count (Neutrophils, Lymphocytes, Eosinophils, Basophils, and Monocytes)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 13, 26, 52, 78, and 103 w
- Animals fasted: Yes, overnight
- How many animals: 10 males and 10 females from Groups 1 (Control) and 4 (I0000 ppm)
- Parameters checked: Plasma Urea nitrogen; Plasma Glucose; Serum alkaline phosphatase (SAP); Serum glutamic-pyruvic transaminase (SGPT)

URINALYSIS: Yes
- Time schedule for collection of urine: After 13, 26, 52, 78, and 103 w
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked: pH; Specific gravity (SG); Protein; Reducing substances; Glucose; Ketones; Bile pigments; Urobilin; Blood pigments; Microscopy of spun deposit

OTHER: On completion of the treatment period, all surviving rats were killed by carbon dioxide euthanasia and subjected to autopsy

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see below)
HISTOPATHOLOGY: Yes (see below)

Statistics:

Student's 't' test was employed to assess significance of intergroup differences where the data suggested a treatment-related response.
Differences in tumour incidence were compared by a 2 x 2 contingency test used as a one-tailed test, computing the exact probability of the observed tumour distribution occurring or one which is more extreme.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no overt signs of reaction to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The survival rates among treated rats were comparable with those of the controls.
Mortality and time to death: 31/50, 26/50, 28/50 and 24/50 for males and 29/50, 30/50, 33/50 and 30/50 for females at 0, 1000, 3000 and 10000 ppm, respectively. No relationship between dose and time of death.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

During the first 24 weeks of treatment, bodyweight gain of treated rats was comparable with that of the controls. Between weeks 24 and 52, bodyweight gain of males receiving test material at 10000 ppm was significantly lower than that of the controls (P<0.05), although subsequent performance was comparable with that of the controls. Between weeks 80 and 104, bodyweight gain of males receiving 1000 ppm was significantly higher than that of the controls, although since this finding was not dose related in degree it is of doubtful biological significance. Bodyweight gains among other treated groups were not disturbed by dietary administration.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

With the exception of a small decrease In the total mean food Intake among all treated rats between weeks 53 and 80, food Intake was not disturbed by treatment.

Food efficiency:

no effects observed

Description (incidence and severity):

No treatment-related change in the efficiency of food utilization was recorded during the period of fastest growth i . e . the first 24 weeks of treatment.

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No changes attributable to treatment were observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Although a small number of differences between controls and rats receiving 10000 ppm attained a level of statistical significance during the course of the study, all values obtained were within normal limits for the strain of rat employed. Therefore, it was concluded that treatment with the test item had no effect on the haematological parameters measured.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At week 13, a decrease in the mean urea nitrogen level and an increase In the mean glucose level were recorded in male rats receiving the test item at 10000 ppm, although values for these parameters in females receiving a similar dosage level were comparable with those of the control females. However, the Individual values In treated male rats were within the 'normal' range for the age and strain of rat employed and the differences were not considered to be of biological significance. No intergroup differences, attributable to treatment, were recorded at week 26.
The marginal Increase In SAP and SGPT values at week 52 for female rats receiving 10000 ppm; the marginal Increase In the SGPT values In treated males at week 78, and the marginal decrease In the SAP values In treated males at week 103, although attaining a level of statistical significance were considered to be of no biological significance. It was concluded that treatment with the test item was without effect on the blood chemistry parameters measured.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No evidence of treatment-related change was seen in investigations made at weeks 13 and 26. Urinalysis carried out after 52 and 78 weeks of treatment revealed a small increase In the alkalinity of the urine excreted by male rats receiving the test item at 10000 ppm, although the values obtained were within the 'normal' range for the strain of rat employed. Values among female rats receiving 10000 ppm were comparable with those of the controls. No evidence of treatment-related change was seen In the Investigations made at week 103.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Organ weight analysis performed on rats killed after 104 weeks of treatment revealed no evidence of a treatment-related effect on organ weight. Although differences from control values in adrenal, thyroid and kidney weights attained
levels of statistical significance, the differences were related to the intergroup disparity in bodyweight and were not dose related in degree. It was, therefore, concluded that the differences were of no toxicological significance and probably arose fortuitously.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The prime cause of death among rats dying during the course of the study showed no relation to treatment. Macroscopic examination of decedents and rats killed after 104 weeks of treatment showed pathology which was common to animals from control and treated groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

GENERAL HISTOPATHOLOGY OF DECEDENTS
- Lung
chronic respiratory disease, characterized by congestion, peribronchiolar and perivascular lymphoid aggregates or hyperplasia, and aggregates of alveolar macrophages, was common to a number of rats from control and treated groups. Bronchopneumonia was observed In rat 50♂ (Control) and interstitial pneumonitis In rats 11♂ (Control) and 131♂ 3000 ppm).

- Liver
Changes In the liver common to some rats from control and treated groups consisted of varying degrees of hepatocytlc vacuolation, occasional aggregates of chronic Inflammatory cells, focal areas of necrosis, minimal bile duct hyperplasia ard minimal periportal fibrosis. A hyperplastic nodule was observed in rat 199♂ (10000 ppm).

- Kidney
Varying degrees of progressive glomerulonephrosis, characterised by cystically dilated nephrons, Interstitial fibrosis, lymphocytic infiltration and associated glomerular changes, were seen In the kidneys of the majority of rats from control and treated groups. A haematoma was observed In the kidney of rat 488♀ (3000ppm).

- Urinary bladder
Eosinophilic cases were observed In the urinary bladder of rats 25♂ (Control) and 82♂ (1000 ppm) and slight hyperplasia of the transitional epithelium of rat 135♂ (3000ppm).

- Cardiovascular svstem
A minor degree of myocarditis and myocardial fibrosis was seen in rats 4♂ and 385 (Control), 115♂ (3000ppm) and 173♂ (10000ppm), myocardial mineralization in rat 56♂ (1000ppm) and mineralization of the coronary arteries In rat 115♂ (3000ppm), an auricular thrombus In rat 438♀ (1000ppm), mineralization of the aortic media in rats 4♂, 18♂ (Control), 79♂ (1000ppm), 155♂ and 513♀ (1000ppm). Aneurysms with or without thrombus formation were observed In the blood vessels of rats 68♂, 70♂, 419♀ and 432♀ (1000ppm), 116♂ (3000ppm) and 186♂ (1000ppm) and an aneurysm with perl-arteritic change In rat 545♀ (1000ppm).

- Reproductive system
Atrophic changes In the testes were common to the occasional male rat from both control and treated groups and an area of haemorrhage was observed in the testis of rat 164♂ (10000ppm). Distended or enlarged seminal vesicles were observed in rats 11♂ and 36♂ (Control), prostatitis andminimal squamous metaplasia In rat 79♂ (1000ppm) and cellular Infiltration of the prostate of rat 32♂ (Control). Cysts were observed In the ovaries of a proportion of rats from control and treated groups. Endometritis in rats 420♀ (1000ppm), 517♀ and 540♀ (10000ppm); pyometritis in rat 417♀ (1000ppm); uterine congestion in rat 428♀ (1000ppm) and endometrial polyps In rats 417♀, 420♀, 423♀ and 427♀ (1000ppm), 459♀ and 473♀ (3000ppm).

- Adrenal
Vacuolation of zona fasciculata cells and moderate to severe cystic degeneration of cortical cells and haemorrhage were common to a number of rats from both control and treated groups. Minimal cortical hyperplasia In rat 432♀ (1000ppm), a hyperplastic nodule In the zona fasciculata 417♀ (1000ppm) and focal hyperplasia In rat 482♀ (3000ppm). The above changes are commonly found In the laboratory rat and were considered to be without toxicological significance.


GENERAL HISTOPATHOLOGY OF RATS KILLED AFTER 104 WEEKS OF TREATMENT

- Trachea
A proportion of animals from both the control and treated groups had small aggregations of chronic Inflammatory cells beneath the respiratory epithelium.

- Lung
The majority of animals from both treated and control groups showed evidence of minimal chronic respiratory disease. The morphological changes encountered Included the followlng:
A minimal degree of peribronchiolar and perivascular lymphoid hyperplasia, and occasional parenchymal collections of alveolar macrophages. Rat 15♂ (Control) had small areas of chronic Interstitial pneumonitis and rat 157♂ (10000ppm) had a moderately large area of bronchopneumonia. These changes are commonly encountered In the laboratory rat and were considered to be of no toxicological significance.

- Liver
No significant group related changes were Identified. Many animals from both the control and treated groups showed varying degrees of hepatocytic vacuolation. In addition, a proportion of animals from both groups showed minimal bile duct hyperplasia, minimal periportal fibrosis and occasional small parenchymal aggregations of chronic Inflammatory cells. Rats 174♂, 176♂ (10000ppm) had occasional small groups of hepatocytes with altered cytoplasmic staining and rat 165♂ (10000ppm) contained numerous small aggregations of hyperplastic hepatocytes. These findings are not uncommon In the aged laboratory rat and were considered to be without toxicological significance.

- Kidney
Varying degrees of progressive glomerulonephrosis were seen in the kidneys of the majority of rats from both control and treated groups. These changes consisted mainly of cystically dilated nephrons with interstitial fibrosis, moderate lymphocytic infiltrations, and occasional glomerular changes. These findings are an age-related change in the laboratory rat and were
therefore considered to be of no toxicological significance. Rat 15♂ (Control) contained moderately large cystic structures In Its kidneys. This change in a single control animal was without significance.

- Cardlovasular svstem
A minor degree of myocarditis and myocardial fibrosis was seen In the ventricles of a small proportion of animals from both the control and treated groups. In rat 28♂ (Control) dystrophic mineralization was noted in the media of the aorta and In the blood vessels surrounding the sciatic nerves, and minimal hyaline degeneration was seen In the testicular arteries. In rat 158♂ (10000ppm) the pancreatic blood vessels were dilated and contained organised thrombi and the testicular blood vessels showed minimal perlarterltlc change. These changes In the vascular system are age-related changes commonly found In the laboratory rat, and were therefore considered to be of no toxicological significance.

- Pancreas
A few animals from both groups showed changes which Included minimal atrophy and loss of zymogen granules in the exocrine portion and minimal fibroblastic reaction In and around occasional islets of Langerhans. Rats 176♂ and 505♀ (10000ppm) each had a small nodule of hyperplastic exocrine tissue. These changes are commonly encountered In old rats and were considered to be without toxicological significance.

- Reproductive system
The following changes were seen and were considered to be of no toxicological slgnlflcance: Testicular atrophy In rats 26♂ (Control), 160♂ and I76♂ (10000ppm); degenerative changes In the tubular epithelium of the epididymis in rat 26♂ (Control). Prostatitis in rats 2♂, 24♂ (Control), 158♂, 161♂, 174♂, (10000ppm); ovarian follicular cysts In rats 351♀ (Control), 509♀ (10000ppm); Uterine polyps in rats 351♀, 362♀, 365♀ (Control); endometritis in rats 511♀, 512♀ (10000ppm); galactoceles In the mammary tissue of rat 359♀ (Control).

- Adrenals
Changes, which consisted of vacuolation of zona fasciculata cells and moderate to severe cystic degeneration of cortical cells with haemorrhage were encountered in a proportion of animals from both treated and control groups. Rat 157♂ (10000ppm) contained a small area of cortical hyperplasia, and rat 504♀ (10000ppm) had a small cyst-like structure with a fibrous capsule attached to one adrenal. These changes are commonly found In the old laboratory rat and were considered ta be without significance.

Other morphological abnormalities which were seen but not considered to be of toxicological importance Included: small areas of necrosis or haemorrhage In the brains of rats 2♂, lO♂, 23♂ (Control); minimal degenerative changes In the pituitary of rats 157♂, 158♂ (10000ppm) and a small cyst In the pars Intermedia of rat 167♂ (10000ppm); minimal hyperplasia of the parathyroids of rats 2♂, 23♂, 28♂ (Control); small aggregations of chronic Inflammatory cells In the skeletal muscle of rat 368♀ (Control); aggregations of inflammatory cells in the submucosa of the stomachs of rats 2♂ and 362♀ (Control); aggregations of acute and chronic Inflammatory cells In the submucosa of the small intestine of rat 23♂ (Control); small vacuoles with minimal myellnophage activity In the spinal cord or cauda equina of rats l0♂, 351♀, 354♀, 360♀, 362♀, 373♀ (Control), 162♂, 507♀, 508♀, 509♀, 516♀ (10000ppm); minimal perivascular gliosis In the spinal cord of rat 2♂' (Control) and occasional haemorrhages In the spinal cord of rat 15♂ (Control); an epidermoid cyst In the subcutaneous tissues of rat 172♂ (10000ppm) and collagen masses with chronic granulation tissue In the subcutaneous tissues of rat 15♂ (Control); marked demyelinization of the sciatic nerve of rat 15♂ (Control); minimal extramedullary haemopoiesis in the spleens of rats 363♀, 365♀ and 373♀ (Control), 505♀, 508♀, 512♀, 516♀, 520♀ (10000ppm); minimal keratitis In the eyes of rats 28♂, 368♀ (Control), and retinal degeneration In rat 507♀ (10000ppm); a reduction In cellularlty and evidence of myelofibrosis in rat 28♂ (Control) and 520♀ (10000ppm); cystic degeneration In a lymph node In rat 158♂ (10000ppm) and numerous pigment-laden macrophages in a thoracic lymph node of rat 161♂ (10000ppm).

CONCLUSION
No morphological abnormality or variation from normal was seen in any of the tissues examined which was considered to be due to the administration of TK 10 042.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- Lymphoreticular svstem
The low Incidence of tumours of the lymphoreticular system showed no evidence to suggest a treatment-related disturbance.

- Lung
The lung tumour incidence was well within the spontaneous incidence for rats of the strain employed. One lung tumour (Grade 2) was recorded In rat 181♂ (10000ppm) and metastatic deposits of squamous cell carcinoma, probably originating from a subcutaneous tumour in the lungs of rat 60♂ (10000ppm). No tumours were seen in female rats.

- Liver
One liver cell tumour was observed In this study, in rat 6♂ (Control), clearly indicating that the test item was not hepatocarcinogenic to rats.

- Kidney
A renal adenoma was observed In rat 186♂ (10000ppm), a squamous cell carcinoma In the kidney of rat 517♀ (10000ppm) and an adenocarcinoma In rat 365♀ (Control). These incidences are well within the normal spontaneous
incidence for CFY rats and, therefore, show no evidence of treatment-related nephrocarclnogenicity.

- Reproductive system
There was nb evidence of a tumorigenlc effect on the reproductive system. One Interstitial cell adenoma was observed In the testes of rat 40♂ (Control). A liposarcoma of the adipose tissue adjacent to the ovary was observed In rat 365♀ (Control). A deciduoma was observed In the uterus of rat 508♀ (10000ppm), a fibrosarcoma in rat 420♀ (1000ppm) and a lipoma in rat 462♀ (3000ppm). Neither the incidence nor the multiplicity of mammary tissue tumours, consisting of flbro-adenomas, adenomas and adenocarcinomas showed any evidence of a treatment-related increase.

- Endocrine glands
The control incidence of pancreatic tumours was hlgher than previously recorded In CFY rats although the increased incidence of tumours In rats treated with 10000ppm was not statistically different from the control Incidence. Following an extensive literature search. Roe and Roberts, 1973*, concluded that with three possible exceptions, i. e . ionizing radiation, an alkaloid of the pyrrollzidine group and streptozotocin with nicotinamide, there was no Information on how tumours of the pancreas may be induced by chemical means. As these tumour types occur in old rats, the incidence recorded In the present study was considered to have arisen by chance. The distribution of tumours of the other endocrine glands showed no treatment-related disturbance.

- Subcutaneous tissues
There was no evidence of a treatment-related effect on the incidence of connective tissue tumours found in subcutaneous locations.

- Miscellaneous tumours
The Individual tumour types and number of tumours recorded fall within the spontaneous range for the strain of rat employed.

- Total tumour incidence
There were significantly fewer tumour-bearing rats among males that had received 1000ppm and were killed at termination (P = 0,0033),
The Incidence of tumour-bearing rats In other treated groups was not significantly different from the control incidence

CONCLUSION
The administration of the test compound was not associated with any evidence of an effect on the spontaneous tumour incidence of the CFY rat.

Dose descriptor:

NOEL

Effect level:

10 000 ppm (analytical)

Sex:

male/female

Basis for effect level:

other: no carcinogenic effects observed

Remarks on result:

other: Corresponds to 446 mg/kg bw for females and 547 mg/kg bw for males

Dose descriptor:

NOAEL

Effect level:

10 000 ppm (analytical)

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Corresponds to 446 mg/kg bw for females and 547 mg/kg bw for males

No increased tumour incidence in treated rats compared to control rats: number of tumour bearing rats: males 30/44, 19/45, 25/43 and 26/47, females 43/49, 46/49, 45/49 and 43/50 at 0, 1000, 3000 and 10000 ppm; tumours found for males were mainly pituitary adenomas (14/37, 5/5, 11/11, 6/37) and pancreas islet cell adenomas (2/42, 0/3, 0/2, 7/44, no statistically significant increase); tumours found for females were mainly pituitary adenomas (29/46, 15/17, 21/24 and 22/39) and mammary fibro-adenomas (35/50, 36/41, 34/36 and 31/34).

The incidence of pancreatic islet cell adenomas, in male rats from the 10,000 ppm dosage group was slightly above that of the control group . However, the increase was not statistically significant  and considered unlikely to be of biological importance. No effect was seen in female rats.

The incidence and distribution of other neoplasms in this study were within the normal tumour profile for laboratory maintained rats of this strain. Briefly : mammary tumours were common in females; pituitary tumours in both sexes; with a lower incidence of subcutaneous tumours predominantly in males.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

107 mg/kg bw/day

Study duration:

chronic

Species:

mouse

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, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for carcinogenicity under Regulation (EC) No. 1272/2008.

Additional information

The carcinogenic potential was investigated in two feeding studies. The study in mice is chosen as key study as it was specifically designed as a carcinogenicity study. Its procedure is equivalent to the OECD testing guideline 451, which was published a few months prior to finalization of that study. Its performance was supervised by the internal quality assurance unit which is certified in the study report. Four hundred MAGf (SPF) mice (50 males and 50 females per dose group) were treated at dose level of 0, 100, 300 and 1000 ppm (11.7, 40.5 and 125.9 mg/kg body weight per day for males and 10.5, 35.0 and 106.8 mg/kg body weight per day for females) for 24 months. No clinical symptoms and no substance-related mortality were observed. A mean daily intake of 1000 ppm did not produce non-neoplastic or neoplastic lesions and was considered to be the NOEL. The study is valid with the following minor limitations: The study pre-dates GLP requirements. A summary table containing non-neoplastic lesions was not provided. The analysis procedures/methods for the test item determination in the feed not reported and no analysis for homogeneity. The accuracy of preparation deviates more than 20% (7 times in total at all dose levels (4 times at 300 ppm).

The study in rats was designed as a chronic feeding study in rats, but in regard to the number of animals and mortality, it is similarly suitable to assess the endpoint of carcinogenicity. Its design is comparable to that of OECD testing guideline 453. Due to the overall low toxicity of this substance, high doses were tested. No indication of an increased tumor incidence or preneoplastic lesions was recorded. A total of 500 CFY rats (hysterectomy-derived strain of Sprague-Dawley rats; 50 males and 50 females per dose group) were treated with the test article in the diet, at dose levels of 0, 1000, 3000 and 10000 ppm in the diet per day for 104 weeks. The doses equal 45, 135 and 446 mg/kg bw/d for males and 55, 166 and 547 mg/kg bw/d for females based on food conversion. The study is valid with the following restrictions: It pre-dates GLP requirements and little details on the test item are available. No analysis for accuracy of preparation, homogeneity and stability were reported. The major part of the histopathological examinations was performed 10 years after the finalization of the study under supervision of the quality assurance unit of the test institute. The NOEL for carcinogenicity was equal or higher than 10000 ppm. Regarding toxicity, a NOAEL was set at 10000 ppm. An inferior bodyweight gain among males between weeks 24 and 52 was not considered related to treatment since no corroborative evidence was seen.