Ziram

Carcinogenicity

Description of key information

Key, report number ZIR 9/942098, combined chronic toxicity/carcinogenicity studies (52/104 weeks, rat, dietary exposure):

NOAEL (general systemic toxicity) no NOAEL could be set

LOAEL (general systemic toxicity) 60 ppm corresponding to 2.5 mg/kg bw/day for males and 3.4 mg/kg bw/day for females

NOAEL (carcinogenicity) 180 ppm corresponding to 7.7 mg/kg bw/day for males

NOAEL (carcinogenicity) 540 ppm corresponding to 34.6 mg/kg bw/day for females

Key, report number 83-5121, chronic/carcinogenicity (2 years, rat, dietary exposure):

NOAEL (general systemic toxicity): 16 ppm, corresponding to 0.56 and 0.66 mg/kg bw/day for males and females, respectively.

NOAEL (carcinogenicity) ≥ 2000 ppm corresponding to 74 mg/kg bw/day for males and 91 mg/kg bw/day for females

 

Key, report number ZIR 12/932311, carcinogenicity studies (80 weeks, mice, dietary exposure):

NOAEL (general systemic toxicity) no NOAEL could be set

LOAEL (general systemic toxicity) 25 ppm corresponding to 3 mg/kg bw/day for males and 4 mg/kg bw/day for females

NOAEL (carcinogenicity) ≥ 675 ppm corresponding to 82 mg/kg bw/day for males and 95 mg/kg bw/day for females

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:

25 Sep 1991 - 01 Oct 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

EPA OPP 83-5 (Combined Chronic Toxicity / Carcinogenicity)

Version / remarks:

adopted in 1984

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

adopted in 2018

Deviations:

yes

Remarks:

The lowest dose level produced toxicity and was therefore too high.

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source : Charles River Breeding Laboratories, Michigan, USA
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 157 - 192 g (males), 114 - 175 (females)
- Housing: 5 animals of the same sex were housed in stainless steel, wire-mesh cages. Each cage measured 36.5 cm wide, 55 cm deep and 25 cm high.
- Diet: SDS Rat and Mouse No. 1 modified maintenance diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 6 day acclimation period after delivery, further 7 days after allocation of the animals in groups and before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
A pre-mix was prepared each week by grinding the test substance directly into untreated sieved basal diet and mixing in a Turbula Mixer for a minimum period of 2 minutes. The required concentrations were then prepared by direct dilution of the pre-mix with further quantities of untreated diet; homogeneity being achieved by further mixing in a double-cone blender for a minimum period of 7 min. The total volumes of diets required were such (i.e. so large) that two mixes (a and b) for each inclusion level were made from one pre-mix. The concentration of test material in the low dose level was increased by 10% above nominal in order to compensate for losses during storage observed in pre-dosing analytical chemistry. On completion of the mixing procedure for each group, the diets to be fed at each level were divided into 8 aliquots labelled, either "a" or "b", for daily use during the following week. All aliquots were transferred as quickly as possible to storage at +4 °C until immediately before feeding. An aliquot was removed daily from +4 °C storage, allowed time to reach ambient temperature and fed to the animals. Whether diet fed was from mix "a" or "b" was documented.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dietary samples were analysed (spectrophotometry at 435 nm after carbon disulphide extraction) before start of treatment as well as with dietary samples from the present study.

1) Prior to the start of treatment the proposed diet mixing procedures were checked by chemical analysis to confirm that the method was acceptable and the homogeneity and stability of the formulation was satisfactory under the conditions of the study. This analysis revealed that the test material was not sufficiently stable in rodent diet to permit storage at room temperature for twice weekly feeding. Therefore, diets were prepared weekly and stored at +4 °C prior to use.

2) Samples of diets of the present study prepared in Weeks 1, 3, 13, 26, 39, 41, 52, 65, 78, 91 and 104 were also analysed to check the accuracy of preparation. Samples were taken at preparation and further samples stored for 7 days at +4 °C followed by exposure at room temperature for 24 h. Samples were taken from both mixes a and b at each dose level. Either mix a or b was analysed and the other was retained frozen for possible further analysis. Analysis of the samples taken showed good agreement with nominal values at the start of each sampling week. Tolerable agreement was achieved for samples obtained at the end of the sampling weeks indicating that the fortification procedure applied to the lowest dietary inclusion level was successful in compensating for degradation. Mean results in the post-dose samples were all within +6% and -17% of nominal concentrations. Further, the test substance was confirmed to be homogeneously blended in rodent diet. The stability in rodent diet showed a maximum concentration loss of 23% at 25 ppm and 13% at 50 ppm during the 8-day storage period. In total, the results indicate that the formulations were homogeneous and stable from the time of preparation to completion of dosing under the study conditions.

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

Continuously via diet

Dose / conc.:

60 ppm

Remarks:

The concentration of test material in the low dose level was increased by 10% above nominal (66 ppm) to compensate losses during storage. In total this corresponded to 2.5 mg/kg bw/day for males and 3.4 mg/kg bw/day for females.

Dose / conc.:

180 ppm

Remarks:

corresponding to 7.7 mg/kg bw/day for males and 10.2 mg/kg bw/day for females

Dose / conc.:

540 ppm

Remarks:

corresponding to 23.7 mg/kg bw/day for males and 34.6 mg/kg bw/day for females

No. of animals per sex per dose:

50 per sex (104 week sacrifice)
20 per sex (52 week sacrifice)

Control animals:

yes, plain diet

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for the first 4 weeks thereafter once weekly for any signs of behavioural changes, reaction to treatment or ill health; twice daily for moribund or dead animals
- Cage side observations checked: signs of behavioural changes, reaction to treatment or ill health an d or moribund or dead animals

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION (if dermal study): No

BODY WEIGHT: Yes
- Time schedule for examinations: at the time of allocation of animals to groups, on the day of commencement of treatment and once a week 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: The quantity of food consumed by each cage of rats was recorded daily and reported on a weekly basis. Food consumption was calculated from the raw data using the total amount of food given to and left by each cage in each group and the number of rats surviving in each cage and presented as weekly food consumption in g/rat/week.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Substance intake (mg/kg bw/day) was calculated from the group mean body weight and food consumption and the nominal dietary inclusion levels of the test material.

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, but food conversion ratios were calculated, where possible, over the period Weeks 1 to 26 from the body weight and food consumption data as weight of food consumed per unit gain in body weight.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Daily by visual appraisal of the water bottles and measured by weight for 7 consecutive days during Weeks 12, 25 and 51 for all satellite group cages.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups that were examined: Before treatment (all animals), during Week 52 (satellite and main group) and at termination (main group only during Week 104), the eyes of all surviving control and high dose level group animals were examined.
Prior to examination, the pupils of each animal were dilated using a Tropicamide ophthalmic solution.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during Weeks 13, 26, 52 and 78 and at termination
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes, overnight
- How many animals: 10 male and 10 female animals from each group
- Parameters checked: Packed cell volume (PCV), Haemoglobin (Hb), Red blood cell count (RBC), Total white cell count (WBC Total), Platelet count (Plts), mean corpuscular haemoglobin concentration (MCHC), Mean corpuscular volume (MCV), Thrombotest (TT), Differential WBC counts and Cell morphology.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during Weeks 13, 26, 52 and 78 and at termination
- Animals fasted: Yes, overnight
- How many animals: 10 male and 10 female animals from each group
- Parameters checked: Total protein, albumin (Alb), globulin (Glob), urea nitrogen, creatinine, sodium (Na), Potassium (K), Calcium (Ca), inorganic phosphorus, chloride (Cl), total cholesterol (Chol), alkaline phosphatase (AP), total bilirubin (bilirubin), glucose, glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), tri-iodothyronine (T3), thyroxine (T4), thyroid stimulating hormone (TSH).

URINALYSIS: Yes
- Time schedule for collection of urine: Weeks 13, 26, 52, 78, 104
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes, overnight
- Parameters checked: Volume, pH, specific gravity and protein content were determined quantitatively. Furthermore, total reducing substances (TRS), glucose, ketones bile pigments, urobilinogen and haem pigments were determined qualitatively. The deposit of a urine sample after centrifugation was examined microscopically for the presence of different cell types and organisms.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On completion of the treatment period all surviving main group animals were sacrificed. Since the terminal procedures took 10 days to complete, the treated animals continued to receive test substance in their diet until the day on which they were sacrificed. All animals were sacrificed by carbon dioxide asphyxiation. The weights of major organs of individual animals dying or sacrificed during the study were recorded at the discretion of the pathologist. The following organs from all animals killed at the scheduled sacrifices were dissected free of fat and weighed: adrenals, kidneys, pituitary, brain, liver, testes (with epididymides), heart, ovaries and thyroid. All superficial as well as subcutaneous tissues and all organs were visually examined.

HISTOPATHOLOGY: Yes
The samples of the following tissues from all animals were preserved and histopathologically examined: Adrenals, aorta (arch and abdominal), bones (sternum and femur), bone marrow (sternum), brain (cerebral cortex, thalamic nuclei, mid-brain, medulla and cerebrum), caecum, colon, duodenum, eyes, femur, heart, jejunum, ileum, kidneys, liver, lungs (all lobes and mainstem bronchi), lymph nodes, mammary gland, other macroscopic abnormalities, oesophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal column, spleen, sternum, stomach, testes (with epididymides), thymus, thyroids/parathyroids, trachea, urinary bladder and uterus (corpus and cervix).

Tissues were preserved in buffered 10% formalin (except eyes, which were preserved in Davidson's fixative).Further tissues were embedded in paraffin wax and sections were cut at 4 µm and stained with haematoxylin and eosin. Frozen sections of the liver were fixed in buffered formalin and cut on a cyrostat at 12 µm and stained for fat with Oil Red O. Liver sections were also stained using PAS for glycogen.

Statistics:

All analyses were carried out both together and separately for male and female.
Data relating to food and water consumption were analysed on a cage basis. For all other parameters, analyses were carried out using the individual animal as the basic experimental unit.
Food consumption data were analysed using cumulative totals and water consumption data were analysed as the total recorded intake over selected time periods, expressed on a weekly basis. Bodyweight data were analysed using weight gains.
The following tests were used for food consumption, bodyweight, organ weight and clinical pathology data:
- If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%), the proportion of animals with values different from the mode was analysed by Fisher and Mantel. Otherwise:
- Bartlett’s test was applied to test for heterogeneity of variance between treatments. Where significant (at the 1% level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
- If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out. If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks was used.
- Analyses of variance were followed by Student’s ‘t’ test and Williams’ test for a dose-related response. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the ‘t’ test and Williams’ test (Shirleys’ test).
Where appropriate, analysis of covariance was used in place of analysis of variance. Mortality was analysed using log rank methods (Mantel, 1966).

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs clearly indicative of a reaction to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no indication of an adverse treatment-related effect when considering the incidence or distribution of decedents throughout treatment. The mortalities observed in the present study were as follows:
Main groups:
Control: 19/50 (males) and 34/50 (females)
60 ppm: 22/50 (males) and 29/50 (females)
180 ppm: 27/50 (males) and 25/50 (females)
540 ppm: 19/50 (males) and 26/50 (females)

Satellite groups:
Control: 2/20 (males) and 0/20 (females)
60 ppm: 1/20 (males) and 1/20 (females)
180 ppm: 1/20 (males) and 1/20 (females)
540 ppm: 3/20 (males) and 0/20 (females)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the first week of treatment, a marked dosage-related reduction in body weight gain was noted for males and females receiving 180 or 540 ppm and this was associated with a reduction in food consumption. Subsequently, body weight gain of males and females receiving 540 ppm, improved noticeably but continued to be lower than that of controls so that absolute mean body weight remained substantially depressed throughout the study. After Week 1, males receiving 180 ppm had a mean body weight gain comparable to that of controls for the majority of the dosing period. Intergroup differences in absolute mean body weight arising from the sacrifice of satellite animals were noted at Week 52 but on statistical evaluation were not of toxicological significance. In the final weeks of the study, however, the mean body weight of this group dropped to a greater extent than that of the concurrent controls resulting mainly from differences in group mean weights caused by decedents and large weight losses prior to their deaths. Females receiving 180 ppm showed a lower mean body weight gain than controls throughout the majority of the study period. In the final weeks of the study, the mean body weight gain of this group was comparable to that of controls. The body weight gain of males and females receiving 60 ppm showed no adverse effect of treatment.
For details, please refer to attachment 1 and 2 under "Overall remarks, attachments".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Consistent with the aforementioned reduction in body weight gain, food intake by animals receiving 540 ppm, and to a lesser degree, those receiving 180 ppm was lower than that of controls during the first week of treatment. Subsequently, the food intake of males and females receiving 540 ppm and females receiving 180 ppm, improved although it continued to be generally lower than that of controls. After Week 1, the food intake of males receiving 180 ppm improved, such that overall mean food intake was considered to be essentially similar to that of controls. The food intake of males and females receiving 60 ppm showed no adverse effect of treatment.
For details, please refer to attachment 3 and 4 under "Overall remarks, attachments".

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food utilisation as assessed by the food conversion ratios was assessed over the first 26 weeks of treatment, the period of fastest growth. Consistent with the effect on body weight gain, which was more marked than the reduction in food intake during Week 1, food utilisation was impaired for both males and females receiving 540 or 180 ppm. Although body weight and food intakes for rats given 540 ppm remained lower than controls, the percentage differences were such that an overall effect on food utilisation was not apparent.
For details, please refer to attachment 3 under "Overall remarks, attachments".

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

no effects observed

Description (incidence and severity):

The measured water consumption during Weeks 12, 25 and 51 of treatment did not reveal any clear treatment-related effects, variations were considered to largely reflect effects on food consumption.
For details, please refer to attachment 3 under "Overall remarks, attachments".

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No lesions likely to prejudice the later evaluation of any toxic effect were noted in the eyes of the rats when an ophthalmoscopic examination was performed prior to the commencement of treatment. No lesions indicative of a reaction to treatment were noted in the eyes of rats receiving 540 ppm in comparison with the controls at the Week 52 or 104 examinations, the lesions seen were those common to the age and strain of rat employed.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematological investigations revealed a reduction in erythrocyte numbers in all treated groups of animals at the first investigation (Week 13) with the effect persisting in females given 60 ppm to Week 26 and in those given 180 or 540 ppm to termination at Week 104. Consequent to these changes in erythrocytes, a reduced packed cell volume was apparent for females given 180 ppm in Week 26 and 52 and in those receiving 540 ppm at Weeks 26, 52, 78 and 104. Additionally, reduced haemoglobin concentrations were apparent for males given 540 ppm at Week 13 only and females of this group from Week 26 to termination. Females given 180 ppm were also affected at Weeks 26 and 52. Changes in the calculated indices, consistent with the effects noted above, were also apparent. Reductions in thrombotest times were noted for treated groups of males at Weeks 13 and 26 only.
Other statistically significant differences noted from control were assessed as random occurrences and considered to be of doubtful toxicological importance at this stage.
In summary, consistent changes in haematological parameters were observed in both males and females receiving 180 or 540 ppm. At 60 ppm, an isolated statistically significant change was observed in males only. Since all values were within background ranges, the small intergroup differences seen for this dose level were considered not to be of toxicological significance.
For details, please refer to attachment 5 under "Overall remarks, attachments".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Measurement of T3, T4 and TSH in Weeks 4, 13, 26, 52, 78 and 104 revealed a dose-related reduction in T3 and T4 values at the Week 4 investigations. The reduction in T4 values was still apparent for treated males at Weeks 13 and 26 but no consistent differences in any of these parameters were apparent at later investigations. Full biochemical investigations in Weeks 13, 26, 52, 78 and 104 showed reductions in albumin values at Week 13 amongst males and females receiving 180 or 540 ppm with a corresponding reduction in total protein (although not statistically significant for females receiving 180 ppm). A similar effect was apparent at Week 26 but limited to males and females receiving 540 ppm. At the Week 52 and subsequent investigations there was a marginal decrease in total protein (not statistically significant in Week 104) in males and a lower protein albumin fraction in females receiving 540 ppm (except in Week 104). Blood urea nitrogen was increased at Week 13 amongst males and females receiving 540 ppm (not statistically significant for females) and at Week 26 for males receiving 180 or 540 ppm. At the Week 52 and subsequent investigations an increase in urea nitrogen was apparent for females receiving 540 ppm (not statistically significant in Week
104). Decreased GPT was noted at the Week 13 and subsequent investigations to termination for males and females receiving 540 ppm (not always statistically significant) and at Weeks 13 and 26 for males given 180 ppm. Increased alkaline phosphatase values were noted at the Week 13 and subsequent investigations to termination for all treated groups of males and females receiving 180 or 540 ppm (although rarely attaining a formal level of statistical significance). Reduced calcium values were apparent at Weeks 13 and 26 for males receiving 540 ppm and females receiving 180 or 540 ppm. Although calcium levels in Week 52 were again apparently lower for females receiving 540 ppm, the importance of this finding at this investigation is debatable, since the value recorded for females receiving 180 ppm exceeded that of controls for this same parameter. A slight, but statistically significant reduction was also noted in calcium levels of treated males in Week 78 (not statistically significant for males receiving 60 ppm). At the Week 13 and subsequent investigations of the study increased chloride values were apparent among females, lower doses being affected at Week 13 and 26 but only those given 540 ppm being affected at Weeks 52 and 78. However, this finding was considered not to be of toxicological significance as intergroup differences were slight and with in background ranges. Values at Week 104 were similar to control. The magnitude of these effects appeared to generally decrease as the actual achieved intake decreased. Other statistically significant differences noted from control were minor in degree and considered not to be of toxicological importance at this stage.
In summary, minor but consistent changes in biochemical parameters occurred in both males and females receiving 180 or 540 ppm. At 60 ppm, values were within the expected ranges and changes were considered not to represent toxicological significance.
For details, please refer to attachment 6 under "Overall remarks, attachments".

Endocrine findings:

not specified

Description (incidence and severity):

For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinalysis revealed a slight increase in urinary pH amongst males receiving 540 ppm with a similar finding noted at 180 ppm during Weeks 26 and 52. Among females a statistically significant increase in urinary pH was only attained in Week 78 for those receiving 540 ppm. A slightly lower volume of urine was noted for females receiving 540 ppm during Week 52. Protein concentrations appeared to be decreased in all treated groups in Week 52, however, upon closer examination this was seen to be caused by a few control animals having very high values. In Week 104, however, there was a statistically significant decrease in protein concentrations of females receiving 540 ppm.
In summary effects on the urinalysis parameters measured were seen at 180 or 540 ppm, were noted at all investigations, but was not considered to be associated with any histopathological changes in the kidney. At 60 ppm no changes were observed.
For details, please refer to attachment 7 under "Overall remarks, attachments".

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A decrease in group mean adjusted adrenal weights of males receiving 180 or 540 ppm was noted at the interim kill, with reductions in mean absolute adrenal weight seen in all treated males at termination. These decreases at Week 105 were not strictly dose-related in degree and achieved statistical significance in males receiving 540 ppm only. The group mean adjusted liver and thyroid weights of all treated groups of females showed a generally dose-related increase, compared to controls, at the interim and terminal kills (although not always statistically significant). Although not attaining statistical significance, a greater mean thyroid weight was noted for males receiving 540 ppm. Additionally, at the interim kill, a dose-related increase in heart weight was apparent for females receiving 180 or 540 ppm, and at the terminal kill decreases were noted for pituitary and kidney weights of males receiving 540 ppm. Changes in relative organ weights reflected those seen in absolute values. Other statistical differences were a reflection of the lower body weights of the animals for organs not sensitive to body weight changes e.g. brain and heart.
For details, please refer to attachment 8 under "Overall remarks, attachments".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic examination of main group rats found dead, sacrificed in extremis or killed at termination revealed the following changes:

Forestomach:
Depressions were observed in a greater number of male and female rats treated with 540 ppm, and male rats treated with 60 or 180 ppm compared with the control rats. Raised areas were observed in 4/50 male rats treated with 540 ppm and 3/50 treated with 180 ppm compared with 0/50 control rats. Thickening was observed in a greater number of terminal male rats treated with 540 ppm, and in a greater number of female rats treated with 540 ppm, compared with the control rats. White discolouration was observed in a greater number of female rats treated with 540 ppm, compared with the control rats.

Stomach antrum mucosa:
Depressions were observed in 8/50 female rats treated with 540 ppm, compared with 2/50 control rats.

Adrenals:
Cysts/cystic adrenals were observed in 4/24 female rats, killed at termination, treated with 540 ppm, compared with 0/16 control rats killed at termination. Cystic enlargement was observed in 2/24 female rats, killed at termination, treated with 540 ppm, compared with 0/16 control rats.

Skeletal muscle:
At termination the incidence of atrophied hind limbs was greater among males given 60 or 540 ppm and females given 540 ppm than controls.

Ovaries:
A greater number of terminal female rats treated with 540 ppm had no corpora lutea visible compared with control rats. There were no statistically significant histopathological findings as this finding is considered not to be of toxicological importance. The incidence and distribution of the remaining macroscopically observed lesions, chiefly attributed to senility, early neoplastic change, habitat and changes secondary to chronic disease and neoplasia were within the expected background range, and therefore were considered to be unrelated to treatment with the test substance.

For details, please refer to attachment 9 and 10 under "Overall remarks, attachments".

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Skeletal muscle:
An increase in the incidence of adipose replacement and narrowing of myofibres in peripheral muscle bundles was recorded in both male and female animals receiving 540 and 180 ppm, which was considered to be related to the treatment with the test substance. Some increase in the incidence of narrowed myofibres was also noted in male animals receiving 60 ppm. At the low dose level of 60 ppm similar changes were observed in a small number of females. Although they were absent in female controls, the incidence at 60 ppm was comparable to the control incidence for males and it is unlikely that a treatment-related effect was present for females at this low dose level. In occasional control and treated animals similar, but minimal, changes were seen as part of the normal background pathology of ageing, in which instances the changes were considered to be generally more diffuse in nature and not confined to the peripheral bundles.

Sciatic nerve/spinal cord:
An increase in the incidence of foci or areas of axonal degeneration was seen in the sciatic nerves of female animals receiving 540 ppm. An increase was also noted in male animals of this group but statistical significance was not achieved. However, the axonal degeneration in sciatic nerves observed at 540 ppm was considered to be ralted to the treatment with the test substance. It is probable that the increase in sciatic nerve lesions was associated with the increase in skeletal muscle changes reported above. Minor degrees of axonal degeneration comparable to that seen as part of the background pathology of ageing in control animals was noted in the sciatic nerves of rats from the low and intermediate dosage groups. In the spinal cords of animals at all dosage levels some degree of minor axonal degeneration was noted, but there was no clear dose relationship in degree or incidence and it was not considered that any treatment effect was involved.

Spleen:
An increase in the incidence and amount of Perls' positive brown pigment aggregation (haemosiderosis) was seen in the spleen of both sexes at 540 ppm and in males receiving 180 and 60 ppm. This pigment is a common background finding in a proportion of ageing animals, however this represents an exacerbation of the background occurrence and was therefore considered to be related to the treatment with the test substance. The effect was generally more pronounced in males than in females.

Liver:
A treatment-related and significantly increased incidence of rats with accumulation of brown pigment in a small number of sinusoidal cells was recorded in both sexes at the 540 and the 180 ppm dose levels when compared to the control levels. Occasional animals from the 60 ppm group also possessed similar pigment at comparable levels of incidence to the control group animals. Sample sections were demonstrated to stain positive by Perls' method for iron indicating that this pigment was probably haemosiderin and thus similar to the pigment seen in the spleens. It is noted that although there was an increase in the number of animals in which this was seen the actual amount of pigment and the number of involved sites/cells in individual animals was in no instance particularly extensive. Bile duct hyperplasia was seen in both control and treated animals at all dose levels but there was a statistically significantly increased incidence in both sexes only at the 540 ppm dose level. An increase was also noted in male animals of the 180 ppm dose group, but this increase did not achieve statistical significance. In almost all cases the degree of hyperplasia was minimal to moderate in both control and treated rats. The increased liver weights reported at the terminal kill may be associated with the above observations.

Kidneys:
An increase in the incidence of brown pigment was seen in the cortical tubular epithelial cells of kidneys in female rats receiving 540 ppm. Although occasionally seen in male animals at this dose level and in both male and female animals at lower dose levels as well as in control animals these other groups showed broadly comparable occurrences. In the affected animals from the highest dose level the amount of pigment was not pronounced. This pigment was (spleen and liver) negative with Perls' stain but was positive with Schmorl's stain for lipofuscin. The incidence of lipofuscin was considered to be related to treatment with the test substance. These minor changes are unlikely to be associated with the urinalysis effects reported above.

Pancreas:
An increase in the incidence and severity of adipose tissue replacement of exocrine pancreas was noted in a proportion of male animals at the 540 and 180 ppm dose levels. A similar increase in male animals of the 60 ppm dose level did not quite achieve statistical significance. No such effect was seen in female animals. There was no concomitant treatment-related effect seen in respect of exocrine atrophy.

Adrenals:
An exacerbation and increase in the incidence and severity of adrenal cortical hypertrophy with varying degrees of cellular vacuolation or, more often, degeneration was seen at all dose levels. Hypertrophic and degenerative lesions of the adrenal cortex are a normal occurrence in ageing rats of this strain and are particularly evident in female animals. This effect was particularly evident in female animals at the 540 ppm dose level where an increase in the incidence and the severity of the lesion from hypertrophy with degeneration to full cystic degeneration was characteristic. An increase, albeit not statistically significant, was also seen in females at the 180 ppm level. In male animals at all dose levels the effect was confined to an increase in the number of animals with cortical hypertrophy and vacuolation. These changes probably represent an exacerbation of normal age-related lesions commonly encountered in rats of this strain and age range.

Thyroids:
An increase in the incidence and prominence of ultimobranchial cysts of the thyroid glands was noted in both sexes at the 540 ppm and 180 ppm dose levels and also in female animals receiving 60 ppm. Small numbers of control animals and males receiving 60 ppm also showed this change. A small number of high dosage animals only showed this change at the 52 week interim stage. The ultimobranchial cyst is a developmental anomaly occasionally seen in a proportion of normal rat thyroid glands and represents the remnants of the embryonic ultimobranchial body, responsible for the distribution of calcitonin secreting C-cells in the thyroid. A small increase in C-cell hyperplasia was seen in occasional male animals (primarily at termination) at the 540 ppm dose level but was not detected in females and thus the aetiology of this change remains equivocal. No treatment-related effects were detected in thyroid follicular tissue.

Stomach:
Increased incidence and severity of hyperplastic and/or ulcerative lesions of the nonglandular stomach epithelium with associated changes, such as subepithelial oedema, were noted in both sexes at the 540 and 180 ppm dose levels and in male animals receiving 60 ppm. In general these changes were more pronounced in male than in female animals. These changes accord with the macroscopic observations for this tissue. These gastric epithelial changes are thought to be the consequence of the irritant action of the compound.

All other changes were within the normal background range of incidence for rats of this strain and age range in this laboratory and were not considered to be of any toxicological significance.
For details, please refer to attachment 11 and 12 under "Overall remarks, attachments".

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Mesenteric lymph nodes:
Benign haemangiomata were seen in the mesenteric lymph nodes (draining the intestinal tract) of five male terminal kill rats receiving 540 ppm. This incidence was statistically significant in comparison to the control group.

Spleen:
A haemangioma was seen in the spleen of a single male decedent animal from the same 540 ppm dose group. Haemangiomata were not observed in other tissues in these animals. They were not seen in any male rats receiving lower doses of the test substance, in any of the female rats, nor in control rats. A haemangiosarcoma was seen in the spleen of a single female intercurrent death animal of the control group. Angiectasis was not an associated finding. Statistical analysis of the relative group incidence for this tumour type by the time-to-tumour methods recommended by the International Agency for Research on Cancer (IARC) demonstrated a statistically significant dose-related trend in the number of animals with tumours and a significant difference between the control and the 540 ppm dosage group. Haemangiomata are benign multilocular tumours occasionally seen in low numbers in rats of this strain and age range, mesenteric lymph nodes and the spleen being the commonest sites for their detection. The incidence which was recorded in the 540 ppm dose group of this study of some 5/50 animals in single site (6/50 in total) is outside the normal background incidence for this laboratory where a range from 0/50 in the majority of instances to a maximum of 2/50 in control animals is recorded for a series of recent and concurrent studies utilising this strain of rat. It is, therefore, considered that the presence of these haemangiomata was a consequence of the administration of the test substance at a dose level of 540 ppm to male animals.

All other changes were within the normal background range of incidence for rats of this strain and age range in this laboratory and were not considered to be of any toxicological significance.
For details, please refer to attachment 11 and 12 under "Overall remarks, attachments".

Key result

Dose descriptor:

LOAEL

Remarks:

general systemic toxicity

Effect level:

60 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

other: additionally, changes in body weight, food consumption and efficiency, and urinalysis were observed at the next higher dose level.

Remarks on result:

other: corresponding to 2.5 mg/kg bw/day for males and 3.4 mg/kg bw/day for females

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

540 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no tumorigenic effects observed at this dose level

Remarks on result:

other: corresponding to 34.6 mg/kg bw/day for females

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

180 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no tumorigenic effects observed at this dose level

Remarks on result:

other: corresponding to 7.7 mg/kg bw/day for males

Key result

Dose descriptor:

LOAEL

Remarks:

carcinogenicity

Effect level:

540 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: corresponding to 23.7 mg/kg bw/day for males

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

540 ppm

System:

other: lymphatic system

Organ:

mesenteric lymph node

spleen

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Table 1: Body weight, haematology and organ weights
Parameter  / Dose	Control		60 ppm		180 ppm		540 ppm
	♂	♀	♂	♀	♂	♀	♂	♀
Body weight			(↓)	(↓)	(↓)	(↓)	(↓)	(↓)
Body weight gain							↓ 14%	↓ 26%
Haematology
Haematocrit (PCV)								↓
Haemoglobin								↓
RBC								↓
Organ weight (rel.)
Testes + epididymides					↑ 12%		↑ 26%
Adrenals							↓ 34%	↑ 56%
Liver								↑ 12%
Brain								↑ 19%
Thyroid								↑ 33%
Heart								↑ 11%
↓ ↑: statistical significance;  (↓ ↑): no statistical significance Table 2: Macroscopic pathology after 104 weeks
Parameter  / Dose Control 60 ppm 180 ppm 540 ppm Dose-response +/– ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ No of animals examined 50 50 50 50 50 50 50 50 Forestomach Depressions 11 8 16 7 20 14 20 31 – + Raised areas 0 0 0 1 3 1 4 0 – – Thickening 5 3 11 3 11 8 12 12 – + White discolouration 4 1 8 1 7 4 6 9 – + Stomach Antrum mucosa depression 6 2 4 3 6 6 4
Table 3:  Micropathology after 104 weeks

Parameter  / Dose	Control		60 ppm			180 ppm		540 ppm		Dose-response +/–
	♂	♀	♂	♀		♂	♀	♂	♀	♂	♀
No of animals examined	50	50	50		50	50	50	50	50
Neoplastic changes
Spleen
Haemangioma	0	0	0	0		0	0	1	0	–	–
Lymphnodes
Haemangioma	0	0	0	0		0	0	5*	0	–	–
Non-neoplastic changes
Spleen
Haemosiderosis	10	24	22*	29		29*	38*	23*	39*	–	–
Skeletal muscle
Adipose replacement	5	0	10	4		27*	21*	43*	26*	+	+
Narrowing of fibres	2	0	10*	4		30*	15*	37*	22*	+	+
Sciatic nerve
Axonal degeneration	14	3	12	5		15	4	22	16*	–	–
Liver
Bile duct hyperplasia	7	5	7	6		13	9	15	17*	+	+
Pigmented sinusoidal cells	2	0	5	3		22*	13*	26*	15*	+	+
Kidneys
Brown pigment in tubular epithelial cells	2	5	6	8		6	9	4	19*	–	–
Pancreas
Replacement by adipose tissue	7	0	14	2		15*	4	21*	3	+	–
Adrenals
Vacuolation	4	1	11*	1		11*	0	12*	2	–	–
Cortical cystic degeneration	2	7	3	9		1	12	2	29*	–	+
Thyroid
Prominent ultimobranchial cyst	4	3	5	12*		14*	22*	15*	27*	–	+
C-cell hyperplasia	1	5	3	5		5	5	8*	3	+	–
Forestomach
Non-glandular hyperplasia	6	7	18*	6		25*	15*	25*	37*	–	+
Non-glandular ulceration or oedema	5	3	8	5		14*	6	14*	12*	–	–
* statistical significance (Fisher’s exact test)

Conclusions:

The present study was conducted to assess the carcinogenicity of the test substance in rats when given for a time frame of approximately 104 weeks. The study was similar to the OECD guideline 453 and was performed under GLP conditions. The test substance was administered via dietary exposure to groups of 50 male and 50 female rats at dose levels of 0, 60, 180, and 540 ppm corresponding to approximately 0, 2.5, 7.7 and 23.7 mg/kg bw/day for males and 0, 3.4, 10.2 and 34.6 mg/kg bw/day for females. Under the conditions of the study, the test substance caused general toxicity as evident by lower body weight gains and food intake, disturbances in food utilisation, haematological, biochemical, urine and organ weight parameters as well as gross pathological and histopathological findings at 60 ppm and/or above. Therefore, no NOAEL with regards to general toxicity was set. Tumorigenic effects were only observed in male animals receiving 540 ppm of the test substance and were limited to haemangiomata in the spleen and mesenteric lymph nodes with no evidence of malignancy. Thus, the NOAEL for carcinogenicity in male rats was set to 180 ppm corresponding to 7.7 mg/kg bw/day and to 540 ppm corresponding to 34.6 mg/kg bw/day for female rats.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Apr 1980 - 01 May 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

adopted 2018

Deviations:

yes

Remarks:

no analytical data on achieved doses, only 8 animals/sex/dose for chronic phase, 10/14 h dark/light cycle, no detailed clinical observations, blood and urine not sampled at 3 month time point, continued under "Principles of method if other than guideline"

Principles of method if other than guideline:

Deviations to the OECD guideline 453:
some blood parameters and histopathology missing (prothrombin time, activated partial thromboplastin time, creatine value, histopathology of vagina and cervix)

GLP compliance:

yes

Species:

rat

Strain:

Fischer 344

Remarks:

(F344/DuCrj) (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc.
- Age at study initiation: approximately 4 weeks
- Weight at study initiation: 55 - 65 g (males) and 55 - 70 g (females)
- Housing: 5 animals of the same sex in iron mesh-type cage, W310 x D440 x H230 mm
- Diet: Powdered food M, Oriental Yeast Co., Ltd., ad libitum
- Water: Tap water provided, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 - 25
- Humidity (%): 50 - 60
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 10/14

IN-LIFE DATES:
Males: From: 16 Apr 1980 To: 22 Apr 1982
Females: From: 23 Apr 1980 To: 01 May 1982

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
The dietary admixture was prepared every week. The food used in this study was analyzed regularly for contaminants which might affect the results of the study during the study period.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

104 weeks (interims-sacrifice after 26, 52 and 78 weeks)

Frequency of treatment:

continuously via diet

Dose / conc.:

20 ppm

Remarks:

corresponding to 0.70 mg/kg bw/day for males and 0.83 mg/kg bw/day for females

Dose / conc.:

200 ppm

Remarks:

corresponding to 6.9 mg/kg bw/day for males and 8.5 mg/kg bw/day for females

Dose / conc.:

2 000 ppm

Remarks:

corresponding to 74 mg/kg bw/day for males and 91 mg/kg bw/day for females

No. of animals per sex per dose:

80/sex/dose

Control animals:

yes, plain diet

Details on study design:

The males and females of each group were sacrificed by eights in Week 26, 52 or 78 of administration to conduct haematological, blood biochemical and pathological examinations. At the end of the study (Week 104) the haematological examination and blood biochemical examination were performed in 10 males and 10 females of each group.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General signs were observed daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: once a week from Day 1 to Week 26 of administration and thereafter once every other week until Week 104 of administration during the study period.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: calculated as g food/rat/day
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Food consumption was measured in males and females twice a week for 8 cages of each dose group specified at the start of the study.

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: Yes
- Time schedule for examinations: Water consumption was measured in males and females twice a week for 8 cages of each dose group specified at the start of the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 26, 52, 78 and 104
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: 8 males and 8 females of each group in Week 26, 52 and 78 of administration and in 10 males and 10 females in week 104 of administration
- Parameters checked: haematocrit (Ht), haemoglobin (Hb), red blood cell count (RBC), mean corpuscular volume (MCY), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count (Platelet) and white blood cell count (WBC). Differential count was determined by multiplying differential white blood cell count (obtained by blood smear specimen stained by May-Grunwald Giemsa method) by WBC count. Reticulocyte count (Retics) was calculated by the blood smear specimens stained with ultravital stain for males in Week 78 of administration and for females in Week 52 and 78 of administration.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 26, 52, 78 and 104
- Animals fasted: Not specified
- How many animals: 8 males and 8 females of each group in Week 26, 52 and 78 of administration and in 10 males and 10 females in week 104 of administration
- Parameters checked: alkaline phosphatase (AL-P), lactate dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), gamma-Glutamyl Transpeptidase (y-GTP, GGTP), total protein (TP), albumin (Alb), globulin (Glob), albumin/globulin ratio (A/G ratio), total bilirubin (T. Bil), direct bilirubin (D. Bil), blood glucose (Glucose), blood urea nitrogen (BUN), total cholesterol (T. Chol) and calcium (Ca). The globulin concentration was obtained by subtracting the albumin concentration from the total protein concentration and albumin/globulin ratio was found from the value. The serum concentrations of sodium (Na) and potassium (K) were measured. Bilirubin was measured by the alkaline azobilirubin method at the time of the examination in Week 26 and 52 of administration and at the time of the examination in Week 78 and 104 of administration by stabilizing diazonium salt method.

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes
- Time schedule for collection of urine: Week 26, 52, 78 and 104
- Metabolism cages used for collection of urine: No, the urine collected by pressing the groin
- Animals fasted: Not specified
- Parameters checked: specific gravity, pH, protein, glucose and occult blood

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The last necropsy in this study was performed on Days 728 (Week 104), and 729 and 730 (Week 105) for males and on Days 728 (Week 104), and 730 and 731 (Week 105) for females. The dead animals were necropsied immediately after they were found dead and the animals killed by moribundity were necropsied immediately after they were killed during the study period. Samples of all the tissues listed below (under histopathology) from all animals were preserved.
After necropsy, the following organ weights were measured in all the animals killed according the killing schedule and in all the surviving animals at the end of the study: brain, pituitary gland, thyroid (including parathyroid gland), thymus, heart, liver, spleen, kidney, adrenal, gonad (testis and ovary), skeletal muscle (crural triceps). However, since the thymus is physiologically retracted severely with ageing, it was not weighed from Week 52 of administration and collected for histological observation only.

HISTOPATHOLOGY: Yes
In histopathological examination, in addition to the above organs (including the thymus), spinal cord (cervical part, chest and lumbar region), sciatic nerve, bone/bone marrow (sternum, femur and knee joint and spine), lymph node (cervical and mesenteric), aorta, tongue, pharynx, salivary gland, oesophagus, stomach, pancreas, duodenum, jejunum, ileum, caecum, colon, head (nasal cavity accessory nasal sinus and middle ear), trachea, pharynx, lung, diaphragm, urinary bladder, epididymis, prostate, seminal vesicle and coagulation gland, uterus, eye ball and accessory gland, skin (dorsolumber part), mammary gland (abdomen) and other organs showing microscopic abnormalities were removed as far as possible from all the animals used and fixed in 10% buffered neutral formalin.
Thereafter the fixed organs and tissues were embedded in paraffin in the usual manner, stained with haematoxylin and eosin and examined histopathologically. The liver of the part of animals killed in week 52 of administration according to the killing schedule was examined by electron microscopy. The removed hepatic tissues were pre-fixed in 3% glutaraldehyde solution and post-fixed in 2% osmic acid solution and embedded in epon in the usual manner. The ultrathin sections were prepared with a ultramicrotome and pre-fixed in uranyl acetate and post-fixed in lead citrate and examined with an electron microscope.

Statistics:

The test of significance was analyzed between the control and treated groups in each test values by Student's t-test. The frequency of histological lesions was analyzed by Fisher's direct probability calculation method.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Abnormal changes in the hindlimbs were observed in 3/56 males of the 2000 ppm group at the terminal stage of the study. In the hindlimbs of these animals, loss of vigor, especially from the knee joint to the crural part and paralytic-like signs were observed. Since changes in hindlimbs were only observed at the highest dose level in one sex and only in 3/56 animals, this observation cannot clearly be related to the treatment with the test substance.
No further treatment-related clinical signs were noted in any of the treated groups.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The cumulative death rates were as follows:
Control: 15/56 males and 21/56 females
20 ppm: 12/56 males and 17/56 females
200 ppm: 15/56 males and 23/55 femlaes
2000 ppm: 22/56 males and 22/56 females
The final cumulative death rate (39%) was slightly increased in males of the 2000 ppm group when compared to that of the control group (27%). No noticable differences were observed for the female rats between the cumulative death rate of the high dose group and the control group. The increase in mortality of males was mainly due to the increase in dead animals due to foreign body pneumonia (and rhinitis) when compared to the control group. There were no differences in the mortality rates between males and females given 200 ppm or less and those of the control group.

For details, please refer to attachment 1 under "Overall remarks, attachments".

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

20 ppm: There were no treatment-related changes in body weight between males and females and those of the control group.
200 ppm: A significant suppression of body weight gain or a tendency to suppress the body weight gain was observed in females from Week 11 to 68 of administration, but thereafter they recovered. No treatment-related changes in body weight were observed in males, when compared to the control group.
2000 ppm: A clear suppression of body weight gain was observed in males and females throughout the study period when compared to the control groups.

For details, please refer to attachment 2 under "Overall remarks, attachments".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

20 ppm: No difference in food consumption was observed in male and female rats relative to the control animals.
200 ppm: A statistically significant decrease in food consumption was sporadically observed in females in the early stage of the study period. Afterwards they recovered and no noticeable differences in food consumption relative to the control group were observed.
2000 ppm: A decrease in food consumption was observed in males and females almost throughout the entire study period and the total mean values decreased by 9% for males and by 17% for females when compared with the respective control group.

For details, please refer to attachment 3 under "Overall remarks, attachments".

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

20 ppm: There were no treatment-related differences in food efficiency compared to the control group.
200 ppm: There were no treatment-related differences in food efficiency compared to the control group.
2000 ppm: The total mean value of food efficiency decreased by 11% for males and by 24% for females, as compared with those in the control group.

For details, please refer to attachment 3 under "Overall remarks, attachments".

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

effects observed, non-treatment-related

Description (incidence and severity):

20 ppm: There were not treatment-related differences in water consumption compared to the control group.
200 ppm: A statistically significant increase in water consumption of males was observed sporadically in some weeks during the study period. However, there were no treatment-related differences in the total mean value for water consumption in males and females when compared to the respective
control groups.
2000 ppm: The total mean values for water consumption for male rats were increased about 13% relative to the control group. A statistically significant increase in water consumption was also observed in females from week 14 to 48 of administration, however, there were no statistically significant differences in the overall mean values for water consumption in either males or females compared to the respective control animals.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

20 ppm: There were no treatment-related changes in haematological parameters observed in males when comapred to the control group. A slight decrease in RBC count in Week 52 of administration and slight decreases in haematocrit and RBC count in Week 78 of administration were observed for female rats relative to the control group, but these changes were extremely slight and therefore not considered toxicologically relevant. There were no further treatment-related changes observed in female rats when compared to the control animals.
200 ppm: No treatment-related changes in haematological parameters were observed in males when compared to the respective control group. In females, decreases in haematocrit, haemoglobin (only week 78 of administration), decrease in RBC count, and increases in mean corpuscular volume and mean corpuscular haemoglobin were observed in week 52 and 78 of administration relative to the control group.
2000 ppm: Slight but statistical significant decreases in haematocrit, haemoglobin and RBC count were observed in males in Week 78 of administration and anemic tendency was also observed when compared to the control group. In females, statistically significant decreases in haematocrit, haemoglobin and RBC count were observed in week 52 and 78 of administration, whereas mean corpuscular volume, mean corpuscular haemoglobin and reticulocyte count were increased relative to the control group. However, most of the changes observed in females recovered almost completely in Week 104 of administration with exception of the decrease in RBC count and the increase in mean corpuscular volume. The increase in mean corpuscular volume observed in females was considered to be due to the increase in regenerated immature erythrocytes.

For details, please refer to attachment 5 under "Overall remarks, attachments".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

20 ppm: Statistically significant increases in GOT and GPT were observed in male rats after 52 Weeks of treatment when compared to the control group. These changes were neither observed in females nor at the other examination time points. Therefore, there was no clear correlation between these changes and the treatment with the test article.
200 ppm: Statistically significant increases in GOT and GPT were observed in male and female rats 26 and 52 weeks after treatment and additionally in males 78 weeks after treatment when compared with the control animals. However, there was no clear dose-dependency as these changes were more marked in the 200 ppm group than in the high dose group, thus there was no clear correlation between these changes and the test article administration.
2000 ppm: Blood glucose, total cholesterol, and calcium were decreased in males at each examination point from Week 52 of administration and in females at each examination point from Week 26 of administration until the end of the treatment period, as compared with those in the control group. This reduction reached statistical significance in most cases. The statistically significant reduction of calcium correlated with observed histopathological findings in the thyroid and bone and was therefore considered to be related to the treatment with the test material. In contrast, the decreases in blood glucose and total cholesterol observed in males and females were considered to reflect the low nutrient state of animals, which showed a marked suppression of body weight gain.

Statistically significant changes were also observed in other examination parameters, but they were not considered to be related to the treatment with the test substance.

For details, please refer to attachment 6 under "Overall remarks, attachments".

Endocrine findings:

not specified

Description (incidence and severity):

For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

20 ppm: No treatment-related differences were observed in males or females when compared to the respective control group.
200 ppm: No treatment-related differences were observed in males or females when compared to the respective control group.
2000 ppm: A statistically significant decrease in urinary specific gravity was observed in males from Week 52 to 104 and in females in Week 26 and 52 of administration when compared to the control animals.

For details, please refer to attachment 4 under "Overall remarks, attachments".

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

20 ppm: No treatment-related changes were observed in male or female rats during the study.
200 ppm: Statistically significant decreases in the absolute and relative weights of the crural muscle were observed in males from Week 52 of administration and in females from Week 78 of administration when compared to the control animals. Furthermore, statistically significant increases in the absolute and relative weights of the spleen were observed in males in Week 52 and in females in Weeks 52 and 78 of administration when compared to the control animals. However, the absolute and relative spleen weights of male and female rats recovered and were similar to those of the control animals at Week 104 after treatment, but it is important to note that the standard deviations of the absolute spleen weight for female rats were very high at this examination time point.
2000 ppm: Statistically significant increase in the absolute and relative weights of the thyroid were observed in females from Week 52 of administration relative to the control group. A statistically significant decrease in the absolute and relative weights of the crural muscle was noted for males and females from Week 52 when compared to the control animals. The relative weight of the thyroid of males and the absolute weight and/or relative weight of the liver and spleen of males and females statistically significantly increased at each examination point when compared to the control group.
In addition to the changes noted above, further statistically significant changes were observed, but they were not considered to be related to the treatment with the test substance.

For details, please refer to attachment 7 under "Overall remarks, attachments".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

20 ppm: No treatment-related gross pathological findings were observed in male or female rats.
200 ppm: No treatment-related gross pathological findings were observed in male or female rats.
2000 ppm: Hypertrophy of the thyroid and atrophy of the crural muscle were observed with a high frequency in males and females at sacrifice schedule in Week 52 and 104 of administration as compared with those in the control group. In males of the same group, proximal arcuation of the tibia and extension deficiency of the knee joint were observed sporadically in some of the animals at the end of the examination. In the soft X-ray examination of the diseased limbs of animals with severe changes, clear extension of the tibia, proximal arcuation of the knee joint, and dilatation of the epiphysis were observed.

For details, please refer to attachment 8 under "Overall remarks, attachments".

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

20 ppm: There were no treatment-related histopathological findings in males and females with regards to non-neoplastic lesions.
200 ppm: The total frequency of follicular hypertrophy of the thyroid (females only), the mucosal cornification of the anterior stomach and the frequency (classified by period) of atrophy of the crural muscle statistically significantly increased from Week 78 of administration when compared to the control group.
2000 ppm: There were statistically significant increases in frequency of various non-neoplastic lesions in the thyroid, knee-joint, crural muscle, sciatic nerve, stomach, bone marrow, spleen, liver, adrenal, nasal cavity, lung and esophagus when compared to the control group. In the thyroid, retention of colloid substance and follicular hypertrophy characterized by squamatization of the epithelial cells increased or tended to increase from Week 26 of administration. The total incidence of these changes were 80% in males and females. Inner bronchial groove remnant of the thyroid also increased statistically significantly, but was not considered to be related to the treatment with the test article. In the knee-joint, excessive remnant of chrondrial plate of the epiphysis (delay in closure of chondrial plate of epiphysis), which is usually closed with aging were observed in males and females. The same lesions were observed mainly in the tibia of males and in both the tibia and femur. Furthermore, chondrial plate of epiphysis of the tibia was hypertrophied in part of the males because of hyperplasia of cartilage cells and hyperplasia of neo-bone trabecula was observed. In the crural muscle, focal muscular atrophy was observed from Week 52 of administration and in the sciatic nerve
, nerve fiber degeneration was observed in males and females with a high frequency in Week 104 of administration. In the stomach, increased mucosal cornification of the anterior stomach increased or tended to increase in males and females from Week 26 of administration. In bone marrow, hematogenesis tended to increase in males and females of the dead animals and animals sacrificed due to moribundity from Week 79 of administration and in females extramedullary hematopoiesis of the spleen increased significantly in some periods. In the liver, frequency of small granuloma increased in males, but there were no further noticeable histological changes and no treatment-related histoptahological changes were observed in hepatic cells using an electron microscope. Therefore, the changes in the liver were not considered to be related to the treatment with the test substance. Frequency (classified by period) of focal sinus dilatation (males and females) associated with blood retention in the adrenal cortex, foreign body rhinitis (males and females) and pneumonia (males) were statistically significantly increased from Week 79 of administration. In males, dilatation of the esophageal cavity, which was considered to be correlated to onset of the above foreign body nasal catarrh and pneumonia were statistically significantly increased in the same period.
In total, follicular hypertrophy of the thyroid, abnormal knee-joint, atrophy of the crural muscle, degeneration of the sciatic nerve, increased mucosal cornification of the anterior stomach and increased hematogenesis of the spleen and bone marrow observed in the high dose groups were considered to be changes caused by the treatment with the test article.

For details, please refer to attachment 9 under "Overall remarks, attachments".

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No neoplastic lesions were observed which were considered to be related to the treatment with the test substance.

Key result

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

20 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: corresponding to 0.70 mg/kg bw/day for males and 0.83 mg/kg bw/day for females

Key result

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

16 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: the NOAEL of 0.7 and 0.83 mg/kg bw/day was converted using a correction factor of 20% based on the assumed instability of the test substance in the diet. Consequently this corresponded to 0.56 and 0.66 mg/kg bw/day for males and females, respectively.

Key result

Dose descriptor:

LOAEL

Remarks:

general systemic toxicity

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

other: additionally, changes in body weight, food consumption and efficiency, haematology, clinical biochemistry, urinalysis, and gross pathology were observed in both sexes at the higher dose level.

Remarks on result:

other: corresponding to 6.9 mg/kg bw/day for males and 8.5 mg/kg bw/day for females

Key result

Dose descriptor:

LOAEL

Remarks:

general systemic toxicity

Effect level:

160 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

other: additionally, changes in body weight, food consumption and efficiency, haematology, clinical biochemistry, urinalysis, and gross pathology were observed in both sexes at the higher dose level.

Remarks on result:

other: the LOAEL of 6.9 and 8.5 mg/kg bw/day was converted using a correction factor of 20% based on the assumed instability of the test substance in the diet. Consequently this corresponded to 5.5 and 6.8 mg/kg bw/day for males and females, respectively.

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

>= 2 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no tumorigenic effects observed at the highest dose level

Remarks on result:

other: corresponding to 74 mg/kg bw/day for males and 91 mg/kg bw/day for females

Key result

Critical effects observed:

no

Conclusions:

The present study was conducted to assess the chronic toxicity and carcinogenicity of the test substance in rats when given for a time frame of approximately 104 weeks. The study was similar to the OECD guideline 453 and was performed under GLP conditions. Fischer 334 rats were given the test substance as a diet admixture at doses levels of 0, 20, 200 or 2000 ppm for 24 months (104 weeks of administration). These dose levels corresponded to 0.7, 6.9 and 74 mg/kg bw/day for male and to 0.83, 8.5 and 91 mg/kg bw/day for female rats. As shown by subsequent repeated-dose studies, the mid- and high-dose level needed to be converted using a correction factor of 20% based on the insufficient stability of the test substance in the diet. Retrospective recalculation of the dose levels 20 and 200 ppm corresponded therefore to achieved doses of 0.56 and 5.52 mg/kg bw/day for males and 0.66 and 6.8 mg/kg bw/day for females.
In the males and females of the 2000 ppm group, marked suppression of body weight gain, decrease in food consumption, decrease in food efficiency, anemia, decrease in serum calcium, increases in the absolute and relative weights of the thyroid, and decreases in the absolute and relative weights of the crural muscle were observed. In histopathological examination, the frequency of follicular hypertrophy of the thyroid, delay in closure of chondrial plate of the tibial and femoral epiphysis and atrophy of the crural muscle increased significantly. In the 200 ppm group, decreases in absolute and relative weights of the crural muscle were observed in the males and females and atrophic changes in the crural muscle were observed histologically with a high frequency, as compared with those in the control group. Furthermore, anemia was observed in females. The frequency of follicular hypertrophy of the thyroid increased significantly. In the 20 ppm group, no abnormal changes related to the treatment with the test article were observed in males or females. According to these results, the NOAEL for general systemic toxicity was set to 20 ppm, which equals a dose of 16 ppm using a correction factor of 20% (corresponding to 0.56 and 0.66 mg/kg bw/day in male and female rats, respectively). Tumorigenic effects of the test substance were not observed during the study period and thus, the NOAEL for carcinogenicity was set to 2000 ppm corresponding to 74 and 91 mg/kg bw/day for male and female rats, respectively.

Reference 3

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Dec 1991 - 15 Jul 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 83-2 (Carcinogenicity)

Version / remarks:

adopted in 1984

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

adopted in 2018

Deviations:

yes

Remarks:

The lowest dose level produced toxicity and was therefore too high. Clinical signs were examined daily only up to week 4.

GLP compliance:

yes

Species:

mouse

Strain:

CD-1

Remarks:

Crl:CD-1 (ICR) BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Michigan, USA
- Age at study initiation: 6 weeks
- Weight at study initiation: 24 - 34 g (males), 18 - 27 g (females)
- Housing: 2 mice per sex were housed in solid bottom polypropylen cages with stainless steel wire tops and autoclaved sifted saydust as bedding.
- Diet: Ground SDS Rat and Mouse No. 1 modified maintenance diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
A pre-mix was prepared each week by grinding the test substance directly into untreated basal diet and mixing in a Turbula Mixer for a minimum period of 2 min. The required concentrations were then prepared by direct dilution of the pre-mix with further quantities of untreated diet, homogeneity being achieved by further mixing in a double cone blender for a period of 7 min. The concentration of test substance in the low dose level was increased by 15% above nominal and the low intermediate by 10% in order to compensate for losses during storage observed in pre-dosing analytical chemistry. On completion of the mixing procedure for each group the diets to be fed at each level were divided into 7 aliquots for daily use during the following week. All aliquots were transferred as quickly as possible to storage at +4 °C until immediately before feeding. An aliquot was removed daily from +4 °C storage and fed to the animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

1) Prior to the start of treatment the proposed diet mixing procedures were checked by chemical analysis to confirm that the method was acceptable and the homogeneity and stability of the formulation was satisfactory under the conditions of the study. This analysis revealed that the test material was not sufficiently stable in rodent diet to permit storage at room temperature for weekly or twice weekly feeding. Therefore, diets should be prepared weekly and stored at +4 °C prior to use with 15% fortification of the low inclusion level and 10% fortification of the low intermediate inclusion level of the test substance.

2) Samples of diets of the present study prepared in Weeks 1, 13, 26, 39, 52, 65, 73 and 78 were also analysed to check the accuracy of preparation. Samples were taken at preparation and further samples stored for 6 days at +4 °C followed by exposure at room temperature for 24 h. The mean results for post-dose samples were all within +5 and -17% of nominal concentrations with the exception of one result, which was 48% below the nominal values. Further, the test substance was confirmed to be homogeneously blended in rodent diet. The stability in rodent diet showed a maximum concentration loss of 23% at 25 ppm and 13% at 50 ppm during the 8-day storage period.
In total, the results indicate that the formulations were homogeneous and stable from the time of preparation to completion of dosing under the study conditions.

Duration of treatment / exposure:

80 weeks

Frequency of treatment:

Daily

Post exposure period:

continuously via diet

Dose / conc.:

25 ppm

Remarks:

The concentration of test material in the low dose level was increased by 15% above nominal (29 ppm) to compensate losses during storage. In total this corresponded to 3 mg/kg bw/day for males and 4 mg/kg bw/day for females.

Dose / conc.:

75 ppm

Remarks:

The concentration of test material in the low dose level was increased by 10% above nominal (83 ppm) to compensate losses during storage. In total this corresponded to 9 mg/kg bw/day for males and 11 mg/kg bw/day for females.

Dose / conc.:

225 ppm

Remarks:

corresponding to 27 mg/kg bw/day for males and 33 mg/kg bw/day for females

Dose / conc.:

675 ppm

Remarks:

corresponding to 82 mg/kg bw/day for males and 95 mg/kg bw/day for females

No. of animals per sex per dose:

50

Control animals:

yes, plain diet

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for the first 4 weeks thereafter once weekly for any signs of behavioural changes, reaction to treatment or ill health; twice daily for moribund or dead animals
- Cage side observations checked: signs of behavioural changes, reaction to treatment or ill health an d or moribund or dead animals

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily for the first 4 weeks thereafter once weekly
- Cage side observations checked: detailed palpations

DERMAL IRRITATION (if dermal study): No

BODY WEIGHT: Yes
- Time schedule for examinations: at the time of allocation of animals to groups, one week before commencement of treatment, on the day of commencement of treatment and once a week thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as gfood/kg body weight/day: The quantity of food consumed by each cage of mice was recorded daily and reported on a weekly basis. Food consumption was calculated from the raw data using the total amount of food given to and left by each cage in each group and the number of rats surviving in each cage and presented as weekly food consumption in g/mouse/week.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Substance intake (mg/kg bw/day) was calculated from the group mean body weight and food consumption and the nominal dietary inclusion levels of the test material.

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, but food conversion ratios were calculated, where possible, over the period Weeks 1 to 26 from the body weight and food consumption data as weight of food consumed per unit gain in body weight.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily by visual appraisal of the water bottles

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during Week 52 and immediately prior to termination
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: 10 male and 10 female animals from each group
- Parameters checked: Differential leukocyte count, cell morphology

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On completion of 80 weeks of treatment, all surviving animals were killed. Since the terminal procedures took 15 days to complete, the treated animals continued to receive test substance in their diet until the day on which they were killed. All animals were killed by carbon dioxide asphyxiation, weighed and then subjected to necropsy procedure. Samples of all the tissues listed below from all animals were dissected out and weighed, paired organs being weighed separately: brain, kidneys, liver, testes (with epididymides).

HISTOPATHOLOGY: Yes
Samples of tissues from all animals were preserved in neutral buffered 10% formalin (except eyes, which were preserved in Davidson's fixative). Additional pieces of liver and kidney were placed in formol calcium.
The following organs and tissues were examined for histopathology: Adrenals, aorta, bones (sternum and femur), bone marrow (sternum), brain, caecum, colon, duodenum, eyes, gall bladder, heart, jejunum, ileum, kidneys, liver, lungs, lymph nodes, mammary gland, other macroscopic abnormalities, oesophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal column, spleen, stomach, testes (with epididymides), thymus, thyroids/parathyroids, trachea, urinary bladder and uterus.
These tissues were embedded in paraffin wax and sections cut at 4 µm were stained with haematoxylin and eosin. Frozen sections of liver, fixed in buffered formalin were cut on a cryostat at 12 µm and stained for fat with Oil Red O. Sections of kidney were stained with Oil Red O or periodic acid-Schiff reagent.

Statistics:

All analyses were carried out separately for male and female.
Data relating to food consumption were analysed on a cage basis. For all other parameters, analyses were carried out using the individual animal as the basic experimental unit.
Food consumption data were analysed using cumulative totals and bodyweight data were analysed using weight gains.
The following tests were used for food consumption, bodyweight, blood smear and organ weight data:
- If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%), the proportion of animals with values different from the mode was analysed by Fisher and Mantel. Otherwise:
- Bartlett’s test was applied to test for heterogeneity of variance between treatments. Where significant (at the 1% level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
- If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out. If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks was used.
- Analyses of variance were followed by Student’s ‘t’ test and Williams’ test for a dose-related response. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the ‘t’ test and Williams’ test (Shirleys’ test).
Where appropriate, analysis of covariance was used in place of analysis of variance. Mortality was analysed using log rank methods, Mantel.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed which were considered to be related to treatment with the test substance.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no indication of an adverse treatment-related effect when considering the incidence or distribution of decedents throughout treatment. The mortalities observed in the present study were as follows:
Control: 16/50 (males) and 12/50 (females)
25 ppm: 16/50 (males) and 15/50 (females)
75 ppm: 9/50 (males) and 10/50 (females)
225 ppm: 12/50 (males) and 10/50 (females)
675 ppm: 17/50 (males) and 15/50 (females)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the first week of treatment a dose-related increase in body weight gain was noted for males receiving 75, 225 or 675 ppm. Treated females had body weight gains comparable to controls. These observations were not accompanied by concomitant effects on food consumption. After Week 1, however, a dose-related reduction in body weight gain among males and females receiving 225 or 675 ppm was apparent. After 80 weeks, with the exception of the gain of females receiving 225 ppm, these reductions in gain, compared to controls, were statistically significant. The body weight gain of males receiving 25 ppm and females receiving 25 or 75 ppm showed no adverse effect of treatment.
For details, please refer to attachment 1 under "Overall remarks, attachments".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A dose-related, statistically significant reduction in food intake was apparent for males and females receiving 225 or 675 ppm, compared to controls. In the absence of any body weight changes for males receiving 75 ppm and females receiving 25 or 75 ppm, the statistically significant reductions in food intake, compared to controls, are considered not to be of any toxicological significance. The food intake of males receiving 25 ppm was unaffected by treatment.
For details, please refer to attachment 2 under "Overall remarks, attachments".

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

During the first week of treatment, food conversion ratios calculated for males receiving 75, 225 or 675 ppm were superior to that of the control value, reflecting the increased body weight gain, independent of an effect on food consumption, for these groups. Food conversion ratios calculated for treated groups of males were generally inferior to those of controls, while those of treated females were highly variable with no consistent trend apparent.
For details, please refer to attachment 2 under "Overall remarks, attachments".

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

no effects observed

Description (incidence and severity):

Visual appraisal of the water bottles did not reveal any treatment-related effects.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

From the examination of blood smears from control and high dose animals, it was considered that there were no treatment-related effects on the haematological parameters measured. The statistically significant differences from control were minor in degree and within background ranges and were considered therefore not to be of toxicological significance.

Clinical biochemistry findings:

not examined

Endocrine 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, treatment-related

Description (incidence and severity):

Analysis of absolute organ weights or organ weights adjusted for final body weight as covariate showed a statistically significant, dose-related decrease in absolute brain weight of males receiving 225 or 675 ppm (with associated increase in body weight-related brain weight) and adjusted brain weight of females receiving 75, 225 or 675 ppm. The mean liver (both absolute and relative to body weight) weights of all treated groups was unaffected by treatment, even though histopathologically centrilobular enlargement of hepatocytes was seen in males and females from all groups. The mean body weight-related kidney weight of males receiving 675 ppm and testes with epididymides weight of males receiving 225 or 675 ppm were higher in comparison with controls. As these changes were not associated with any intergroup differences in absolute kidney or testes with epididymides weights they were considered not to be of toxicological significance but to be an effect of the lower mean body weight of these groups.
For details, please refer to attachment 3 under "Overall remarks, attachments".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The macroscopic examination of all decedents and animals killed at termination of the study revealed the following changes:

Adipose tissue:
A reduction in adipose tissue was observed in a greater number of decedent male mice (11/17) treated with 675 ppm, compared with the control mice (5/16).

Forestomach:
Roughened forestomachs were observed in a greater number of female mice (6/50) treated with 675 ppm, compared with the control mice (2/50). White discolouration was observed in a greater number of female mice (10/50) treated with 675 ppm, compared with control mice (5/50).

Kidneys:
Irregular cortical scarring was observed in a greater number of terminal male mice (14/33) treated with 675 ppm, compared with the control mice (5/34). Brown kidneys were observed in 5/50 male mice treated with 675 ppm, compared with none in the control mice.

These macroscopic changes however, were not related to any histopathological findings and therefore were considered not to be of toxicological significance. The incidence and distribution of the remaining macroscopically observed lesions, chiefly attributed to senility, early neoplastic change, habitat and changes secondary to chronic disease and neoplasia, were within the expected background range and therefore, were considered to be unrelated to treatment.
For details, please refer to attachment 4 under "Overall remarks, attachments".

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver:
An increased incidence of centrilobular enlargement of hepatocytes, sometimes with vacuolation, and/or generalised enlargement of hepatocytes was seen in males and females from all treated groups when compared to controls. This did not show any clear dose relationship. The severity of this change was only minimal in most of the cases and this change was not associated with liver weight changes or macroscopic observations.

Urinary bladder:
An increased incidence of epithelial hyperplasia was seen in male and female mice receiving 675 ppm and in males receiving 225 ppm when compared to control mice. The severity of this change was only minimal in most of the cases reported.

For details, please refer to attachment 5 and 6 under "Overall remarks, attachments".

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Neoplastic lesions reported in the study did not reveal any treatment-related effect, either on the incidence of any tumour type or on the total number of mice with benign or malignant neoplasms per group. The incidence of the various neoplasms observed in this study showed no significant deviation from the expected tumour profile of laboratory-maintained mice of this strain. In this study lymphoid tumours and pulmonary tumours among male and female mice and liver tumours among male mice were among the most frequently observed tumours. Other types of tumours were of low incidence.
For details, please refer to attachment 5 and 6 under "Overall remarks, attachments".

Key result

Dose descriptor:

LOAEL

Remarks:

general systemic toxicity

Effect level:

25 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

other: additionally, changes in body weight, food consumption, organ weights and gross pathology were observed at higher dose levels.

Remarks on result:

other: corresponding to 3 mg/kg bw/day for males and 4 mg/kg bw/day for females

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

675 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no tumorigenic effect observed at the highest dose level tested

Remarks on result:

other: corresponding to 82 mg/kg bw/day for males and 95 mg/kg bw/day for females

Key result

Critical effects observed:

no

Table 7.7 -B1   Histopathology of the relevant tissues
Parameter  / Dose	Control		25 ppm		75 ppm		225 ppm		675 ppm		Dose-response +/–
	♂	♀	♂	♀	♂	♀	♂	♀	♂	♀	♂	♀
Liver / number examined	50	50	50	50	50	50	50	50	50	50
Centrilobular hepatocyte enlargement	2	0	21	12	21	19	25	16	19	13	–	–
Centrilobular hepatocyte enlargement and  vacuolation	0	0	7	0	9	2	5	1	5	1	–	–
Generalised enlargement	1	1	9	10	12	10	15	7	6	7	–	–
Urinary bladder / number examined	50	48	50	50	46	48	50	49	50	49
Epithelial hyperplasia	7	0	7	5	9	1	20	5	31	14	+	–

Conclusions:

The present study was conducted to assess the carcinogenicity of the test substance in mice when given for a time frame of approximately 80 weeks. The study was similar to the OECD guideline 451 and was performed under GLP conditions. The test substance was administered via dietary exposure to groups of 50 male and 50 female mice at dose levels of 0, 25, 75, 225 and 675 ppm corresponding to approximately 0, 3, 9, 27 and 82 mg/kg bw/day for males and 0, 4, 11, 33 and 95 mg/kg bw/day for females. Under the conditions of the test, the test substance caused general toxicity as evident by lower body weight gains and food intake, changes in organ weight parameters as well as gross pathological and histopathological findings at 25 ppm and/or above. Therefore, no NOAEL with regards to general toxicity was set. Tumorigenic effects of the test substance were not observed during the study period and thus, the NOAEL for carcinogenicity was set to 675 ppm corresponding to 82 and 95 mg/kg bw/day for male and female mice, respectively.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

7.7 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The quality of the database is good comprising three carcinogenicity studies in rodent species (rat and mouse), similar to OECD TG 453 and 451, respectively, under GLP conditions. All studies are considered of reliable quality and validity, fulfilling the criteria of a key study. Thus, all three studies are suitable for assessment of the present endpoint.

System:

other: lymphatic system

Organ:

spleen

other: mesenteric lymphnodes

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

No data available.

Justification for classification or non-classification

The available data on the carcinogenicity of the substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.

Additional information

Three key studies were conducted to assess the carcinogenic potential of the test substance, two in rats and one in mice. All three studies were performed under GLP conditions according to OECD guidelines.

Carcinogenicity study in rats:

A combined chronic and carcinogenicity study on rats was considered as key, as the study was conducted similar to OECD guideline 453 and under GLP conditions (Report number: ZIR 9/942098). The test substance was administered via dietary exposure to male and female Sprague-Dawley rats. As also observed in the short- and long-term repeated dose studies (please refer to the respective end point summary under repeated dose toxicity), the test substance caused general toxicity as evident by lower body weight gains and food intake, disturbances in food utilization as well as changes in blood parameters. Changes in blood parameters (haematological and clinical biochemistry) consisted of a dose-related reduction in T4 serum and minor, but consistent changes were observed for the number of erythrocytes, packed cell volume, haemoglobin concentration, thrombotest times (males), albumin, total protein, urea nitrogen, glutamic-oxaloacetic transaminase, alkaline phosphatase and calcium. The decrease of erythrocytes parameters were suggestive of anemia. Further, there were effects on organ weight, gross pathological and histopathology, the latter including haemosiderin deposits in spleen and liver, cortical degeneration of adrenals (males), increased prominence of ultimobronchial cysts in the thyroid and C-cell hyperplasia (females), degenerative/atrophic skeletal muscles (males), axonal degeneration on spinal cord and sciatic nerves, adipose replacement of exocrine pancreas (males), lipofuschin accumulation in kidneys (females), bile duct hyperplasia in the liver and hyperplastic erosive lesions of the non-glandular epithelium in the stomach. Beside these treatment-related non-neoplastic lesions, also neoplastic histopathological findings were observed, however solely in male rats treated with the highest dose level (540 ppm, corresponding to 23.7 mg/kg bw/day). The treatment-related neoplastic findings observed in males referred to haemangiomata in mesenteric lymph nodes and spleen. These haemangiomata were not pronounced, did not show evidence of malignancy and were confined to this dose group and sex only. However, the NOAEL for carcinogenicity in male rats was set to 180 ppm corresponding to 7.7 mg/kg bw/day and to 540 ppm corresponding to 34.6 mg/kg bw/day for female rats.

A previously conducted carcinogenicity study on rats was also considered as key study (1983, Report number: 83-51-21). The study was conducted similar to OECD guideline 453 and according to GLP. Fischer 344 rats were given the test substance as a diet admixture at four dose levels of 0, 20, 200 or 2000 ppm for 24 months (104 weeks of administration). These dose levels corresponded to 0.7, 6.9 and 74 mg/kg bw/day for male and to 0.83, 8.5 and 91 mg/kg bw/day for female rats. As shown by subsequent repeated-dose studies, the low- and mid-dose level need to be converted using a correction factor of 20% based on the insufficient stability of the test substance in the diet. Retrospective recalculation of the dose levels 20 and 200 ppm corresponded therefore to achieved doses of 0.56 and 5.52 mg/kg bw/day for males and 0.66 and 6.8 mg/kg bw/day for females. The results of this combined chronic and carcinogenicity study mostly support the results of the previously mentioned key study (Report number: ZIR 9/942098). After dietary exposure of the test substance to male and female Fischer 344 rats, general toxicity was observed by decreased body weight gain, food consumption, food efficiency and changes in blood parameters. In contrast to the previously described key study, no treatment-related neoplastic findings were observed either in male nor female rats in the present study. Non-neoplastic histopathological findings considered to be related to the treatment with the test substance included follicular hypertrophy of the thyroid, abnormal knee-joint, atrophy of the crural muscle, degeneration of the sciatic nerve, increased mucosal cornification of the anterior stomach and increased hematogenesis of the spleen and bone marrow at the high dose group. These histopathological findings were accompanied by organ weight changes (crural muscle, spleen and thyroid) and/or gross pathological findings. As no tumorigenic effects were observed in the present study, the NOAEL for carcinogenicity was set to ≥ 2000 ppm, which was the highest dose level tested and corresponded to 74 mg/kg bw/day for males and 91 mg/kg bw/day for females.

Carcinogenicity study in mice:

To assess the carcinogenicity of the test substance in mice, a carcinogenicity study was conducted similar to the OECD guideline 451 and under GLP conditions. The test substance was administered via dietary exposure to male and female CD-1 mice for a time frame of approximately 80 weeks. Under the conditions of the test, the test substance caused general toxicity as evident by lower body weight gains and food intake, changes in organ weight parameters as well as gross pathological and histopathological findings at the lowest dose tested. Changes in organ weights and macroscopic changes were not accompanied by histopathological effects, which included increased incidences of centrilobular and/or generalised hepatocellular enlargement and urinary bladder epithelial hyperplasia. Tumorigenic effects of the test substance were not observed during the study period and thus, the NOAEL for carcinogenicity was set to the highest dose tested (675 ppm corresponding to 82 and 95 mg/kg bw/day for male and female mice, respectively).

Conclusion:

Overall, the lowest NOAEL with respect to carcinogenicity was set to 180 ppm corresponding to 7.7 mg/kg bw/day in male rats based on observed haemangiomata in mesenteric lymph nodes and spleen. However, it is important to note, that the haemangiomata were not pronounced, did not show evidence of malignancy, were only confined to the highest dose tested and in one sex, and were not verified in rat, even at considerably higher dose levels, or mouse by additional carcinogenicity studies. Thus, the available data on the carcinogenicity of the substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.