Ethylenebis(oxyethylene) bis[3-(5-tert-butyl-4-hydroxy-m-tolyl)propionate]

Carcinogenicity

Description of key information

The substance was tested for carcinogenicity in rats in a GLP-compliant study following OECD testing guideline 453. Rats were treated with the test article in the diet, corresponding to 0, 5, 15, 50 and 100 mg/kg body weight per day for 24 months. Enlarged thyroid glands of all high dose group animals were accompanied by slightly increased incidences of hyperplasia and/or cystic dilatation of the thyroid follicles and an increased incidence of thyroid follicular adenomas. Mechanistic investigations and studies with dogs and monkeys show that this is caused by a rodent specific stimulation of the pituitary-thyroid-liver axis. In addition, no genotoxicity was observed in-vitro and in vivo. The thyroid adenomas observed in rats are not relevant for human health hazard assessment.

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:

16.3.1984 to 8.7.1988

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 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

reliability scoring based on 1981 guideline

Deviations:

yes

Remarks:

10 rats/group selected for hematology.

GLP compliance:

yes

Species:

rat

Strain:

other: Tif; RAIf (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Production CIBA-Geigy LTD. 4332 Stein, Switzerland
- Age at study initiation: approx. 4 to 5 weeks
- Weight at study initiation: 127.3 g (males) and 112.5 g (females)
- Housing: housed in groups of 5 in macrolon cages type 4 with standard granulated soft wood bedding
- Diet (e.g. ad libitum): pelleted, certified standard diet Nafag No 890 tox was provided ad libitum
- Water (e.g. ad libitum): tap water was provided ad libitum
- Acclimation period:12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 10
- Air changes (per hr): 16-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: April 5, 1984 To: April 28, 1986

Route of administration:

oral: feed

Type of inhalation exposure (if applicable):

other: not applicable

Vehicle:

other: feed

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): diets were prepared for the first week, and then every 2 to 3 weeks until week 18, after that diets were prepared every 4 weeks.
- Mixing appropriate amounts with (Type of food): pelleted, certified standard diet Nafag No. 890 tox

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Food samples were analysed for concentration, homogeneity and stability periodically with the diet batches applied during the study.

Duration of treatment / exposure:

24 months

Frequency of treatment:

daily

Post exposure period:

None

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

achieved dosage: 5.03 and 5.24 mg/kg bw in males and females, respectively

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

achieved dosage: 15.2 and 15.6 mg/kg bw in males and females, respectively

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

achieved dosage: 49.9 and 51.1 mg/kg bw in males and females, respectively

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

achieved dosage: 96.8 and 100 mg/kg bw in males and females, respectively

No. of animals per sex per dose:

70 animals/sex/dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: were selected on the basis of the results of 3 previous studies (28 days peroral range finding study in rats with NOEL = 300 mg/kg bodyweight; 28 days palatability study in rats with NOEL above 2000 mg/kg bodyweight; and 3 months oral toxicity study in rats with NOEL = 50 mg/kg bodyweight)
Feeding levels were adapted according to the mean food consumption and the mean bodyweight of the animals in order to achieve a daily intake of 0, 5.0, 15.0, 50.0 and 100 mg/kg bodyweight. Thus the concentration of the compound in the diet
was increased successively from 45 to 190, 130 to 600, 430 to 1900 and from 860 to 4000 ppm during the course of the study.

Positive control:

None used

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (am and pm on working days)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (midweek) for the first 3 months, and monthly for the rest of the study period

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: monthly for the first 28-weeks. Since no differences were found, measuring was suspended according to the provision in the protocol.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before (day -6) and on days 79, 177, 261, 351, 444, 542, 634, and 723
- Dose groups that were examined: highest dose group and control

HAEMATOLOGY: Yes
- Time schedule for collection of blood: between 7 am and 11 am on pretest and weeks 13, 27, 39, 52, 65, 79, 92, and 105
- Anaesthetic used for blood collection: Yes; ether
- Animals fasted: Yes
- How many animals: 10/group
- parameters checked: Erythrocyte Count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Leucocyte Count, Differential Leucocyte Count, Thrombocyte Count, Prothrombin Time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: between 7 am and 11 am on pretest and weeks 13, 27, 39, 52, 65, 79, 92, and 105
- Animals fasted: Yes
- How many animals: 10/group
- parameters checked: Glucose, Urea, Creatinine, Total bilirubin, Total protein, Albumin, Globulins, A/G Ratio, Cholesterol, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Sodium, Potassium, Calcium, Chloride, Phosphorus inorganic

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:Hearing test- Time schedule for examinations: before (day -6) and on days 177, 261, 351, 444, 542, 634, and 723
- Dose groups that were examined: highest dose group and control

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At the one year interim sacrifice and at termination of the study, all control and treated animals which survived were bled under ether anesthesia and subjected to a detailed autopsy. Besides the weight of the exsanguinated body the following organs were weighed: adrenals, brain, gonads (testes / ovaries), heart, kidneys, liver

HISTOPATHOLOGY: Yes
The following organs and tissues were preserved in neutral buffered 4% formalin: skin, mammary area (with mammary gland), spleen, mesenteric lymph node, axillary lymph node, popliteal lymph node, sternum with bone marrow, femur with joint, skeletal muscle, trachea, lung, heart, aorta, submandibular salivary gland, liver, pancreas, oesophagus, stomach, small intestine (ileum, jejunum, duodenum), large intestine (colon, caecum, rectum), kidneys, urinary bladder, prostate, seminal vesicle, testis, epididymis, uterus (corpus and cervix), vagina, ovary (with fallopian tubes), pituitary gland, adrenal gland, thyroid with parathyroid gland, thymus, peripheral nerve, brain, spinal cord (2 levels), eye with optic nerve (and continguous, Harderian gland), orbital gland extraorbital lacrimal gland, muzzle, organs and tissues showing macroscopical changes.
Unless prevented by advanced autolysis or cannibalism, complete autopsy with tissue preservation was performed also on animals which died during the test period or were sacrificed in moribund condition. After the fixation, all above listed organ samples from each control and test animal were taken, embedded in paraplast, sectioned at 3-5 microns, stained with hematoxylin and eosin and subjected to microscopic examination, with exception of popliteal lymph node, which was not requested for histopathological examination.

Statistics:

For each time point and parameter a univariate statistical analysis was conducted. Due to the routine manner of the analysis system, parameter free (nonparametric) methods were applied. Each treated group was compared to the control group in respect of dispersion and displacement. This test is a combination of Wilcoxon's and Ansari-Bradley's statistics, i.e. a combined test for location and variation. In addition a trend test was applied considering all groups. Survival analysis was performed by the regression model (partial likelihood) introduced by Cox in order to compare survival experience of treated animals with the control and versus treated group 2.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Among males, a slightly inferior survival was recorded for the group receiving 100 mg/kg. However, analysis revealed that the difference from the control group was not of statistical significance and no common causal pathology was evident among decedents. Mortality among other treated males was similar to that of the controls. Among females, rats treated with 100 mg/kg had a superior survival to that of the controls; the difference attaining a level of statistical significance (P=0.0067). Females treated with 50 mg/kg showed a similar tendency although the difference from controls was not statistically significant. The survival rate of female treated with 5 or 15 mg/kg was virtually identical with that of the controls.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the first 80 weeks of the study, group 5 males (100 mg/kg) attained slightly higher mean bodyweights than the controls; the difference attaining levels of statistical significance between weeks 16 and 51. During the final 6 months of the study, mean bodyweights became similar to those of the controls. Bodyweights of other treated male groups were not disturbed by treatment. For females receiving 15, 50 or 100 mg/kg, bodyweight gains were slightly higher than those of the controls during the first seven weeks of the study. Thereafter, the weight gain of females receiving 100 mg/kg was less than that of the controls with mean bodyweights differing significantly from control values. The weight gain of females receiving 15 or 50 mg/kg became similar to control values for the remainder of the treatment period. Bodyweight gain of females receiving 5 mg/kg wais not disturbed by treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the first year of the study, males receiving 50 mg/kg consumed slightly less food than the controls ( 95.6% of the control intake). Thereafter, their food intake was similar to that of the controls. Males receiving 100 mg/kg consumed a similar quantity of food to that of the controls during the first year of the study. However, over the final 12 months of treatment their food intake decreased to an overall value of 92% of the control intake. Among females receiving 50 or 100 mg/kg there was a consistent dosage related reduction in the quantity of food consumed when compared with control values. Overall intakes for the treatment period were 93.7% and 89.2% respectively of the control value. Food intakes of males and females receiving 5 or 15 mg/kg were not disturbed by treatment.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Males receiving 100 mg/kg had consistently lower food consumption ratios when compared with control ratios, reflecting the higher mean bodyweights recorded for this group. Females receiving 15, 50 or 100 mg/kg had slightly lower ratios than the controls during the first 12 weeks of treatment, reflecting the higher mean bodyweights recorded during the early stage of the study. Thereafter, the food consumption ratios for these female groups were similar to control values. Males receiving 15 or 50 mg/kg and males and females receiving 5 mg/kg had similar ratios to those of the controls throughout the study.

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

no effects observed

Description (incidence and severity):

Among males, measurement of water intake during week 4 revealed similar values for treated and control groups. Further measurements during weeks 8 to 20 revealed lower values for males receiving 50 or 100 mg/kg although the differences were not dosage related. During weeks 24 and 28 the differences between treated and control values were of insufficient magnitude to be of toxicological relevance, therefore, measurements were discontinued. Measurements among female groups revealed no indication of a treatment related effect on water intake.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The results of the examinations gave no indication that treatment with the test article was detrimental to the appearance or function of the eye.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The investigations performed from week 65 through to termination revealed lower values for hematocrit, red blood cell count and hemoglobin among males receiving 100 mg/kg. Lower values for these parameters were also recorded at week 105 for male rats No. 211 and 263 from the group receiving 50 mg/kg. Values recorded for treated females and for males receiving 5 or 15 mg/kg were similar to those of the controls throughout the study. Other differences between treated and control values which attained a level of statistical significance on some occasions were considered to have arisen fortuitously and to be of no toxicological significance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There was a dosage related increase in cholesterol levels in treated males and females throughout the study. All rats receiving 50 or 100 mg/kg had higher than expected values on most occasions whereas for rats receiving 5 or 15 mg/kg not all rats were affected on each occasion. For the majority of males and females receiving 50 or 100 mg/kg, triglyceride levels were towards the lower end of the control range on most occasions. No consistent change was recorded for rats receiving 5 or 15 mg/kg although male rat 200 (15 mg/kg) had an elevated level until death in week 96. Higher values for phospholipids were recorded on most occasions for males and females receiving 100 mg/kg. Among rats receiving 5, 15 or 50 mg/kg some rats had phospholipid levels at the upper end of the control range on most occasions. ASAT, ALAT, ALP or GGT enzyme activities were elevated for the majority of males receiving 100 mg/kg although within the group, as for males receiving 50 mg/kg, some individuals had transiently high values. ALAT enzyme activity was at the upper end of the control range for females receiving 100 mg/kg whereas ASAT activity was similar to control values except for rat 683 in week 105 in which all enzyme activities were elevated. A few females receiving 50 mg/kg had a transient increase of ALAT enzyme activity although no consistent trend emerged. Mean values for ALP and GGT among females receiving 100 mg/kg were influenced by 3 or 4 rats with values at the upper end of the control range. The remainder of the group had activities similar to control values. ALP values for some females receiving 50 mg/kg were transiently elevated whereas GGT activities for 4 out of 10 rats were consistently elevated from week 39/52. Enzyme activities for males and females receiving 5 or 15 mg/kg were not disturbed by treatment. From week 39, inorganic phosphate levels in some males receiving 100 mg/kg tended towards the upper end of the expected range, However, among females of this dosage group, apparent fluctuations in mean values were a reflection of the large within group variation among controls. As values for females were within the expected range, these differences were not considered to be of toxicological relevance. Protein levels in females receiving 100 mg/kg were towards the upper end of the expected range with both higher albumin and globulin values recorded for these rats. Hence, the A/G ratios were not disturbed. Values for other treated females and for treated males were within the expected range. Other differences between control and treated values which attained a level of statistical significance were inconsistent or resulting from the abnormal value for a single animal in the group. Therefore, these differences were considered to be of doubtful toxicological relevance.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At the interim sacrifice, mean liver and kidney weights relative to bodyweight of males and females treated with 100 mg/kg were heavier than those of the controls. A level of statistical significance was attained by the difference from control values except for the kidney weights of females. In addition, males treated with 5, 15 or 50 mg/kg had higher mean values for relative kidney weights at the interim sacrifice. However, in the absence of related pathology, these differences are of doubtful toxicological relevance. The high mean value for adrenal weight of males treated with 100 mg/kg was influenced by the weight recorded for rat 343 (307.5 mg), subsequently diagnosed as bearing a benign medullary tumor.
At termination, liver and kidney weights relative to bodyweight for males and females treated with 100 mg/kg were significantly heavier than control values. No relevance is attached to the higher group mean values for relative liver weight of other treated females as in the groups treated with 5 or 50 mg/kg the higher than expected liver weight for one rat in each group had influenced the mean value. The mean relative kidney weight for females treated with 5 mg/kg and for males and females treated with 15 or 50 mg/kg were also heavier than control values. Three females treated with 5 mg/kg (rats 435, 460 and 479), one female and four males treated with 15 mg/kg (female rat 305) and (male rats 180, 183, 185 and 188) and three females and two males treated with 50 mg/kg (female rats 569, 575 and 584) and (male rats 226 and 230) had kidney weights outside the control range. Recalculation of mean values excluding these rats produced mean values similar to those of the controls. Therefore, the fragility of these data suggests the differences arose by chance. The higher mean ovarian weight for females treated with 100 mg/kg was a reflection of the ovarian weight for rat 636 (10.3 g). Histopathology revealed a malignant granulosa/theca cell tumor. The ovarian weights of other rats in the group were similar to those of the control. The weights of other organs from treated rats were similar to control values at both the interim and terminal sacrifices.
In conclusion, at both the interim and terminal sacrifices the heavier liver and kidney weights of group 5 animals (100 mg/kg) were considered to be related to treatment although no histopathological change was associated with the kidney weight change. The weights of other organs from treated rats were not influenced by treatment.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopical examination revealed an increased incidence of liver cysts or masses in male groups 4 and 5 (50 and 100 mg/kg), and an increased incidence of rats with enlarged thyroid glands in both sexes from group 5 (100 mg/kg).

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Higher numbers of group 4 and 5 males (50 and 100 mg/kg) presented with focal liver change, the main feature of which was cystic dilatation of the liver sinusoids. In its early stages this lesion appeared as focal hepatocellular hypertrophy and/or hyperplasia with varying degrees of sinusoid dilatation. In more advanced stages the sinusoidal cystic dilatation was predominant and sometimes associated with small foci of hepatocellular necrosis. This change accounted for the majority of cysts and masses found at the autopsy. A very low incidence of liver sinusoid cystic dilatation was found in female groups 4 and 5 (50 and 100 mg/kg) - 4/70 and 3/70 respectively against 0/70 in the control group. A higher number of females from group 5 (100 mg/kg) presented with biliary cyst.
The statistical analysis indicated that there is a significant dose related increase with regard to the concurrent control in the incidence of hyperplasia and cystic dilatation of the thyroid follicles in male rats of groups 3, 4 and 5 (15, 50 and
100 mg/kg), and in female rats of groups 4 and 5 (50 and 100 mg/kg). However, a comparison of the incidence encountered in this study to the historical data base derived from untreated controls of our rat strain shows that only the values occurring in groups 4 and 5 (50 and 100 mg/kg) in both sexes are out of range. In view of the intra-strain variability encountered, the results in groups 4 and 5 (50 and 100 mg/kg) are considered to be of experimental significance.
There was a significant dose related increase with regard to the concurrent control in the incidence of occurrence of foam cells within the lung alveoli in male rats of groups 3, 4 and 5 (15, 50 and 100 mg/kg), and in females of group 5 (100 mg/kg). However, a comparison of the incidence encountered in this study to the historical data base derived from untreated control rats showed that only the values occurring in female rats from group 5 (100 mg/kg) are out of range. In view of the intra-strain variability encountered, the
results in female group 5 (100 mg/kg) are considered to be of experimental significance.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

An increased incidence of follicular cell adenomas occurred in males treated with 50 and 100 mg/kg as well as in females treated at 100 mg/kg. In addition, follicular carcinoma incidence was slightly increased at these doses.
There was an apparent increase in the number of animals with adrenal cortical adenoma in the group 5 (100 mg/kg) when compared with the controls (4/70 versus 1/70 in males and 2/70 versus 0/70 in females). However, historical data derived from untreated controls of our rat strain indicate a spontaneous adrenal cortical tumour incidence of 0 to 2.9% for males and 0 to 5.7% for females. Therefore, the similarity of the incidence encountered in this study to that defined by the historical data suggests the group differences arose by chance and are not related to treatment.
A variety of different neoplastic and non-neoplastic lesions found in this study, commonly occur in our colony of rats and neither their incidence nor the distribution gave any indication of a treatment related association. In particular, atrophy of the seminal vesicles occurring in slightly higher number in male group 5 (100 mg/kg), was attributed either to incidentally decreased amount of secretory material or occurred in rats with testicular atrophy or reduced spermatogenesis which was not dose-dependent.

Other effects:

no effects observed

Description (incidence and severity):

HEARING TEST
During the first 91 weeks of the study, rats treated with test material exhibited a reaction to the emitted sound on each test occasion. At the terminal test in week 104, between 1 to 3 rats in both treated and control groups failed to respond. This incidence is however typical for rats of this age and strain and reflects approaching senescence. These data clearly indicate that the test article is not ototoxic to rats.

Dose descriptor:

NOAEL

Effect level:

15 mg/kg diet

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

ORGAN WEIGHTS

Absolute organ weights

		dose (mg/kg)	0	5	15	50	100
week 55	males	heart (g)	1.733	1.814	1.826	1.636	1.807
		liver (g)	25.08	26.15	27.1	26.24	30.1*
		kidney (g)	4.058	4.358	4.502	4.471	5.588**
		adrenal (mg)	84.99	97.6	85.75	68.75	105.9
		testis (mg)	4.308	4.629	4.473	4.261	4.415
	females	heart (g)	1.177	1.15	1.167	0.998	1.037
		liver (g)	16.02	15.5	14.19	14.56	15.37
		kidney (g)	2.886	2.795	2.96	2.717	2.83
		adrenal (mg)	102.6	91.28	91.96	86.48	83.85
		ovary (mg)	184.6	214	183.8	184.8	184.7
Week 105	males	heart (g)	2.162	2.175	2.181	2.066	2.225
		liver (g)	23.88	24.29	24.66	25.35	31.03**
		kidney (g)	5.05	5.21	5.63	5.661	6.675**
		adrenal (mg)	96.5	128.8	187.4	176.9	118.4
		testis (mg)	5.217	4.741	4.615	4.19	4.764
	females	heart (g)	1.458	1.502	1.466	1.392	1.283**
		liver (g)	18.84	19.11	20.14	19.55	17.98
		kidney (g)	3.285	3.646	4.221	3.607*	3.342
		adrenal (mg)	100.9	145.1	105	93.93	97.96
		ovary (mg)	186.9	185.4	209.6	171.1	371.5*

Organ body weight ratio

		dose (mg/kg)	0	5	15	50	100
week 55	males	heart (g)	2.363	2.561	2.442	2.296	2.303
		liver (g)	33.79	37.11	36.17*	36.36	38.21*
		kidney (g)	5.478	6.202*	6.019*	6.205**	7.125**
		adrenal (mg)	0.116	0.137	0.115	0.122	0.133
		testis (mg)	5.873	6.547	6.031	5.962	5.628
	females	heart (g)	2.685	2.624	2.822	2.666	2.768
		liver (g)	36.31	35.52	34.42	38.57	40.7*
		kidney (g)	6.681	6.38	7.161*	7.233	7.518
		adrenal (mg)	0.237	0.21	0.223	0.232	0.223
		ovary (mg)	0.42	0.494	0.438	0.493	0.493
Week 105	males	heart (g)	2.918	2.903	3.126	2.884	3.04
		liver (g)	31.8	31.86	35.09	34.07	41.93**
		kidney (g)	6.825	6.956	8.105	7.894*	9.119**
		adrenal (mg)	0.131	0.168	0.286	0.243	0.163
		testis (mg)	6.803	6.212	6.472	5.676	6.31
	females	heart (g)	2.898	3.092	2.788	2.811	3.049
		liver (g)	36.68	38.1	37.71	38.49	41.32
		kidney (g)	6.566	7.590 *	7.931	7.465*	7.933**
		adrenal (mg)	0.202	0.322	0.199	0.192	0.233
		ovary (mg)	0.377	0.392	0.389	0.353*	0.79

HISTOPATHOLOGY

Occurrence of sinusoid cystic dilatation in males

Group	1	2	3	4	5
Dose (mg/kg)	0	5	15	50	100
Animals initially in study	70	70	70	70	70
Livers examined	70	70	70	70	70
Males with sinusoid cystic dilatation	21	16	16	40	51

Occurrence of biliary cysts in females

Group	1	2	3	4	5
Dose (mg/kg)	0	5	15	50	100
Animals initially in study	70	70	70	70	70
Livers examined	68	69	69	70	70
Females with biliary cysts	1	1	3	2	5

Occurrence of cystic dilatation or hyperplasia of thyroid follicles in males

Group	1	2	3	4	5
Dose (mg/kg)	0	5	15	50	100
Animals initially in study	70	70	70	70	70
Thyroid glands examined	68	66	70	67	64
Males with thyroid follicles cystic dilatation	2	1	4	5	5
Males with thyroid follicles hyperplasia	2	1	5	20	22

Occurrence of cystic dilatation or hyperplasia of thyroid follicles in females

Group	1	2	3	4	5
Dose (mg/kg)	0	5	15	50	100
Animals initially in study	70	70	70	70	70
Thyroid glands examined	68	66	68	70	70
Males with thyroid follicles cystic dilatation	1	1	1	10	6
Males with thyroid follicles hyperplasia	0	2	1	7	19

Occurrence of foam cells within the lunq alveoli in males

Group	1	2	3	4	5
Dose (mg/kg)	0	5	15	50	100
Animals initially in study	70	70	70	70	70
Lungs examined	70	70	70	70	70
Males with foam cells within the lung alveoli	13	8	19	26	22

Occurrence of foam cells within the lunq alveoli in females

Group	1	2	3	4	5
Dose (mg/kg)	0	5	15	50	100
Animals initially in study	70	70	70	70	70
Lungs examined	68	69	69	70	70
Females with foam cells within the lung alveoli	15	17	18	21	34

NEOPLATIC LESION

Occurrence of neoplastic lesions in thyroid gland in males

Group	1	2	3	4	5
Dose (mg/kg)	0	5	15	50	100
Animals initially in study	70	70	70	70	70
Thyroid glands examined	68	66	70	67	64
Males with follicular adenoma	0	1	0	4	16
Males with follicular carcinoma	0	0	1	1	8

Occurrence of neoplastic lesions in thyroid qland in females

Group	1	2	3	4	5
Dose (mg/kg)	0	5	15	50	100
Animals initially in study	70	70	70	70	70
Thyroid glands examined	68	66	68	70	70
Females with follicular adenoma	0	1	0	0	21
Females with follicular carcinoma	1	0	0	0	3

Executive summary:

In the present study a total of 700 albino rats, 70 males and 70 females per dosage group was used. The test article was administered in the diet at dosages of 0, 5.0, 15, 50 and 100 mg/kg bodyweight per day for a period of 24 months. Average achieved intakes corrected for the quantity of test material determined by frequent chemical analysis of the diets were 5.03, 15.2, 49.9 and 96.8 mg/kg bodyweight per day for males and 5.24, 15.6, 51.1 and 100 mg/kg bodyweight per day for females of groups 2, 3, 4 and 5 respectively. Treatment was without effect on the appearance and behaviour of rats receiving the test article. Treatment had no adverse effect on survival. On the contrary, females of group 5 (100 mg/kg) had a superior survival compared with controls. Mean bodyweights of males of group 5 (100 mg/kg) were higher than control values during the first 80 weeks of treatment. Thereafter, mean bodyweights were similar to those of the controls. Mean bodyweights of female groups 3, 4 and 5 (15, 50 and 100 mg/kg) were slightly higher than control values during the first seven weeks of treatment. Thereafter, mean bodyweights of females of group 5 (100 mg/kg) were significantly lower than control values, whereas female groups 3 and 4 (15 and 50 mg/kg) had similar bodyweights to those of the controls. Bodyweight gains in group 2 (5 mg/kg) and in males of group 3 and 4 (15 and 50 mg/kg) were not disturbed by treatment. Males of group 5 (100 mg/kg) ate approximately 8% less food than the controls during the second year of treatment, whereas males of group 4 (50 mg/kg) ate slightly less food during the first year of treatment (4% less). At other times, intakes were similar to control values. A consistent dosage related reduction in the quantity of food consumed by females of group 4 and 5 (50 and 100 mg/kg) was recorded throughout the treatment period. The food intake of groups 2 and 3 (5 or 15 mg/kg) was not disturbed by treatment. Consistently lower food consumption ratios reflected the higher mean bodyweights and lower food intakes recorded for males of group 5 (100 mg/kg). Lower food consumption ratios were also recorded during the first 12 weeks of treatment for female groups 3, 4 and 5 (15, 50 and 100 mg/kg), reflecting the higher mean bodyweights during this period. Thereafter, ratios were similar to those of the controls indicating that the lower bodyweights recorded for females of group 5 (100 mg/kg) were directly related to the lower food intakes. Measurements at intervals during the first 28 weeks of the study gave

no indication of a treatment related effect. Treatment had no effect on hearing ability or on the appearance or function of the eyes. Slightly lower values for red blood cell count, hemoglobin and hematocrit were recorded from week 65 for males of group 5 (100 mg/kg). Lower values for these parameters were also recorded at week 105 for 2/10 males of group 4 (50 mg/kg). Values for female and other treated male groups were similar to those of the controls. There was a dosage related increase in cholesterol levels of treated males and females throughout the treatment period. High phospholipid levels were also recorded for rats of group 5 (100 mg/kg), and for some rats of other treated groups, phospholipid levels at the upper end of the control range were recorded on most occasions. For rats of group 4 and 5 (50 and 100 mg/kg), triglyceride levels were towards the lower end of the control range on most occasions. Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and gamma-glutamyl transpeptidase activities were elevated or the majority of males of group 5 (100 mg/kg) and transiently higher for some males of group 4 (50 mg/kg). Females of group 5 (100 mg/kg) had ALAT activities at the upper end of the control range. AIP and GGT activities were elevated for 3 or 4 rats of the group whereas ASAT activity was not disturbed by treatment. Among females of group 4 (50 mg/kg), transient increases in ALAT activity were recorded and GGT activity was consistently high in 4 rats of the group. Inorganic phosphate levels tended towards the upper limit in some males of group 5 (100 mg/kg), and higher protein, albumin and globulin levels were recorded among females of this group. At both the interim and terminal sacrifices the heavier liver and kidney weights of group 5 animals (100 mg/kg) were considered to be related to treatment although no histopathological change was associated with the kidney weight change. The weights of other organs from treated rats were not influenced by treatment. Macroscopical examination revealed an increased incidence of liver cysts or masses in male groups 4 and 5 (50 and 100 mg/kg), and an increased incidence of rats with enlarged thyroid glands in both sexes from group 5 (100 mg/kg). Histopathological examination revealed the following changes which were considered to be related to treatment: An increased incidence of liver sinusoid cystic dilatation in male groups 4 and 5 (50 and 100 mg/kg). This change accounted for the majority of liver masses or cysts found at autopsy. In female group 5 (100 mg/kg) there was a slight increase in the number of liver biliary cysts and a slight increase in the number of liver sinusoid cystic dilatation in female groups 4 and 5 (50 and 100 mg/kg). An increased incidence of hyperplasia or cystic dilatation of the thyroid follicles in both sexes from groups 4 and 5 (50 and 100 mg/kg); and an increased incidence of thyroid gland follicular adenoma and carcinoma in both sexes from group 5 (100 mg/kg). A slight increase of foam cell accumulations within the lung alveoli occurred in female group 5 (100 mg/kg).

In conclusion, the test item had no adverse effect on the appearance, behaviour or survival of treated rats, nor was there a direct effect on bodyweight. Treatment induced hepatotropic effects, the evidence of which were disturbance of enzyme activities related to liver function, of blood lipid parameters and non-neoplastic liver pathology at the higher dose levels. In addition, a slightly increased accumulation of foam cells in the lungs occurred. Neoplastic changes related to treatment were confined to the thyroid. An increased incidence of follicular cell adenomas occurred in males treated with 50 and 100 mg/kg as well as in females treated at 100 mg/kg. In addition, follicular carcinoma incidence was slightly increased at these doses. Furthermore, cystic dilatation and follicular hyperplasia was observed in higher numbers in animals treated at 50 and 100 mg/kg.

Endpoint conclusion

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

The neoplastic changes observed have been shown in a variety of special studies to be of secondary nature due to thyroid stimulating hormone (TSH) imbalance and subsequent proliferation of thyroid follicular cells. Stimulation of the thyroid gland was not observed in a 28-days study with cynomolgus monkeys and a three-months study with dogs. Therefore, the increased incidence of thyroid follicular adenomas observed in the rat carcinogenicity study is not relevant for human health hazard assessment and the substance is not classified for carcinogenicity under Directive 67/548/EEC, as amended for the 28th time in Directive 2001/59/EC as well as under Regulation (EC) No. 1272/2008.

Additional information

In a 24-months carcinogenicity and chronic toxicity study, a total of 700 albino rats (70 males and 70 females per dose group) were treated with the test article in the diet, corresponding to 0, 5, 15, 50 and 100 mg/kg body weight per day for 24 months. No clinical symptoms and no substance-related mortality were observed. Except for a small dose-related reduction of food consumption and body weight gain in females of the upper two dose groups, no further effect on food consumption and body weight gain was observed. Slight anemia of the high dose males was the only hematological change observed. Investigations of the blood chemistry revealed increased levels of cholesterol and phospholipid in all high dose animals, and to a lesser extent, in both sexes of the 50 mg dose group. Activities of alanine amino transferase, aspartate amino transferase, alkaline phosphatase and gamma-glutamyl transpeptidase were increased in both sexes of the high dose, and to a lesser extent, also in the 50 mg dose group. Inorganic phosphate, total protein, albumin and globulins were slightly reduced in females of the high dose group. The activated state of the liver, as shown by the increased activities of some key enzymes, was further expressed by increased liver weight of the high dose group animals of both sexes, accompanied by histological changes such as increased incidence of liver cysts (sinusoid cystic dilatation) in animals of the 50 and 100 mg dose group as well as liver biliary cysts in females of the high dose group. In the absence of any histological changes the slightly increased kidney weights of the high dose group animals was considered to be of rather minor importance.

Enlarged thyroid glands of all high dose group animals were accompanied by slightly increased incidences of hyperplasia and/or cystic dilatation of the thyroid follicles and an increased incidence of thyroid follicular adenomas. The NOEL was determined to be 15 mg/kg bw/day.

The neoplastic changes observed have been shown in a variety of special studies to be of secondary nature due to thyroid stimulating hormone (TSH) imbalance and subsequent proliferation of thyroid follicular cells. Stimulation of the thyroid gland was not observed in a 28-days study with cynomolgus monkeys (CIT, 1997) and a three-months study with dogs (Ciba-Geigy, 1982). Therefore, the increased incidence of thyroid follicular adenomas observed in the rat carcinogenicity study is not relevant for human health hazard assessment. For details it is referred to the section on specific investigations.