Thiram

Carcinogenicity

Description of key information

Key, report number HLA 6111-113, combined chronic toxicity/carcinogenicity studies (52/104 weeks, rat):

NOAEL (systemic toxicity) 30 ppm, corresponding to 1.5 mg/kg bw/day for males and 1.8 mg/kg bw/day for females)

NOAEL (carcinogenicity) 30 ppm, corresponding to 1.5 mg/kg bw/day for males and 1.8 mg/kg bw/day for females

 

Key, report number 798-223, carcinogenicity studies (97 weeks, mice):

NOAEL (systemic toxicity) 30 ppm, corresponding to 3 mg/kg bw/day for males and females

NOAEL (carcinogenicity) 300 ppm, corresponding to 50 mg/kg bw/day for males

NOAEL (carcinogenicity) 600 ppm, corresponding to 112 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:

20 Jul 1989 - 3 Jun 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

adopted in 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-2 (Carcinogenicity)

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 Laboratories, Raleigh, North Carolina
- Age at study initiation: approx. 7.5 weeks
- Weight at study initiation: 27.8 +/- 2.2 g; Females: 21.2 +/- 1.7 g
- Fasting period before study: not applicable
- Housing: maximum of 2 animals of the same sex per cage, stainless-steel cages with wire-mesh floors
- Diet: Purina Certified Rodent Laboratory Chow #5002, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 24 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9 - 25.6
- Humidity (%): 30 - 70
- Air changes (per hr): minimum of 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 Jul 1989 To: 3 Jun 1991

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with (basal diet): the test material was ground using a mortar and pestle until a fine powder was achieved. For each dietary level, the required amount of test article was premixed in a Waring blender using approx. 200 g of basal feed. This premix was then mixed with appropriate amounts of the basal diet.
- Storage temperature of food: frozen until used, then room temperature
New food was presented to the mice every afternoon and the remaining old feed was removed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method used to assay the level of test substance in the basal diet involved extraction of the test article from the feed mixture using an acetone/triethylene solvent and analysis via HPLC.
Homogeneity and stability was assessed prior to initiation of treatment and in previous studies. From Week 1 mixes, additional samples were collected to assess stability under the conditions of the test (the feed was analysed on Day 0 after preparation and on Day 1 after exposure to room temperature as well as at Day 7 after being frozen for 6 days and kept at room temperature for 1 day). Stability at Day 0 ranged from 77.9 to 102% of the target concentration. Samples of Day 1 reached 50.7 (15 ppm) - 97.2% (600 ppm) of the target concentration and samples assayed on Day 7 (after 6 days of freezing and 1 day at room temperature) reached 55.2 (15 ppm) - 96.6% (600 ppm) of the target concentration.

Homogeneity was also determined in samples from Week 1 and was considered satisfactory.

Routine assays for test diet concentrations were performed in Week 1, 2, 3, 4 and monthly (either 4- or 5-week intervals) through the in-life phase of the study. The mean recoveries were 88 - 101% of the target concentrations.

Duration of treatment / exposure:

97 weeks (extended from planned 78 weeks due to high survival)

Frequency of treatment:

continuously via the diet

Post exposure period:

none

Dose / conc.:

15 ppm

Remarks:

males, Group 2, corresponding to approx. 2.5 mg/kg bw/day.

Dose / conc.:

150 ppm

Remarks:

males, Group 3, corresponding to approx. 24 mg/kg bw/day.

Dose / conc.:

300 ppm

Remarks:

males, Group 4, corresponding to approx. 50 mg/kg bw/day.

Dose / conc.:

15 ppm

Remarks:

females, Group 2, corresponding to approx. 3 mg/kg bw/day.

Dose / conc.:

300 ppm

Remarks:

females, Group 3, corresponding to approx. 57 mg/kg bw/day.

Dose / conc.:

600 ppm

Remarks:

females, Group 4, corresponding to approx. 112 mg/kg bw/day.

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: twice daily for mortality and moribundity, and daily for signs of ill-health

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice weekly

BODY WEIGHT: Yes
- Time schedule for examinations: prior to the first dosing (Week 0), weekly through Week 14, once every 4th week thereafter through Week 94, and during Week 97

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean weekly diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Food consumption was measured daily throughout Week 14 and daily for a 1-week period every 4th week through Week 94.

FOOD EFFICIENCY: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Weeks 53 and 97/98
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: 10 per sex and dose (Week 53), all surviving animals of all groups (Week 97/98), leukocyte differentials, cell morphology, reticulocyte count were initially assessed only for control and high dose animals. Only if differences between the groups were found, low and mid dose animals were examined for these parameters.
- Parameters checked: leukocyte count (WBC), erythrocyte count (RBC), haemoglobin (HGB), haematocrit count (HCT), platelet count (PLATELET), mean cell volume (MCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), leukocyte differentials, cell morphology, reticulocyte count, myeloid/erythroid ratios (for all females).

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All surviving animals at scheduled sacrifice or on all mice that died or were killed in moribund condition.
Examination of external surface, all orifices, cranial cavity, carcass, external surfaces of the brain (at necropsy), external surface of spinal cord and cut surfaces of brain and spinal cord (at time of tissue processing), nasal cavity and paranasal sinuses, thoracic, abdominal, and pelvic cavities and their viscera, cervical tissues and organs.
Additional, bone marrow smears (proximal femur) were prepared from all animals at scheduled sacrifice.

Organs weighed: Brain (including brainstem), kidneys, liver (with gall bladder), testes (with epididymides), ovaries (postfixation)
- How many animals: 10/sex/dose

HISTOPATHOLOGY: Yes
- Tissues collected: Masses and associated tissues, lesions, brain with brainstem (medulla/pons, cerebellar cortex, and cerebral cortex), adrenals, aorta (thoracic), bones (sternum and femur), bone marrow (sternum and femur), caecum, colon, duodenum, oesophagus, eyes, gall bladder, heart, jejunum, ileum, kidneys, liver, lungs, mammary gland, mesenteric lymph node, mandibular lymph node, ovaries, pancreas, pituitary, prostate, rectum, salivary gland (mandibular), sciatic nerve, seminal vesicles, skeletal muscle, skin, mid-thoracic spinal cord, lumbar spinal cord, spleen, stomach, testes (with epididymides), thymus, thyroids/parathyroids, trachea, urinary bladder, uterus with vagina and cervix
- Fixative: 10% neutral-buffered formalin
- Embedding media: Paraffin
- Staining: haematoxylin and eosin
- How many animals: all animals of all the high and control groups, all animals that died or were sacrificed during the study, gross lesions as well as lung, liver, nonglandular stomach, eyes, urinary bladder and kidneys were examined from all dose groups. The spleen and adrenal cortex from females of the low and mid dose group were also examined.

Statistics:

Statistical analyses (body weight, food consumption, clinical pathology data (except cell morphology gradings), terminal body weights, and organ weight data) were performed by one-way analysis of variance (ANOVA) techniques and trend. If variances of untransformed data were heterogeneous, transformations were performed on the data to achieve variance heterogeneity. When the series of transformations was not successful in achieving variance homogeneity, analyses were performed on rank-transformed data.
Group comparisons were performed routinely at the 5% two-tailed probability level, or at the 5% one-tailed probability level. All trend statistics were evaluated at the one-tailed 5% probability level.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Many control and test substance animals exhibited skin lesions (scores or reddened areas on ears, head, various body areas) consistent with bacterial dermatitis. This finding is often observed in mice. There was no significance difference between control and treatment animals. However, the generally consistent pattern of these findings (males - throughout the majority of the study; females - primarily during the later phase of the study) suggested a test-substance related increased incidence of skin lesions in the highest dose group.

Summarized results can be found in Attachments 2 (Summary of results) in the attached background material.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No treatment-related effects were observed. Survival rates at termination were 60, 50, 64 and 58% for males and 60, 57, 58, and 54% for females (Group 1, 2, 3, and 4, respectively).

Summarized results can be found in Attachment 1 (Overview of relevant data) and Attachments 2 (Summary of results) in the attached background material.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Group 2: No effects were observed in the males and females of the low-dose group compared to the control group.
- Group 3: mean body weights of males and females were statistically significantly decreased, beginning at Week 4 for males and Week 5 for females. A significant negative trend was seen at Week 3 and 4 in males and females, respectively.
- Group 4: mean body weights of males and females were statistically significantly decreased, beginning at Week 4 for males and Week 5 for females. A significant negative trend was seen at Week 3 and 4 in males and females, respectively.

Summarized results can be found in Attachment 1 (Overview of relevant data) and Attachments 2 (Summary of results) in the attached background material.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Group 2: No effects were observed in the males and females of the low-dose group compared to the control group.
- Group 3: A significant negative trend and statistically significantly decreased mean food consumption values were observed. The data are consistent with the decreases observed in the body weight development of the groups.
- Group 4: A significant negative trend and statistically significantly decreased mean food consumption values were observed. The data are consistent with the decreases observed in the body weight development of the groups.

Summarized results can be found in Attachment 1 (Overview of relevant data) and Attachments 2 (Summary of results) in the attached background material.

Food efficiency:

no effects observed

Description (incidence and severity):

No effects were observed. In general, food efficiency was high varaibility.

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- Group 2: No treatment-related effects in males and females.
- Group 3: In males, statistically significantly increased MCV (+6%) and MCH (+3.9%) values, and statistically significantly decreased MCHC (-1.5%) values. Females had statistically significantly reduced RBC values (-7.7%), and statistically significantly increased WBC (max of +105% in Week 53) and platelet values (+18.7%). All values were compared to the control groups and for termination, if not stated otherwise.
- Group 4: MCHC values (-1.2%) were statistically significantly decreased in males. In females platelet counts were statistically significantly increased (+28.2%), whereas the RBC, HGB and HCT values were statistically significantly decreased (-10.8%, -8.2% and -7.7%, respectively), although the mean values and most individual values were within the historical control ranges. Cell morphology data showed no definite compound-related changes. All values were compared to the control groups and for termination, if not stated otherwise.

Summarized results can be found in Attachment 1 (Overview of relevant data) and Attachments 2 (Summary of results) in the attached background material.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study: histopathology of cervix, epididymis, mammary gland, ovary, prostate, seminal vesicles, testis, thyroid, pituitary, uterus, vagina and adrenals, organ weights were recorded for liver, ovary, testis and brain. For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test-substance-related organ weight findings indicative of direct or indirect organ toxicity were observed. Statistically significant decreases were observed in the mean absolute brain weights of the 300 and 600 ppm females, were contributed to the lower body weights in these groups. In addition, the relative organ weight values were not significantly altered in these groups.

Summarized results can be found in Attachments 2 (Summary of results) in the attached background material.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test-substance-related effects were observed on gross tissue changes.

Summarized results can be found in Attachments 2 (Summary of results) in the attached background material.

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following tissues showed abnormalities:

Stomach: Treatment-related hyperkeratosis of the nonglandular stomachs was observed in mid-dose males, and in both sexes of the high-dose group. This finding was characterized by a minimal to moderate increase in keratin in the nonglandular stomach, suggesting a mild irritant effect of the test substance in this region. There was no evidence of degeneration, necrosis, or inflammation.

Eyes: Retinal atrophy was present in the eyes of mid-and high-dose mice, with the severity being the greatest in high-dose females. Microscopic evaluation revealed a decrease to an absence of the outer nuclear cell layer of the retina. This lesion was not specifically associated with cataracts.

Urinary bladder: Intracytoplasmic protein-like droplets were observed in the transitional epithelium (umbrella cells) of both sexes of the mid- and high-dose groups. There was no morphologic evidence of necrosis or inflammation directly associated with the superficial transitional cells and the underlying epithelium was generally not involved.

Adrenals: Decreased pigment was noted in the inner adrenal cortex of mid-and high-dose females. The pigment is morphologically compatible with ceroid and is normally associated with the degeneration of the x-zone that occurs spontaneously. The function of the x-zone in female mice is unknown. Thus, the importance of the decrease in incident and severity of the pigment is unclear and was considered to have little biological significance.

Spleen: Increased pigment was observed in mid- and high-dose females. This pigment was morphologically compatible with hemosiderin and likely represents increased splenic red blood cell turnover associated with test substance administration. This finding is correspondent with the decreased red cell mass (RBC, HGB, HCT) found in mid- and high-dose female mice.

Skin: There was a high incidence of necrosis and suppurative inflammation in the skin of control and treated animals. These findings generally correlated to the ear and body sores observed in-life and at necropsy. Although present in all groups, dermal necrosis and suppurative inflammation were most severe in the mid- and high-dose group. Microscopic evaluation of the lesions revealed epidermal and dermal necrosis with a marked neutrophil infiltrate. Bacterial colonies were often present. These lesions were morphologically compatible with focal staphylococcal dermatitis, which is not an uncommon finding in aged mice.

Summarized results can be found in Attachment 1 (Overview of relevant data) and Attachments 2 (Summary of results) in the attached background material.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No compound-related oncogenic effects were observed.

Summarized results can be found in Attachments 2 (Summary of results) in the attached background material.

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

300 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No oncogenic effects were observed up to and including the highest dose level.

Remarks on result:

other: corresponding to an actual ingested dose of approx. 50 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

600 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No oncogenic effects were observed up to and including the highest dose level.

Remarks on result:

other: corresponding to an actual ingested dose of approx. 112 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

15 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed at this dose level.

Remarks on result:

other: corresponding to an actual ingested dose of approx. 2.5 and 3 mg/kg bw/day in males and females, respectively.

Key result

Dose descriptor:

LOAEL

Remarks:

general systemic toxicity

Effect level:

150 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

haematology

histopathology: non-neoplastic

Remarks on result:

other: corresponding to an actual ingested dose of approx. 24 mg/kg bw/day.

Key result

Dose descriptor:

LOAEL

Remarks:

general systemic toxicity

Effect level:

300 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

haematology

histopathology: non-neoplastic

Remarks on result:

other: corresponding to an actual ingested dose of approx. 57 mg/kg bw/day.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 ppm

System:

haematopoietic

Organ:

liver

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The present study was conducted to assess the carcinogenicity of the test substance on mice when given for a time frame of approximately 97 weeks. The study was similar to the OECD guideline 451 and was performed under GLP conditions.
Based on the study results the NOAEL for oncogenic effects was 300 ppm for males and 600 ppm for females, corresponding to 50 and 112 mg/kg bw/day for male and female mice, respectively, the highest dose tested. Based on body weight data, reduced food consumption, changes in haematology and histopathology, the NOAEL for general systemic toxicity was 15 ppm, corresponding to approx. 3 mg/kg bw/day for males and females.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Jun 1988 - 7 Oct 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

adopted in 2018

Deviations:

yes

Remarks:

Organ weights of adrenals, epididymides, uterus, spleen, and heart were not determined.

Qualifier:

according to guideline

Guideline:

other: EPA 83-5 (1982)

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 Laboratories, Inc., Portage, Michigan, USA
- Age at study initiation: 5 -6 weeks
- Weight at study initiation: Males: 124.5-160.5 g; Females: 114.9-193.4 g
- Fasting period before study: not applicable
- Housing: individually in suspended, stainless steel, screen-bottom cages, with absorbent paper lining the urine- and faeces- collecting pans. Some animals were placed in polycarbonate cages, when health problems dictated.
- Diet: Certified Rodent Chow® #5002 (Purina Mills, Inc.), ad libitum
- Water: Water, treated by reverse osmosis and sterilised by ultraviolet light, ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±1
- Humidity (%): 50±20
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 28 Jun 1988 To: 11 Jul 1990

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with (basal diet): Preparation of test diets was performed weekly during the treatment period by preparing a premix and then blending the remaining amount of diet in a mixer.
- Storage temperature of food: frozen

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diet analyses for thiram were carried out in duplicate using HPLC. Homogeneity was determined at all dose levels, by analysing samples taken from the top, bottom and two opposing sides. The values for the homogeneity assay ranged from 21.8 to 24.2 ppm (73% to 81%), 128 to 138 ppm (85% to 92%) and 255 to 276 ppm (85% to 92%) for the theoretical levels of 30, 150 and 300 ppm, respectively, indicating that the mixing procedure resulted in a homogeneous distribution in the diet.

Concentration at each dose level was analysed from samples taken directly from the mixing bowl. To evaluate the stability of the test material in the basal diet, four sets of samples were taken from all diets to be used in the study. One set was assayed on the day of preparation. One was kept below 0 °C for 6 days, placed in the animal’s room for 1 day, kept below 0°C for 30 days, then assayed. The remaining two sample sets were stored below 0°C and assayed after 2 and 7 weeks. Mean values for the stability assay results for samples of the diets on the day of preparation
were 74%, 92% and 91% of the theoretical values of 30, 150 and 300 ppm, respectively.
For routine analysis, samples of all diets were assayed each week for the first 4 weeks and every 4 weeks thereafter through to Week 104. Through to Week 72 samples of diet were stored below 0°C for 7 days moved to the animal room for 1 day, then assayed within 1 day for test material content. Starting with the diets for Week 72, samples of all diets for routine analysis were assayed within 1 day of mixing and stored below 0°C until assayed with the exception of Week 80 when diets were analysed within 2 days of mixing. Retention samples were assayed for Weeks 3 - 7, 9 - 11, 13 - 15, 17 - 19 and 60. Samples of diets for the 30 ppm level were also assayed in duplicate for Weeks 3 - 8, 10, 16, 20, 24 and 28. Cumulative means for the routine diet analysis for dose confirmation were 54% (16.3 ppm), 79% (119 ppm) and 87% (262 ppm). For the most part, the results of the analyses of diets were lower than target, especially for the 30-ppm level. Assays done for Week 72 showed marked improvement due to the changed storage regime. As demonstrated in another study (HLA 6111-131), animals receiving the diet containing 30 ppm can be expected to receive a systemic exposure to the substance approx. equal to the target amount, even though the expected level could not be verified analytically. Therefore, these data demonstrate that the anticipated levels of the test substance were achieved in the test diets.

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

continuously via the diet

Post exposure period:

no

Dose / conc.:

30 ppm

Remarks:

corresponding to approx. 1.46 and 1.80 mg/kg bw/day for male and female rats, respectively.

Dose / conc.:

150 ppm

Remarks:

corresponding to approx. 7.31 and 8.86 mg/kg bw/day for male and female rats, respectively.

Dose / conc.:

300 ppm

Remarks:

corresponding to approx. 14.66 and 18.57 mg/kg bw/day for male and female rats, respectively.

No. of animals per sex per dose:

60 (50 animals for terminal sacrifice, 10 for interim sacrifice)

Control animals:

yes, plain diet

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

DERMAL IRRITATION: No

BODY WEIGHT: Yes
- Time schedule for examinations: : Before start of treatment, first day of treatment, weekly for Week 1 - 16, then every month (instead done during week 98 instead of 96) and before necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Before start of treatment, first day of treatment, weekly for Week 1-16, then every month (instead done during week 98 instead of 96) and before necropsy

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: : before initiation of treatment and during Week 51 for animals scheduled for the interim necropsy and during Week 103 of treatment before the scheduled terminal necropsy
- Dose groups that were examined: all animals of all dose groups
- Procedure: The pupils were dilated with 0.5% Mydriacyl, the the eyes were examined with an indirect ophthalmoscope.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before initiation of treatment and during Week 51 for animals scheduled for the interim necropsy and during Week 103 of treatment before the scheduled terminal necropsy
- Anaesthetic used for blood collection: Yes (ketamine)
- Animals fasted: Yes (overnight)
- How many animals: 10 animals/sex/dose
- Parameters checked: erythrocyte count (RBC), haemoglobin concentration (Hb), haematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count, prothrombin time (for animals scheduled for sacrifice after 104 weeks), total leukocyte count (WBC), blood cell morphology and differential leukocyte count. Differential leukocyte count included: nucleated erythrocyte count, corrected leukocyte count, segmented neutrophil count, band neutrophil count, lymphocyte count, monocyte count, eosinophil count, and basophil count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before initiation of treatment and during Week 51 for animals scheduled for the interim necropsy and during Week 103 of treatment before the scheduled terminal necropsy
- Anaesthetic used for blood collection: Yes (ketamine)
- Animals fasted: Yes (overnight)
- How many animals: 10 animals/sex/dose
- Parameters checked: glucose, blood urea nitrogen, creatinine, total protein, albumin, globulin, total bilirubin, cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), calcium, inorganic phosphorus, sodium, potassium, and chloride

URINALYSIS: Yes
- Time schedule for collection of urine: during Weeks 27, 53, 79 and 105 of treatment before blood sampling (16 h)
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes (overnight)
- How many animals: 10 animals/sex/dose, the same animals were used at each interval, when possible
- Parameters checked: Appearance, volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, and occult blood were determined on urine samples. The sediment was examined using a microscope.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

Pathology: Yes.
Gross pathology: All animals in the main group, including those found dead or killed in extremis during the treatment period and those surviving after 104 weeks of treatment were subjected to a complete necropsy. 10 animals/sex/group (satellite group) selected at random (not including animals used for clinical pathology) were subjected to interim kills during Week 53. Animals were sacrificed after an overnight fasting period, by exsanguination under methoxyflurane anaesthesia and subjected to macroscopic examination. Macroscopic examination included: examination of the external surface of the body, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the nasal cavity and paranasal sinuses, and the thoracic, abdominal, and pelvic cavities and viscera. Cut surfaces of the brain and spinal cord were examined when tissues were trimmed.

Organ Weights: Yes
- How many animals: 10 animals/sex/dose at weeks 52 and 104.
- Organs weighed: Brain, kidneys, liver, ovaries, testes

Histopathology: Yes
- Tissues examined: Adrenals, aorta, brain, caecum, cervix, colon, duodenum, epididymides, eyes, femur with bone marrow, heart, jejunum, ileum, kidneys, lesions, liver, lungs, lymph nodes (mandibular and mesenteric), mammary gland, other macroscopic abnormalities, muscle (thigh), ovaries, pancreas, pituitary, prostate, rectum, salivary gland (submandibular), sciatic nerve, seminal vesicles, skin, spinal cord (cervical, mid-thoracic, and lumbar), spleen, stomach, testes, thymus, thyroids/parathyroids, trachea, urinary bladder, uterus, vagina. Low-and mid-dose animals: macroscopic lesions, lungs, liver, kidneys, thyroid gland, pancreas, spleen, mesenteric lymph node
- How many animals: all tissues from all animals of the control and high dose group (interim and terminal sacrifice). Macroscopic lesions, lungs, liver and kidneys were examined microscopically from animals in the 30 and 150 ppm group. The thyroid gland, pancreas, spleen and mesenteric lymph node were identified as potential target tissues, these were examined microscopically from animals given 30 and 150 ppm.
- Fixative: 10% phosphate-buffered formalin
- Embedding media: paraffin
- Staining: haematoxylin and eosin

Statistics:

For test of variance homogeneity Levene´s test (1960) was use. If p≤0.05, transformations were used to stabilize variance. ANOVA was done on homogeneous or transformed data. If ANOVA was significant, Dunnett´s test was used for pairwise comparison between groups. When no transformation established variance of homogeneity at p ≤ 0.001, the data were also examined by nonparametric pairwise comparison between groups (Wilcoxon-Mann-Whitney two-sample rank test). One-way ANOVA was used for analysis of body weights, cumulative body weight gains, food consumption, clinical chemistry and haematology values, urine pH, specific gravity, and volume, organ weights, organ-to-body weight percentages, and organ-to-brain weight ratios.

Survival and tumour analyses: Cumulative survival data were analyzed using the NCI package (Thomas et al., 1977). Trend analysis of survival was evaluated at the 5.0% one-tailed probability level. Group comparisons of survival were evaluated at the 5% two-tailed probability level. Any animal that died within the first 6 weeks of treatment was excluded from survival and tumour analysis, unless at least one of their lesions was considered treatment-related. Tumours from terminal sacrifice were analyzed by the Cochran-Armitage test for trend, and the Fisher-Irwin exact test for group comparisons (Thakur et al., 1985). For tumours in females: If no trend or heterogeneity was observed in survival analysis, tumours from all non excluded animals were selected for analysis by the Cochran-Armitage test for trend, and the Fisher-Irwin exact test for group comparisons (Thakur et al., 1985).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Possible test-material related clinical effects in males included swollen nose, soft faeces and opaque eyes. The incidence of swollen nose and opaque eyes was lowest in the controls and increased as the dose level increased (incidence of swollen nose was 18, 22, 25 and 28, incidences of opaque eyes were 1, 5, 6 and 8 for the Control, 30, 150 and 300 ppm group, respectively).

Ophthalmic examinations indicated no test material-related observations. Soft faeces were also observed to decrease as dose levels increased in both males and females. No clinical signs were noted that suggested test material-related neurotoxicity.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Adjusted survival ratios at Week 104 were 21/49 (43%) for control males and 30/50 (60%), 27/50 (54%), and 32/50 (64%) for males given 30, 150, and 300 ppm, respectively. Adjusted survival ratios at Week 104 were 19/49 (39%) for control females and 21/48 (44%), 28/50 (56%), and 24/49 (49%) for females given 30, 150, and 300 ppm, respectively. Adjusted survival at Week 104 was statistically significantly higher for males given 300 ppm. There were no other statistically significant differences for males or females. Trend analysis also indicated a dose-related increase in survival for males through Week 104. During the scheduled sacrifice (after the last data collection at Week 104) one male from the control group and one male from the top dose group died. It is concluded that mortality is comparable among control and treated groups throughout the study.

Neoplasia was the predominant cause of death in unscheduled deaths recorded during the course of the study, being listed 175 times. Pituitary tumours accounted for 117 incidences (67% of all tumours), followed by fibrous histiocytomas (10 incidences) and mammary gland adenocarcinomas (8 incidences). The remaining 40 neoplastic causes of death were variably distributed among 16 different neoplasms. With the exceptions of mammary gland adenocarcinomas (7 female and 1 male) and leiomyosarcomas (4 females), there appeared to be no sex difference in the incidences of these neoplastic causes of death. The test substance did not appear to affect the incidences of the causes of unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 30 ppm group: No treatment-related effects on body weight. At Week 104, the mean cumulative body weight gain for low dosed males was 95% of the mean for the control group. At Week 104, the mean cumulative body weight gain for low dosed females was 108%, 99%. There were no statistically significant differences from the controls for cumulative body weight gains of low dosed males or females.
- 150 ppm group: Body weights were statistically significantly reduced compared to the control group in males for Weeks 1 - 68, and weeks 80-104. For females body weights were statistically significantly reduced for weeks 1-64 compared to the control group. At Week 104, the mean cumulative body weight gain for mid dosed males was 87% of the mean for the control group. Cumulative body weight gains were statistically significantly reduced compared to those of the controls for weeks 1 through 68 and 80 through 104. At Week 104, the mean cumulative body weight gain for mid dosed females was 99% of the mean for the control group. Cumulative body weight gains were statistically significantly reduced compared to those of the controls for weeks 1 through 64.
- 300 ppm group: Body weights were statistically significantly reduced in males for weeks 1-104, and in females for weeks 1-84 compared to the control groups.At Week 104, the mean cumulative body weight gain for high dosed males was 84% of the mean for the control group. Cumulative body weight gains were statistically significantly reduced compared to those of the controls for weeks 1 through 104. At Week 104, the mean cumulative body weight gain for high dosed females was 92% of the mean for the control group. Cumulative body weight gains were statistically significantly reduced compared to those of the controls for weeks 1 through 84.

Summarized results can be found in Attachment 1 under "Any other information on results incl. tables".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- 30 ppm: statistically significantly reduced compared to the control group in males during Weeks 6, 10, 20, 28, and 40, and in females for Weeks 12, 13, and 20. For Weeks 10, and 80-88 food consumption was statistically significantly higher in females compared to the control group.
- 150 ppm: statistically significantly reduced compared to the control group in males for Weeks 1 -28, 36, and 44. In females, food consumption was statistically significantly reduced for Weeks 1-9, 11-28, 36, 40, 52, 56, 64 and 76 compared to the control group.
- 300 ppm: statistically significantly reduced compared to the control group in males for Weeks 1-72 and 84, and in females for Weeks 1-9, 11-44, 52, 56, 64, 72 and 76. In Week 100 food consumption was statistically significantly increased in females compared to the control group.

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

Interim sacrifice:
No treatment-related effects were observed.

Terminal sacrifice:
Eyelid masses in 5 males and 3 females of the high-dose group, and in 1 female of the mid-dose group were observed at the 103-week sacrifice. No eyelid masses were observed at necropsy after week 104. Therefore, it could not be determined whether these findings were test substance related.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- 30 ppm: No treatment related differences were observed between the control and treatment group.
- 150 ppm: statistically significantly decreased red blood cell count (up to approx. -11%), haemoglobin (up to approx. -9.4%) and haematocrit (up to approx. -9.9%) and statistically significantly elevated mean corpuscular volume (up to approx. +3.3%), mean corpuscular haemoglobin (up to approx. +6.4%) and mean corpuscular haemoglobin concentration (up to approx. +4.7%) in females compared to the control group at different time points throughout the study.
- 300 ppm: statistically significantly decreased red blood cell count (up to approx. -18.8%), haemoglobin (up to approx. -14%) and haematocrit (up to approx. -15.8%) and statistically significantly elevated mean corpuscular volume (up to approx. +6.5%) and mean corpuscular haemoglobin (up to approx. +10.5%) and mean corpuscular haemoglobin concentration (up to approx. +5.8%) in females compared to the control group at different time points throughout the study.

Summarized results can be found in Attachment 2 under "Any other information on results incl. tables".

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Clinical chemistry did not show any differences compared to the control group.

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study: histopathology of cervix, epididymis, mammary gland, ovary, prostate, seminal vesicles, testis, thyroid, pituitary, uterus, vagina and adrenals, organ weights were recorded for liver, ovary, testis and brain. For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis did not show any differences compared to the control group.

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Interim sacrifice:
Administration of the test substance was associated with few changes in organ weight. Animals sacrificed at Week 53 showed several organ weight changes that did not follow through to terminal sacrifice.

- 30 and 150 pm: No treatment related differences were observed between the control and treatment groups.
- 300 ppm: At the Week 53 interim sacrifice male animals showed a statistically significant increase in brain-to-body weight percentage, the right kidney-to-brain weight ratio was statistically significantly decreased compared to the control group. Terminal liver-to-body-weights and brain-to-body weights percentages were statistically significantly elevated in females compared to the control group. However, at Week 107 although terminal body weights remained reduced and liver-to-body-weights percentages remained increased, the changes were no longer statistically significant.

Terminal sacrifice:
- 30 ppm: At terminal sacrifice absolute kidney weights and kidney-to-brain weight ratios were statistically significantly decreased compared to the control group (males).
- 150 ppm: At terminal sacrifice absolute kidney weights and kidney-to-brain weight ratios were statistically significantly decreased compared to the control group (males).
- 300 ppm: At terminal sacrifice absolute kidney weights and kidney-to-brain weight ratios were statistically significantly decreased compared to the control group (males). The statistically significantly reduced absolute right ovary weights and right ovary-to-brain weight ratios compared to the control group (females), were probably related to a decreased number of ovarian cysts in that dose group.

Summarized results can be found in Attachment 2 under "Any other information on results incl. tables".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Interim sacrifice:
No treatment-related differences between control and treated animals were observed at Week 53 interim sacrifice.

Terminal sacrifice:
Liver: An increased incidence of red foci/area in the liver was observed at the highest dose applied in males compared to the control group.

Mesenteric Lymph nodes: At a dose of 150 and 300 ppm mottled mesenteric lymph nodes were observed.

Kidneys: Increased incidences of mottled kidneys were seen at 30, 150 and 300 ppm.

Heart: A reduced incidence of light foci/areas in the heart was found in male rats after treatment with 300 ppm.

Adrenals: In females given 30, 150 and 300 ppm there was an increased incidence of enlarged adrenal glands, cystic adrenals were also identified in females treated with 150 and 300 ppm.

Reproductive organs: There was a reduced incidence in ovarian cysts for females given 300 ppm. There was an increased incidence in thickening of the uterine wall at all dose levels applied and an increase in uterine masses at 150 and 300 ppm.

Summarized results can be found in Attachment 4 under "Any other information on results incl. tables".

Neuropathological findings:

not examined

Description (incidence and severity):

There were no clinical signs that suggested test material-related neurotoxicity.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver: An increased incidence of extramedullary haematopoiesis (EMH) in the liver was observed in males given 150 and 300 ppm and in females given 300 ppm. EMH appeared to be increased also in the spleen of females of the mid dose group at interim sacrifice and at terminal sacrifice of females of the mid and high dose group, however, the increase was not dose-related. This increase in EMH might correlate with changes in erythroid parameters seen clinically for high dosed females. The incidence of bile duct hyperplasia showed a statistically significant positive trend in females which had died unscheduled, in the high-dose group reaching statistical significance. Bile duct hyperplasia is known to occur during chronic inflammation or toxic effects in the liver. However, liver enzymes analyses were all within normal ranges.

Spleen: EMH appeared to be increased also in the spleen of females given 150 and 300 ppm, however, the increase was not dose-related. This increase in EMH might correlate with changes in erythroid parameters seen clinically for females at 300 ppm. An increase in sinusoidal pigmentation was observed in the spleen for females given 300 ppm, a change commonly seen with increased EMH and erythroid turnover.

Bone marrow: Myeloid hyperplasia in the bone marrow (femur and sternum) was also increased in
females treated with 300 ppm.

The combined changes in liver, spleen and bone marrow suggest a test material induced effect
on haematopoiesis.

Pancreas: Steatosis/fatty infiltration in the pancreas appeared to be increased for males and females given 150 or 300 ppm. Multifocal acinar atrophy was more common for males given 30, 150 or 300 ppm and for females given 150 ppm. The low incidence for this finding in females given 300 ppm suggests that the effect observed is not test material-related. The increase seen in males given 30 ppm might also be incidental, however, the relevance of the increase seen at higher doses is unclear.

Thyroid: A statistically significant increase in thyroid C-cell hyperplasia was observed in females given 150 ppm and demonstrated a statistically significant positive trend for development in all females and in males sacrificed at termination of the study. However, the incidences for C-cell hyperplasia were well within the historical control data range.

Mesenteric Lymph nodes: The incidences of haemorrhages within the mesenteric lymph nodes were increased for males given 30, 150 and 300 ppm and for females given 150 and 300 ppm. No dose-related effect could be observed for the male animals. The results suggest a test material-related lesion, but the significance of this finding is unclear. Haemorrhage was not a consistent lesion seen in any other tissue.

Summarized results can be found in Attachment 1 under "Any other information on results incl. tables".

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males and females given the test material demonstrated a statistically significant positive trend for the development of hepatocellular adenomas. However, individual group comparisons for both males and females did not show a statistically significant difference between control and treated animals. No effects were seen on the incidence of hepatocellular carcinomas in treated males and females. It is suggested that liver function increased because of adaptive metabolic mechanisms, resulting in the development of adenomas. No effects were seen on the incidence of hepatocellular carcinomas in treated males and females. Compared to historical control data the incidence in the current study of 10% in males of the high dose group was at the upper limit (10%) of the range for the historical control data, while the incidence (8.3%) in females was noted above the upper limit of the range for the historical control data (6.7%). Therefore, hepatocellular adenoma observed at high dose in both sexes and intermediate dose in females were considered related to the administration of the test substance.
Males sacrificed at the end of treatment and all females (unscheduled deaths and terminal sacrifice) given the test material showed a statistically significant positive trend for the development of thyroid C-cell adenomas, but individual group comparisons did not show a statistically significant difference between control and treated animals. The incidences for C-cell adenomas were well within the historical control data range. There was no increase in the incidence of thyroid C-cell carcinoma in animals given the test material.

Summarized results can be found in Attachment 1 and historcial control data under Attachment 3 under "Any other information on results incl. tables".

Relevance of carcinogenic effects / potential:

In this combined chronic/carcinogenicity study in rats hepatocellular adenoma were observed at the high dose in both sexes and at the intermediate dose in females which were considered related to the administration of the test substance. However, the incidence was only slightly above the historical control data range as reported by the test laboratory and no indication of any development of malignancy was observed.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

30 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed at this dose level.

Remarks on result:

other: corresponding to approx. 1.5 and 1.8 mg/kg bw/day for male and female rats, respectively.

Key result

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Effect level:

150 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

Remarks on result:

other: corresponding to approx. 7.3 and 8.9 mg /kg bw/day for male and female rats, respectively.

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

30 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related carcinogenicity was observed.

Remarks on result:

other: corresponding to approx. 1.5 and 1.8 mg/kg bw/day for male and female rats, respectively.

Key result

Dose descriptor:

LOAEL

Remarks:

carcinogenicity

Effect level:

150 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: corresponding to approx. 7.3 and 8.9 mg /kg bw/day for male and female rats, respectively.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The study was conducted similar to OECD guideline 453 and under GLP conditions.
Based on statistically significant decreases in body weights and body weight gain for males and females given 150 or 300 ppm, effects on haematological parameters in mid and high dosed females and thyroid C-cell hyperplasia and bile duct hyperplasia in mid and high dosed males and females the No-Observed-Adverse-Effect-Level (NOAEL) of the test substance in the diet given to male and female rats for chronic toxicity is derived at 30 ppm (corresponding to 1.46 mg/kg/day for males and 1.8 mg/kg/day for females).
The NOAEL for carcinogenicity was established at 30 ppm (corresponding to 1.46 mg/kg/day for males and 1.8 mg/kg/day for females) based on hepatocellular adenomas in the liver and C-cell adenomas in the thyroid.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

14.7 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

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

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The 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

 

Two key studies were conducted to assess the carcinogenic potential of the test substance, one in rats and one in mice. Both 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: HLA 6111-113). The test substance was administered via dietary exposure to male and female Sprague-Dawley rats at dietary concentration of 30, 150 and 300 ppm. As also observed in the subchronic 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 as well as changes in blood parameters. Changes in blood parameters (haematological and clinical biochemistry) consisted of signs of hemolysis, evident by changes for the number of erythrocytes, haemoglobin and haematocrit concentration, as well as increaser of mean corpuscular volume and mean corpuscular haemoglobin. Further, the relative and absolute kidney weights of rats treated with 300 ppm of the test substance (high dose) were decreased compared to the control group. Effects on gross pathologicy and histopathology and histopathology were observed, the latter including extramedullary haematopoeises in the liver and spleen, suggesting a test material induced effect on haematpoesis. Further organs showing abnormalities were the pancreas, the thyroid and the mesenteric lymph nodes.  However, no clear dependency on the test material administration could be established for those.

 

The incidence of benign tumours in liver and thyroid showed a statistically positive trend, although their number was not statistically different between control and treated groups. As no clear relationship to treatment could be established, the trend was considered incidental. The number of hepatic adenomas was also evaluated in an additional statement (Covance 6111 - 113). It was found that the occurrence of hepatocellular adenoma from all animals in the current study, the incidence (10%) in males administered 300 ppm of the test substance was at the upper limit (10%) of the range for the historical control data, while the incidence (8.3%) in females was slightly above the upper limit the range for the historical control data (6.7%).

 

Thus, the NOAEL for carcinogenicity in male and female rats was set to 300 ppm corresponding to 14.7 and 18.6 mg/kg bw/day for male and female rats, respectively. The NOAEL for systemic toxicity was set to 30 ppm, corresponding to 1.46 and 1.8 mg/kg bw/day for male and female rats, respectively.

 

Carcinogenicity study in mice:

To assess the carcinogenicity of the test substance in mice, a carcinogenicity study was conducted similar to the OECD guideline 452 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 97 weeks. Under the conditions of the test, the test substance caused general toxicity as evident by lower body weight gains and food intake. RBC count, Hb and Hct were decreased in both sexes, while platelet count was increased in females. Corresponding to these values, indicating an increased red blood cell turnover, increased pigment was observed in mid- and high-dose females (morphological compatible with hemosiderin). Other organs that showed histopathological changes were the skin, adrenals (female mice only, not considered relevant for humans), urinary bladder, eyes and stomach. 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 (300 ppm in males, corresponding to 50 mg/kg bw/day and 600 ppm in females, corresponding to 112 mg/kg bw/day).

 

 

Conclusion:

Overall, the lowest NOAEL with respect to carcinogenicity was to 1300 ppm corresponding to 14.7 and 18.6 mg/kg bw/day for male and female rats, respectively. The studies show a statistically positive trend in the development of benign tumours, although their number is not statistically different between control and treated groups. There is no increase in malignant tumours. There is no tumour induction in the mouse.

 

In literature, it is suggested that liver function increased because of adaptive metabolic mechanisms, with as a result the development of hyperplasia and adenomas. A positive trend in the induction of C-cell hyperplasia and adenomata is also seen, for which no explanation is given. C-cells, or parafollicular cells, synthesize and secrete the hormone calcitonin in direct response to raised blood calcium levels (see also section 5.8.3). These cells are not under control of the hypohyse as are the thyroid hormone secreting cells. The test substance is not metabolized into ETU which is well known to interfere with thyroid function.

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.

 

Reference:

Covance 6111 -113, 104-week dietary combined chronic toxicity and sarcinogenicity study with thiram in rats, Supplementary Information on Hepatocellular Adenomas, 2016