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Diss Factsheets

Administrative data

Description of key information

Oral toxicity

 

Key, report number HLA 6111-127, subacute (4 weeks, mouse):

NOAEL (oral, females) 300 ppm corresponding to approx. 59 mg/kg bw/day
LOAEL (oral, males) 300 ppm corresponding to approx. 51 mg/kg bw/day (no NOAEL could be set for males

 

Key, report number HLA 6111-110, subchronic (13 weeks, rat):
NOAEL (oral) 50 ppm corresponding to approx. 3.5 mg/kg bw/day (males) and approx. 4 mg/kg bw/day (females)

 

Key, report number HLA 6111-121, subchronic (13 weeks, dog):

NOAEL (oral) 75 ppm, corresponding to approx. 2 mg/kg bw/day (males and females)

 

Key, report number HLA 6111-112, chronic (52 weeks, dog):

NOAEL (oral) 30 ppm for males (corresponding to approx. 0.84 mg/kg bw/day), 90 ppm for females (corresponding to approx. 2.54 mg/kg bw/day)

 

Key, report number HLA 6111-113, combined chronic toxicity/carcinogenicity studies (52/104 weeks, rat):
NOAEL (general systemic toxicity) 30 ppm, corresponding to approx. 1.5 mg/kg bw/day for males and 1.8 mg/kg bw/day for females)

 

Dermal toxicity

Key, report number UCB 421/920767, subacute (21 days, rat):

NOAEL (local, dermal) <100 mg/kg bw/day (females, males)

NOAEL (general systemic toxicity, dermal) 300 mg/kg bw/day (females, males)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Nov - 10 Dec 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
No guideline followed. Range-finding study with mice as test organism, used for a 78 week carcinogenicity study
GLP compliance:
yes
Limit test:
no
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, Inc, Portage, Michigan, USA
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: group mean was 24.3 - 24.8 g for males and 19.0 - 19.3 g for females
- Fasting period before study: not applicable
- Housing: individually in stainless steel, screen-bottomed cages
- Diet: Certified rodent chow #5002 (Purina Mills, Inc), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 - 23.9
- Humidity (%): 30 - 70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 10 Nov 1988 To: 10 Dec 1988
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: the appropriate amount of Certified Rodent Chow #5002 was weighed in to a Hobart mixing bowl. 250 g of food was taken from this bowl and added with the appropriate amount of the test material to a Waring blender. This premix was mixed thoroughly and then trasnferred to the Hobart mixing bowl. Then 100 g of additional food was blended to remove residual test material from the blender.
- Storage temperature of food: frozen until in use
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Data for stability and homogeneity of similar diets were available from a previous toxicity study.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
continuously via the diet
Dose / conc.:
300 ppm
Remarks:
corresponding to approx. 51 – 58 mg/kg bw/day for males and 59 – 66 mg/kg bw/day for females
Dose / conc.:
600 ppm
Remarks:
corresponding to approx. 101 – 115 mg/kg bw/day for males and 111 – 127 mg/kg bw/day for females
Dose / conc.:
1 200 ppm
Remarks:
corresponding to approx. 177 – 226 mg/kg bw/day for males and 221 – 281 mg/kg bw/day for females
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
- Fasting period before blood sampling for clinical biochemistry: yes (overnight)
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for moribundity and mortality and once daily for signs of toxicity

BODY WEIGHT: Yes
- Time schedule for examinations: immediately before start of treatment, on Day 0, weekly thereafter and at termination.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks of treatment
- Anaesthetic used for blood collection: Yes (ketamine)
- Animals fasted: Yes (overnight)
- How many animals: all animals of all groups (treatment and control)
- Parameters checked: red blood cell (RBC) count, haemoglobin (Hb), haematocrit (Hc), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count (PLT), white blood cell count (WBC), differential leucocyte count, including nucleated red blood cell count (NRBC), corrected white blood cell count (COR WBC), segmented neutrophil count (N-SEG), band neutrophil count (N-BAND), lymphocyte count (LYMPH), monocyte count (MONO), eosinophil count (EOSIN), basophil count (BASO), morphology.

CLINICAL CHEMISTRY: Yes
- Anaesthetic used for blood collection: Yes (ketamine)
- Animals fasted: Yes (overnight)
- How many animals: all animals of all groups (treatment and control)
- Parameters checked: Glucose (GLU), urea nitrogen (UN), total protein (T PRO), albumin (ALB), globulin (GLOB), alanine aminotransferase (ALT)

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Animals were anesthetized with methoxyflurane, weighed, exsanguinated and necropsied.
Organs weighed: Brain, kidneys, liver with gallbladder, ovaries, testes

HISTOPATHOLOGY: Yes
- Tissues sampled in 10% buffered formalin: Adrenals, aorta, bone, bone marrow, brain, caecum, cervix, colon, duodenum, epididymides, eyes, femur, oesophagus, gallbladder, gross lesions, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes, mammary gland, muscle, ovaries, pancreas, parathyroid, prostate, sciatic nerve, pituitary, rectum, salivary glands, seminal vesicles, skin, spinal cord, spleen, sternum, stomach, testes, thymus, thyroid gland, trachea, urinary bladder, uterus, vagina.
- embedding media: Paraffin
- Staining: haematoxylin and eosin
- Examined for: all animals of the control and high-dose group. Macroscopic lesions, lungs, liver with gallbladder and kidneys were also examined microscopically from all animals of the low and mid dose group.
Statistics:
Levene's test was applied to test for heterogeneity of variance between treatments. Where significant heterogeneity was found, a logarithmic, square, square root, reciprocal, angular or rank 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 (ANOVA) was carried out. If significance was found, a Dunnett's t-test was used for pairwise comparison.

If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis H-test ANOVA was used. If this was significant, the Nemenyi-Kruskal-Wallis test for multiple comparisons or the Wilcoxon-Mann-Whitney two-sample rank test was used.

Statistical significance was defined at the 5% two tailed probability level.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Control: alopecia (1/10 males)
- 300 ppm: hunched posture (1/10 males), animal was thin (1/10 males), alopecia (1/10 males), squinting of both eyes (1/10 males), bloody crust on the tail (1/10 females), red tail (1/10 females), necrotic tail (1/10 females).
- 600 ppm: animal was thin (1/10 females), alopecia (1/10 females), opaque red eye (1/females)
- 1200 ppm: hunched posture (1/10 males, 2/10 females), animal was thin (1/10 males, 2/10 females), alopecia (1/10 females), lacrimation in the left eye (1/10 males)

The hunched posture and the occurrence of thin animals was considered treatment related. All other signs were considered incidental.
Mortality:
no mortality observed
Description (incidence):
No mortality was observed during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 300 ppm: body weight and body weight gain were statistically significantly lower in males compared to the control group in Week 3 and 4.
- 600 ppm: body weight and body weight gain were statistically significantly lower in males compared to the control group in Week 4.
- 1200 ppm: body weight was statistically significantly lower in males compared to the control group in Week 3 and 4. Body weight gain was statistically significantly lower compared to the control in Weeks 2 - 4 for males and for Weeks 3 and 4 for females.

Summarized results can be found in Attachment 1 in the attached background material.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 300 ppm: food consumption was statistically significantly decreased during the complete treatment period.
- 600 ppm: food consumption was statistically significantly decreased during the complete treatment period.
- 1200 ppm: food consumption was statistically significantly decreased during the complete treatment period.

Summarized results can be found in Attachment 1 in the attached background material.
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:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- 300 ppm: decreased RBC (males -6.5%), slightly decreased Hb (males -6.2%), slightly decreased Hct (males -5.1%), all changes were statistically significantly different from the control group.
- 600 ppm: decreased RBC (males -7%, females -6.2%), slightly decreased Hb (males -5%), slightly decreased Hct (males -5.1%), increased platelet count (females +18.8%), all changes were statistically significantly different from the control group.
- 1200 ppm: decreased RBC (males -7.3%), slightly decreased Hb (males -8.6%), slightly decreased Hct (males -7.7%), increased platelet count (females +27.6%), all changes were statistically significantly different from the control group.

Although these changes were statistically significant and most were treatment-related, they were relatively small and not considered indicative of severe primary pathologic effects.

Summarized results can be found in Attachment 1 in the attached background material.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The only statistically significant difference was decreased glucose in females of the 1200 ppm group. This was not considered treatment-related but incidental. Apart from that, no difference was observed in clinical chemistry between the treatment and control groups.

Summarized results can be found in Attachment 1 in the attached background material.
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, uterus, vagina and adrenals, organ weights were recorded for epididymis, liver, prostate, 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:
effects observed, treatment-related
Description (incidence and severity):
- 300 ppm: the relative brain weight was statistically significantly higher in males compared to the control group.
- 600 ppm: increased relative liver weight (males +12.7%), and kidney weight (males +15.1%), decreased absolute brain weight was observed in males (-6.2%), all differences were statistically significant compared to the control group.
- 1200 ppm: increased relative liver weight (females +11%, males +13.8%), all differences were statistically significant compared to the control group.

These changes are considered secondary to decreased terminal body weight.

Summarized results can be found in Attachment 1 in the attached background material.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related difference was observed between control and treatment groups up to and including the highest dose level.

Summarized results can be found in Attachment 1 in the attached background material.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment related difference was observed between control and treatment groups up to and including the highest dose level.

Summarized results can be found in Attachment 1 in the attached background material.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
LOAEL
Effect level:
300 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
food consumption and compound intake
haematology
organ weights and organ / body weight ratios
Remarks on result:
other: corresponding to 51 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Effect level:
300 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects were observed at this dose level.
Remarks on result:
other: corresponding to 59 mg/kg bw/day.
Key result
Dose descriptor:
LOAEL
Effect level:
600 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
food consumption and compound intake
haematology
organ weights and organ / body weight ratios
Remarks on result:
other: corresponding to 127 mg/kg bw/day.
Key result
Critical effects observed:
no
Conclusions:
The present study was conducted to assess the sub-acute toxicity of test substance on mice when given for a time frame of 4 weeks. The study was not conducted according to any guideline as it served as a dose-range finder. It was performed under GLP conditions. The test substance was administered via dietary exposure to groups of 10 male and female mice at dose levels of 300, 600 and 1200 ppm corresponding to approximately 51-58, 101-115 and 177-226 mg/kg bw/day for males and 59-66, 111-127 and 221-281 mg/kg bw/day for females.

There were no mortalities during the study. Body weights were significantly lower for males in the 300 and 1200 ppm groups during Week 3 and 4 and for males in the 600 ppm group for Week 4. Females showed no differences. Food consumptions were significantly lower for all treated animals throughout the study period. RBC count, Hb and Hc were slightly lower in all treated males. In females RBC count was decreased in the 600 ppm group while platelet count was increased at 600 and 1200 ppm. The only statistically significant alteration in clinical chemistry was the decreased glucose value in females dosed at 1200 ppm. Absolute organ weights alterations were not dose-response-related and regarded as a result of the lower terminal body weight. There were no treatment-related macroscopic or microscopic observations noted.

Based on reduced food consumption and enhanced platelet count, the no-observable-adverse-effect level (NOAEL) for the test substance when fed to female mice for 4 weeks was 300 ppm (corresponding to 59-66 mg/kg bw for females). Due to the effects observed in the low dosed male group, a NOAEL for males could not be determined.
Endpoint:
chronic toxicity: 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
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 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
Version / remarks:
adopted in 1982
GLP compliance:
yes
Limit test:
no
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 oral 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
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, pancre as, 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.
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 under "Any other information on results incl. tables". Historical control data can be found in Attachment 3 under "Any other information on results incl. tables".
Key result
Dose descriptor:
NOAEL
Remarks:
general 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:
general 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
Critical effects observed:
yes
Lowest effective dose / conc.:
150 ppm
System:
other: hepatobiliary, haematopoietic
Organ:
liver
spleen
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 effects regarding carcinogenicity are discussed in the respective RSS.
Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 May 1989 - 21 Jun 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Version / remarks:
adopted in 2018
Deviations:
yes
Remarks:
On several occasions there was only one observation per day for mortality and moribundity. Due to mistakes food consumption values for week 10 reflect 6-day consumption. Blood sampling prior to treatment (week -2) was done on non-fastened animals.
Qualifier:
according to guideline
Guideline:
EPA OPP 83-1 (Chronic Toxicity)
GLP compliance:
yes
Limit test:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Hazleton Research Products
- Age at study initiation: 4 -5 months
- Weight at study initiation: 4.0 - 6.8 kg
- Fasting period before study: not applicable
- Housing: Individually in stainless steel, screen-bottom cages. Animal housing and husbandry complied with standards outlined in the Guidance for the Care and Use of Laboratory Animals.
- Diet: Certified Canine Diet® #5007 (Purina Mills., Ltd.) ad libitum
- Water: Water ad libitum.
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 May 1989 To: 25 May 1990
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: A specified amount of basal diet was weighed into a labelled mixing bowl, from which about 200 g was removed and placed in a blender. The required amount of test material was weighed, added to the blender, overlaid with about 50 g of basal diet from the mixing bowl before blending. This premix was transferred to the mixing bowl. About 100 g of basal diet from the mixing bowl was then transferred to the blender, blended to recover residual test material and returned to the mixing bowl and thoroughly mixed. Samples for dose analyses were taken directly from the mixing bowl. Diets were stored frozen in covered containers until dispensed daily into food containers.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability was confirmed for all test material dietary concentrations used in the study. Samples of diet were analysed on the day of mixing except on holidays when they were analysed within one day of mixing. All samples were stored in the freezer until analysed. Mean values for stability assay results for samples on the diets on the day of preparation were 88%, 93.8% and 96% of the theoretical levels of 30, 90 and 250 ppm, respectively.

Homogeneity was determined at all dose levels, by analysing samples taken from the top, bottom and two opposing sides. The mean values for homogeneity assay results were 83%, 91.6% and 92% of the theoretical levels of 30, 90 and 250 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. Test diet verification analysis demonstrated that the anticipated levels of the test substance were achieved in the test diets.
Duration of treatment / exposure:
52 weeks
Frequency of treatment:
continuously via the diet
Dose / conc.:
30 ppm
Remarks:
corresponding to approx. 0.84 and 0.9 mg/kg bw/day for male and female dogs, respectively.
Dose / conc.:
90 ppm
Remarks:
corresponding to an approx. 2.61 and 2.54 mg/kg bw/day for male and female dogs, respectively.
Dose / conc.:
250 ppm
Remarks:
corresponding to approx. 7.35 and 7.23 mg/kg bw/day for male and female dogs, respectively.
No. of animals per sex per dose:
6
Control animals:
yes, plain diet
Details on study design:
- Fasting period before blood sampling for clinical biochemistry: yes, overnight (except for Week -2)
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at least twice daily for mortality, morbidity and clinical signs.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded weekly before initiation of treatment, on the first day of treatment, weekly from Week 1 until Week 16 of treatment, and every 4 weeks thereafter. In addition, the body weight of each animal was recorded on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No
Food consumption data were recorded daily and reported weekly beginning 1 week before initiation of treatment, for Weeks 1 through 16 and once every four weeks thereafter.

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before initiation of treatment and during Week 51 of treatment
- Dose groups that were examined: The pupils were dilated with 1% Mydriacyl, then the eyes were examined with an indirect ophthalmoscope.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week -2, 13, 26 and 52
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, overnight (except for Week -2)
- How many animals: all animals of all dose groups including controls (for Week 26 and 52: all surviving animals)
- Parameters checked: erythrocyte count (RBC), haemoglobin (Hb), haematocrit (Hc), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), white blood cell count, differential white blood cell count (including nucleated red blood cell count, corrected white blood cell count, segmented neutrophil count, band neutrophil count, lymphocyte count, monocyte count, eosinophil count, basophil count), platelet count, prothrombin time (PT), reticulocyte count, cell morphology

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week -2, 13, 26 and 52
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, overnight (except for Week -2)
- How many animals: all animals of all dose groups including controls (for Week 26 and 52: all surviving animals)
- Parameters checked: glucose, blood urea nitrogen, creatinine, total protein, albumin, globulin, total bilirubin, cholesterol, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatine kinase, calcium, inorganic phosphorus, sodium, potassium, and chloride.

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes
- Time schedule for collection of urine: Week -2, 13, 26 and 52 before blood sampling
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes (food, water was provided ad libitum)
- 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

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals, including two animals killed in extremis during the treatment period and those surviving after 52 weeks of treatment were subjected to a complete necropsy. Animals were sacrificed, after an overnight fasting period, by exsanguination under pentobarbital sodium 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.

Organs weighed (paired organs were weighed separately): brain, kidneys, liver, ovaries, testes, and thyroids with parathyroids. Organ-to-body weight percentages and organ-to-brain weight ratios were calculated.

HISTOPATHOLOGY: Yes
- Tissues collected: adrenals, aorta, brain, caecum, cervix, colon, duodenum, epididymides, oesophagus, eyes, femur and bone marrow (articular surface of the distal end), gallbladder, heart, ileum, jejunum, kidneys, all gross lesions, liver, lungs, lymph node (mandibular and mesenteric), mammary gland (females only), muscle (thigh), ovaries, pancreas, pituitary, prostate, rectum, salivary gland (submandibular), sciatic nerve, skin, spinal cord (cervical, mid-thoracic and lumber), spleen, sternum and bone marrow, stomach, testes, thymus, thyroid with parathyroid, trachea, urinary bladder, uterus, and vagina.
- Fixative: 10% phosphate-buffered formalin (except eyes: Zenker’s solution)
- Embedding media: Paraffin
- Staining: haematoxylin and eosin
- Animals examined: All animals of all dose groups, including controls
Optional endpoint(s):
Bone marrow smears from the sternum were prepared and retained for possible examination.
Statistics:
Levene’s test was performed to test for variance homogeneity. In the case of heterogeneity of variance at p < 0.05, transformations were used to stabilise the variance.
Analysis of variance (ANOVA) was performed on the homogeneous or transformed data. If significant; Dunnett’s t-test, Student’s t-test, or Games and Howell Modified Tukey-Kramer test was used for pairwise comparisons between groups.
One-way ANOVA was used to analyse initial body weights, food consumption, clinical chemistry and haematology values (except red blood cell morphology), urine (pH, volume, and specific gravity), organ weight, organ-to-body weight percentages, and organ-to-brain weight ratios.
One-way analysis of covariance (ANCOVA) was used to analyse body weight, with initial body weight as a covariate. If the ANCOVA was significant, Dunnett’s t-test, Student’s t-test, or Games and Howell Modified Tukey-Kramer test was used for pairwise comparisons between groups.
Group comparisons were evaluated at the 5% two-tailed probability level.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test material related observations that were considered abnormal. No effect on the neurological system was observed.

Summarized results can be found in Attachment 1 in the attached background material.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two animals were sacrificed moribund during the course of the study, but their conditions did not appear to be test material related. One female treated with 250 ppm was observed in clonic seizures with opisthotonos before sacrifice during week 26. This death was attributed pathologically to hydrocephalus. One female treated with 90 ppm was observed in clonic convulsions with pulmonary congestion before sacrifice during week 25. This death was attributed pathologically to meningoencephalitis.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistically significant differences in body weights were observed in males and females at any of the applied doses. At Week 52, mean body weights of the male animals given 30, 90 and 250 ppm were 93%, 105% and 87%, respectively, of the control group mean. Mean body weights of the female animals given 30, 90 and 250 ppm were 108%, 94% and 98%, respectively, of the control group mean.

Summarized results can be found in Attachment 1 in the attached background material.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No statistically significant effects on male food consumption were observed. Food consumption for females given 250 ppm diet was significantly lower than those of the controls during Week 20, but as this was a single occurrence, it was not considered adverse or treatment-related. There were no consistent effects for males or females throughout the study.

Summarized results can be found in Attachment 1 in the attached background material.
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:
no effects observed
Description (incidence and severity):
No ophthalmic lesions were observed for any animal.

Summarized results can be found in Attachment 1 in the attached background material.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- 30 ppm: No adverse effect was observed. MCV was slightly increased in females, but was not considered adverse.
- 90 ppm: increased MCV (males, females), increased MCH (males, females). All changes noted were statistically significantly different compared to the control group.
- 250 ppm: decreased RBC (males, Week 13, 26 and 52, up to approx. -11.4%, females, Week 13, 26 and 52, up to approx. -9.7%), increased MCV (males, up to approx. +9.2%, females, up to approx. +14.5%), increased MCH (males, up to approx. +10.1%, females up to approx. 14.9%). All changes noted were statistically significantly different compared to the control group. However, the data on increased MCV and MCH were considered inconclusive by the study director because of similar differences present in the study animals before initiation of treatment.

Other statistically significant differences were considered to represent normal biological variation and were not test material related.

Summarized results can be found in Attachment 1 in the attached background material.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 30 ppm: No treatment-related differences were observed between the control and treatment group.
- 90 ppm: decreased total protein (males), increased cholesterol (males)
- 250 ppm: decreased total protein (males, up to approx. -11.9%), increased cholesterol (males, up to approx. +55.9%, females, up to approx. +95.5%), decreased albumin (males, up to approx. -15%, females up to approx. -14.3%). These changes were considered to be treatment-related.

Summarized results can be found in Attachment 1 in the attached background material.
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, testis, thyroid, uterus, vagina and adrenals, organ weights were recorded for liver, prostate, 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 revealed no significant differences after treatment with the test substance.

Summarized results can be found in Attachment 1 in the attached background material.
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):
Administration of the test substance was associated with few changes in organ weight. Statistically
significant increases were:
- 30 ppm: statistically significantly increased relative liver weight (males, +17.5%) compared to the control group
- 90 ppm: statistically significantly increased absolute (+26.6%) and relative (+19.9%) liver weight (males) compared to the control group
- 250 ppm: statistically significantly increased absolute (males +29.4%) and relative (males +44.5%, females +28.1%) liver weight compared to the control group

The increase in of ≥20% liver weight (at 90 and 250 ppm) is considered adverse, irrespective of histopathological findings which were also found in control animals and indicate no adverse effect.The significant increase in liver-to-body weight in male animals given 30 ppm was not considered test material related. Additionally, a statistically significant increase in male liver-to-brain weight was observed after treatment with 250 ppm.

Summarized results can be found in Attachment 1 in the attached background material.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic changes found at necropsy.

Summarized results can be found in Attachment 1 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):
There was a variable incidence across all treatment groups (including controls) of extramedullary haematopoiesis (EMH) in the liver, characterised by multifocal accumulations of mixed haematopoietic cells with the sinusoids or around vascular channels. Some higher severity scores for EMH were observed for a few animals. The severity scores for hepatic EMH were minimal in the control animals and most of the animals that received the test material. 1/6 males given 30 ppm and 1/6 males given 250 ppm received a moderate EMH score. 1/6 males given 90 ppm and 2/6 females given 250 ppm received a slight EMH score.
Scattered microgranulomas were observed in liver, but these were similar to the observations made in control animals. 1/6 females given 90 ppm showed some focal hepatic necrosis with minimal severity and the incidence was too low to be considered conclusively treatment related.
All other microscopic findings were considered incidental and unrelated to test material administration.
The conditions of the two animals sacrificed moribund did not appear to be related to the test material.

Summarized results can be found in Attachment 1 in the attached background material.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
NOAEL
Effect level:
30 ppm
Based on:
test mat.
Sex:
male
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 0.84 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Effect level:
90 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects were observed at this dose level.
Remarks on result:
other: corresponding to approx. 2.54 mg/kg bw/day.
Key result
Dose descriptor:
LOAEL
Effect level:
90 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
haematology
organ weights and organ / body weight ratios
Remarks on result:
other: corresponding to approx. 2.61 mg/kg bw/day. The effects on haematology and clinical chemistry were not clearly attributable to treatment, but are included here as a worst-case scenario. At higher doses, histopathological changes were observed.
Key result
Dose descriptor:
LOAEL
Effect level:
250 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical biochemistry
haematology
organ weights and organ / body weight ratios
Remarks on result:
other: corresponding to an actual ingested dose of 7.23 mg/kg bw/day.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
90 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
The present study was conducted to assess the toxicity of test substance on dogs when given for a time frame of approximately 52 weeks. The study was similar to the OECD guideline 452 and was performed under GLP conditions. The test substance was administered via dietary exposure to groups of 6 male and 6 female dogs at dose levels of 30, 90 and 250 ppm corresponding to
approximately 0.84, 2.61 and 7.35 mg/kg bw/day for males and 0.9, 2.54 and 7.23 mg/kg bw/day for
females. Under the conditions of the test, the test substance caused changes in haematology, clinical biochemistry parameters, organs weights and histopathology at 90 and/or 250 ppm in males and at 250 ppm in females. Therefore, the NOAEL was set at 30 ppm for males (0.84 mg/kg bw/day) and at 90 ppm for females (2.54 mg/kg bw/day).
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Jul - 1 Nov 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
Version / remarks:
adopted in 1998
Deviations:
yes
Remarks:
Urinalysis was not performed. Homogeneity samples were analysed 2 days after mixing. No analyses of samples for Week 2.
Qualifier:
according to guideline
Guideline:
EPA OPP 82-1 (90-Day Oral Toxicity)
GLP compliance:
yes
Limit test:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Hazleton Research Products, Virgina, USA
- Age at study initiation: 5 months
- Weight at study initiation: 6.5 - 8.8 kg (males), 5.6 - 9.0 kg (females)
- Fasting period before study: not applicable
- Housing: individually in stainless steel, screen-bottom cages
- Diet: certified canine diet #5007, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 16 days, including a complete physical examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 - 26.1
- Humidity (%): 30 - 70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 28 Jul 1988 To: 1 Nov 1988
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with: Basal diet. A specified amount of basal diet was weighed into a labelled mixing bowl, from which about 200 g was removed and placed in a blender. The required amount of test material was weighed, added to the blender, overlaid with about 50 g of basal diet from the mixing bowl before blending. This premix was transferred to the mixing bowl. About 100 g of basal diet from the mixing bowl was then transferred to the blender, blended to recover residual test material and returned to the mixing bowl and thoroughly mixed. Samples for dose analyses were taken directly from the mixing bowl. Diets were stored frozen in covered containers until dispensed daily into food containers.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity of freshly prepared Week 1 diets was determined for all treated dose levels. One sample each was taken from the top, bottom and two opposite sides of the mixing bowls of the finished diets and assayed in duplicate for test material content. Values for homogeneity were 92-94%, 95-100% and 94-102% of the theoretical levels of 75, 250 and 500 ppm, respectively.
Stability of the test material in the basal diet was determined. Four sets of samples each were taken from all test material dietary concentrations used for the study. One set was analyzed on the day of mixing, one was stored at room temperature and under the same conditions that were expected to be used for the study and then analyzed. The remaining two sets were analyzed after being stored in a freezer for 2 and 7 weeks. Mean results for diets analysed on the day of mixing were 91, 100 and 96% of the theoretical levels of 75, 250 and 500 ppm, respectively. Mean stability results for diets stored frozen for 2 or 7 weeks ranged from 90-97%. Diets analysed after being frozen for 7 days, in the animal room for 1 day, then frozen for 4 weeks were 98, 95 and 96% of the theoretical levels of 75, 250 and 500 ppm, respectively. Thus, the distribution of the test substance in the diet was homogenous and stable.

All diets were assayed each week for Weeks 1, 3 and 4 and every 4 weeks thereafter.
Duration of treatment / exposure:
90 days
Frequency of treatment:
continuously via the diet
Dose / conc.:
75 ppm
Remarks:
corresponding to approx. 1.94 - 2.58 mg/kg bw/day for males and 2.14 - 2.55 mg/kg bw/day for females
Dose / conc.:
250 ppm
Remarks:
corresponding to approx. 6.17 – 7.85 mg/kg bw/day for males and 6.67 – 8.01 mg/kg bw/day for females
Dose / conc.:
500 ppm
Remarks:
corresponding to approx. 10.55 - 14.69 mg/kg bw/day for males and 11.75 - 13.42 mg/kg bw/day for females
No. of animals per sex per dose:
4
Control animals:
yes, plain diet
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were checked twice daily for mortality and morbidity and once daily for signs of toxicity

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: prior to feeding, once a week throughout the pre-dosing and
dosing periods (including the first day of dosing and day of necropsy)

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once before dosing and during Week 13
- Dose groups that were examined: all animals of all dose groups, including the control
Prior to examination, the pupils of each animal were dilated using 1% Mydriacyl. The eyes were examined with an indirect ophthalmoscope.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before dosing (Week -2) and during Weeks 5 and 14
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, overnight
- How many animals: all animals of all dose groups, including the control
- Parameters checked: erythrocyte count (RBC), haemoglobin (Hb), haematocrit (Hc), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), white blood cell count, differential white blood cell count (including nucleated red blood cell count, corrected white blood cell count, segmented neutrophil count, band neutrophil count, lymphocyte count, monocyte count, eosinophil count, basophil count), platelet count, prothrombin time (PT), cell morphology

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before dosing (Week -2) and during Weeks 5 and 14
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, overnight
- How many animals: all animals of all dose groups, including the control
- Parameters checked: alkaline phosphatase activity (AP), alanine amino-transferase activity (ALT), aspartate amino-transferase activity (AST), creatinine concentration, creatine kinase, glucose concentration, total bilirubin concentration, total cholesterol concentration, total protein concentration, globulin, albumin, sodium (Na), potassium (K), chloride (Cl), calcium (Ca) concentrations, inorganic phosphorus concentration (P).

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
After 13 weeks of treatment, all animals were fasted overnight, then weighed, euthanized and necropsied.

The necropsy included a macroscopic examination of the external surface of the body, all orifices, the cranial cavity, external surfaces of the brain and spinal cord, the nasal cavity and paranasal sinuses, the thoracic abdominal and pelvic cavities and viscera. At the scheduled necropsy, the following organs were weighed: brain, kidneys, liver, ovaries, testes, thyroid with parathyroid. Organ-to-body weight percentages and organ-to- brain weight ratios were calculated.

Organs weighed: brain, kidney, liver, ovaries, testes, thyroid with parathyroid.

HISTOPATHOLOGY: Yes
- Tissues collected: adrenals, aorta, brain, caecum, colon, duodenum, epididymides, oesophagus, eyes, femur and bone marrow, gallbladder, heart, ileum, jejunum, kidneys, lesions, liver, lungs, lymph node, mammary gland, muscle, ovaries, pancreas, pituitary gland, prostate, rectum, salivary gland, sciatic nerve, skin, spinal cord, spleen, sternum, stomach, testes, thymus, thyroid with parathyroid, trachea, urinary bladder, uterus.
- Fixative: 10% phosphate-buffered formalin (except for eyes: Zenker’s solution)
- Embedding media: paraffin
- Staining: haematoxylin and eosin
- Animals examined: all animals from all dose groups, including the control group

Optional endpoint(s):
Additional bone marrow smears from the sternum were prepared, stained with Wright’s stain
and retained for possible examination.
Statistics:
Data were tested for homogeneity with Levene’s test. In the case of heterogeneity, data were transformed to stabilise the variance. Analysis of variance (ANOVA) was performed on the homogeneous or ranked data. If significant, Dunnett's t-test, student’s t-test, or Tukey- Kramer test was used for pairwise comparisons between groups. When no transformation established variance homogeneity at p < 0.001, the data were also examined by nonparametric techniques (Kruskal-Wallis H-test ANOVA, Nemenyi-Kruskal-Wallis test for multiple comparisons, Wilcoxon-Mann-Whitney two-sample rank test).
Standard one-way ANOVA was used to analyse initial body weights, food consumption, clinical chemistry and haematology values (except red blood cell morphology), organ weight, organ-to-body weight percentages, and organ-to-brain weight ratios.
Standard one-way analysis of covariance (ANCOVA) was used to analyse body weight, with initial body weight as a covariate. If the ANCOVA was significant, Dunnett’s t-test was used for pairwise comparisons between groups.
Group comparisons were evaluated at the 5% two-tailed probability level.
Clinical signs:
not specified
Description (incidence and severity):
Treatment-related signs were food-like vomitus and low food consumption in the males and females receiving the test substance.

Summarized results can be found in Attachment 1 in the attached background material.
Mortality:
no mortality observed
Description (incidence):
All animals survived the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 75 ppm: There were no statistically significant differences from controls in body weights and body weight gains.
- 250 ppm: Body weights were statistically significantly lower in females compared to the control group for Week 6.
- 500 ppm: Body weights were statistically significantly lower compared to the control group for Weeks 8, 10, 12 and 13 for males and in Weeks 1 - 13 for females. Terminal body weights were 18% (males) and 21% (females) lower compared to the control groups.

Summarized results can be found in Attachment 1 in the attached background material.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 75 ppm: No difference was observed between the control and treatment group.
- 250 ppm: statistically significantly decreased food consumption in females compared to the control group in Week 13 (-18.5%).
- 500 ppm: Statistically significantly lower food consumptions compared to the control group for males during Weeks 9, 11, 12, and 13 (up to approx. -32%), and for females during weeks 5, 6, 8, 10, 11, 12, and 13 (up to approx. -33%).

Summarized results can be found in Attachment 1 in the attached background material.
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:
no effects observed
Description (incidence and severity):
No treatment-related ophthalmic observations were noted at termination.

Summarized results can be found in Attachment 1 in the attached background material.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- 75 ppm: No adverse toxicological differences were observed. MCV was increased in males (+4%) but the difference did not reach statistical significance.
- 250 ppm: decreased RBC (females -14.3%), increased MCV (males +7.7%, females +9.2%), increased MCH (males +8.5%, females +6.6%), higher platelet count (males +26%). All changes noted were statistically significantly different compared to the control group.
- 500 ppm: decreased Hb (females -16.7%), decreased Hct (females, -16.9%), decreased RBC (males -11.8%, females -23.3%), increased MCV (males +7.7%, females +9.2%), decreased MCHC (males –2.1%), higher platelet count (males, +26.3%). All changes noted were statistically significantly different compared to the control group.

Summarized results can be found in Attachment 1 in the attached background material.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 75 ppm: No adverse toxicological differences were observed, the only change of note was decreased albumin levels in males compared to the control group (-16.7%), which was not considered to be adverse.
- 250 ppm: decreased total protein (females -16.1%), decreased albumin (males -13.9%, females -21.1%), higher cholesterol (males +30.6%). All changes noted were statistically significantly different compared to the control group.
- 500 ppm: decreased total protein (females -19.4%), decreased albumin (males -19.4%, females -26.3%), higher cholesterol (males +60.5%, females +41.8%, but the difference did reach statistical significance only for males). Unless stated otherwise, all changes noted were statistically significantly different compared to the control group.

The mild changes in total protein, albumin and cholesterol might be related to changes in food consumption or assimilation. Calcium was statistically significantly lower in females given 500 ppm. This effect did not appear to be adverse.

Summarized results can be found in Attachment 1 in the attached background material.
Endocrine findings:
not specified
Description (incidence and severity):
The following ED-related parameters were investigated in the study: histopathology of epididymis, mammary gland, ovary, prostate, pituitary, testis, thyroid, uterus, vagina and adrenals, organ weights were recorded for liver, prostate, testis, thyroid 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):
There were no treatment-related organ weight differences found at necropsy.

Summarized results can be found in Attachment 1 in the attached background material.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic changes found at necropsy.

Summarized results can be found in Attachment 1 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):
Microscopic changes were found in the livers of several animals. There were two variations of lesions found. One variant was characterised by multifocal accumulations of chronic inflammatory cells, primarily lymphocytes. The second variation was also multifocal in distribution. These changes were characterised by hepatocellular degeneration with granulomatous inflammation, bile duct hyperplasia and bile pigmentation. In some cases, one or more of these components were lacking. Thus, the two variations were not always distinctly separable. The two variations were considered to be differences in degree of development of one basic change, i.e. the second variant represented a more advanced manifestation of the same basic abnormality. The second variant was found in treated animals only (named "Hepatocellular degeneration" in the list below). Treatment appeared to facilitate development and progression of these changes, but they were not considered to be toxicological effects.

In summary, the following effects were noted in the liver:
- 75 ppm: chronic inflammation (2/4 males, 1/4 females vs 1/4 males and 1/4 females in the control group)
- 250 ppm: chronic inflammation (1/4 males, 2/4 females vs 1/4 males and 1/4 females in the control group), hepatocellular degeneration (1/4 males vs 0/4 males in the control group)
- 500 ppm: chronic inflammation (1/4 males, 2/4 females vs 1/4 males and 1/4 females in the control group), hepatocellular degeneration (2/4 males and 2/4 females vs 0/4 males in the control group).

Summarized results can be found in Attachment 1 in the attached background material.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
NOAEL
Effect level:
75 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 approximately 2 mg/kg bw/day.
Key result
Dose descriptor:
LOAEL
Effect level:
250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
haematology
Remarks on result:
other: corresponding to an actual ingested dose of approximately 6 mg/kg bw/day for males and females. At higher doses, changes in histopathology of the liver were also observed.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 ppm
System:
other: hepatobiliary and haematopoietic
Organ:
blood
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
The present study was conducted to assess the sub-chronic toxicity of test substance on dogs when given for a time frame of approximately 13 weeks. The study was similar to the OECD guideline 409 and was performed under GLP conditions. The test substance was administered via dietary exposure to groups of 4 male and 4 female dogs at dose levels of 75, 250 and 500 ppm corresponding to 1.94 - 2.58, 6.17 - 7.85 and 10.55 - 14.69 mg/kg bw/day for males and 2.14 - 2.55, 6.67 - 8.01 and 11.75 - 13.42 mg/kg bw/day for females. Under the conditions of the test, the test substance caused reduced body weights and food consumption and changes in haematology and clinical biochemistry parameters, and histopathology at 250 and/or 500 ppm. Therefore, the NOAEL
was set at 75 ppm for males and females (corresponding to approximately 2 mg/kg bw/day).
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Jun 1987 - 23 Mar 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted in 2018
Deviations:
yes
Remarks:
Mainly missing examinations, for example for sensory reactivity, thyroid hormone, ALP concentrations, blood clotting time/potential, no oestrus cycle was determined and the weight of only some organs was determined.
Qualifier:
according to guideline
Guideline:
EPA OPP 82-1 (90-Day Oral Toxicity)
Deviations:
no
GLP compliance:
yes
Limit test:
no
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, Portage, Michigan, USA
- Age at study initiation: approx. 7 weeks
- Weight at study initiation: mean of groups was 137 - 138.2 g (males), 118.4 - 125.1 g (females)
- Fasting period before study: not applicable
- Housing: individually in suspended, stainless steel, screen-bottom cages
- Diet: Certified Rodent Chow #5002, ad libitum
- Water: partially demineralized and sterilized, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 - 23.9
- Humidity (%): 30 - 70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 22 Jun 1987 To: 23 Sep 1987
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with: basal diet
- Storage temperature of food: refrigerated
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity of the diets was determined for all dose levels from freshly prepared diets for Week 1 (sampled at the top, bottom and sides of the mixing bowls). Homogeneity was 80, 97 and 98% for the theoretical levels of 50, 500 and 1000 ppm, respectively.

The stability of the test material in the diet was analyzed for all dose levels. Three samples per dose level were tested, of these, one was kept at room temperature for 7 days while the others were stored below 0 °C and assayed after 14 and 35 days. For frozen diets, the stability ranged from 81 to 99%. However, after 7 days at room temperature, the actual dietary concentration was 17, 80 and 88% of 50, 500 and 1000 ppm, respectively, indicating a stability problem of the substance in diets placed in the animal room, especially at the low-dose level.

All dose levels were assayed in duplicate for Weeks 2 through 4. Thereafter, one dose level was selected sequentially for analysis each week and analyzed every 4 weeks. Mean actual dietary concentrations were found to be 75, 95 and 97% of the target dietary concentrations (50, 500 and 1000 ppm, respectively) throughout the dosing period.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
continuously via the diet
Dose / conc.:
50 ppm
Remarks:
corresponding to approx. 3.5 mg/kg bw/day for males and 4 mg/kg bw/day for females.
Dose / conc.:
500 ppm
Remarks:
corresponding to approx. 38 mg/kg bw/day for males and females.
Dose / conc.:
1 000 ppm
Remarks:
corresponding to approx. 67 mg/kg bw/day for males and 80 mg/kg bw/day for females.
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Fasting period before blood sampling for clinical biochemistry: yes, overnight
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule and cage side observations checked: Two checks were made on a daily basis for dead or moribund animals. Individual animals were observed for any signs of toxic effects once daily.

DETAILED CLINICAL OBSERVATIONS: 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: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before study initiation and at termination
- Dose groups that were examined: all animals of all groups, including the control group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at study termination (Week 13)
- Anaesthetic used for blood collection: Yes (Ketamine)
- Animals fasted: Yes (overnight)
- How many animals: all animal of all groups, including the control
- Parameters checked: haemoglobin concentration (Hb), erythrocyte count (RBC), total leucocyte count (WBC), differential leucocyte count (WBC, differentiating among nucleated red blood cell count (NRBC), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M) corrected white blood cell count (COR WBC), segmented neutrophil count (N-SEG), band neutrophil cound (N-BAND)), platelet count (PLT), mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at study termination (Week 13)
- Anaesthetic used for blood collection: Yes (Ketamine)
- Animals fasted: Yes (overnight)
- Parameters checked: alanine amino-transferase activity (ALT), aspartate amino-transferase activity (AST), urea nitrogen concentration (UN), creatinine concentration (CREAT), glucose concentration (GLU), total bilirubin concentration (T BILI), total protein concentration (T PRO), albumin/globulin ratio (A/G RATIO), sodium (NA), potassium (K), chloride (CL), calcium (CA) concentrations, inorganic phosphorus concentration (I PHOS).

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
After 13 weeks of treatment, all animals were fasted overnight, then anesthetized using methoxyflurane, weighed, exsanguinated and necropsied. The necropsy included a detailed examination of the external features and orifices, the cranial cavity, carcass, the external surfaces of the brain and spinal cord, the nasal cavity and paranasal sinuses and the thoracic, abdominal and pelvic cavities and their viscera. The external and cut surfaces of the organs and tissues were examined as appropriate. Abnormalities, interactions and changes were recorded and the required tissues preserved in fixative.

Organ weights were recorded for: kidneys, liver, testes with epididymides

HISTOPATHOLOGY: Yes
- Tissues collected: Adrenals, aorta, bone & bone marrow, brain (medulla/cerebellum, thalamus, cerebral cortex, and caudate nucleus/cerebral cortex), caecum, colon, duodenum, epididymides, oesophagus, eyes, gross lesions, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (mesenteric, mandibular, cervical), mammary glands, pancreas, pituitary, rectum, salivary glands, sciatic nerve, spleen, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder, uterus
- Fixative: 10% phosphate buffered formalin (except eyes, which were preserved in Zenker's solution)
- Embedding media: Paraffin
Staining: hematoxylin and eosin
- Animals examined: All tissues from the animals of the control and high-dose group were examined histopathologically. If abnormalities were found (as was the case for the stomach), the mid and low dose group were also examined. In addition, the tissues from lungs, liver and kidneys were examined for all groups, including the control.
Optional endpoint(s):
No
Other examinations:
A bone marrow smear was preserved for possible examination.
Statistics:
Data were processed to give group mean values and standard deviations where appropriate. Haematology, clinical chemistry, organ weight (absolute and relative to terminal bodyweight), food consumption, body weight and weekly bodyweight gain were assessed with one way analysis variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Covariance was additionally assessed for body weight data.

Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s t-test or Games and Howell Modified Tukey-Kramer test.

Where Levene’s test showed unequal variances, the data were analysed using non-parametric methods: Kruskal-Wallis H-test ANOVA, Nemenyi-Kruskal-Wallis test and Wilcoxon-Mann-Whitney two-sample rank test.

All group comparisons found to be statistically significant at the 5% two-tailed probability level are indicated in the results section with an asterisk.
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related observations were noted in any treatment group compared to the control groups.

Summarized results can be found in Attachment 1 in the attached background material.
Mortality:
no mortality observed
Description (incidence):
No deaths occurred during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 50 ppm: No difference was observed between control and treatment groups.
- 500 ppm: Body weight and cumulative body weight gain was significantly lower compared to the control group throughout the study. Terminal body weight was 19 and 17% lower compared to the control group for males and females respectively (statistically significant). Cumulative body weight gains during Week 13 were 26 and 30% lower compared to the control group for males and females respectively (statistically significant).
- 1000 ppm: Body weight and cumulative body weight gain was significantly lower compared to the control group throughout the study. Terminal body weight was 28 and 23% lower compared to the control group for males and females respectively (statistically significant). Cumulative body weight gains during Week 13 were 38 and 37% lower compared to the control group for males and females respectively (statistically significant).

Summarized results can be found in Attachment 1 in the attached background material.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 50 ppm: No difference was observed between control and treatment groups.
- 500 ppm: Food consumption was statistically significantly reduced in Week 1 - 12 (males) and Week 1 - 13 (females) compared to the control group.
- 1000 ppm: Food consumption was statistically significantly reduced in Week 1 - 13 (males) and Week 1 - 8 and Week 10 - 13 (females) compared to the control group.

Summarized results can be found in Attachment 1 in the attached background material.
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:
no effects observed
Description (incidence and severity):
No treatment-related ocular effects were observed.

Summarized results can be found in Attachment 1 in the attached background material.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- 50 ppm: No difference was observed between control and treatment groups.
- 500 ppm: haemolysis was observed and affected the following parameters: decreased RBC (females -9.5%), decreased Hb (females -4.8%), decreased Hct (females -6.6%), increased MCV (males +9.1%), increased MCH (males +8.4%, females +5.4%), higher WBC (females +54.8%) and corrected WBC (females +52.4%), all differences noted were statistically significant compared to the control group.
- 1000 ppm: haemolysis was observed and affected the following parameters: decreased RBC (females -17%), decreased Hb (females -4.8%), decreased Hct (females -8.3%), increased MCV (males +7.3%, females +10.7%), increased MCH (males +7.2%, females +14.8%), higher WBC (females +97.6%) and corrected WBC (females +95.2%), all differences noted were statistically significant compared to the control group.

Statistically significant differences that were present for WBC count, corrected WBC count, absolute neutrophil count, and total bilirubin in males and for creatinine in females were considered to be normal biological variation, not treatment related and not toxicologically meaningful.

The higher WBC count and corrected WBC count observed in female rats at 500 and 1000 ppm, was characterized by a higher absolute neutrophil count, absolute lymphocyte count and absolute monocyte count. This generalized response is considered suggestive of an inflammatory process.


Summarized results can be found in Attachment 1 in the attached background material.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 50 ppm: No difference was observed between control and treatment groups.
- 500 ppm: decreased albumin concentrations (females -8%), decreased total protein concentrations (males -7%, females -6.9%), increased urea nitrogen concentrations (females +19.7%), all differences noted were statistically significant compared to the control group.
- 1000 ppm: decreased glucose concentrations (males -11.1%, females -14.7%, the difference did only reach statistical significance for females), decreased albumin concentrations (females -8%), decreased total protein concentrations (males -7%, females -5.6%, the difference did only reach statistical significance for males), increased urea nitrogen concentrations (females +35.8%), increased chloride concentrations (females +3.8%). If not stated otherwise, all differences noted were statistically significant compared to the control group.

Summarized results can be found in Attachment 1 in the attached background material.
Endocrine findings:
not specified
Description (incidence and severity):
The following ED-related parameters were investigated in the study: epididymis and testes weight, liver weight, histopathology for testis, thyroid, uterus, adrenals and pituitary. 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:
effects observed, treatment-related
Description (incidence and severity):
Animals dosed at 500 or 1000 ppm tended to have lower absolute organ weight, corresponding to the lower terminal body weight, and higher organ-to-body weight percentages. These were considered a secondary effect to the reduced body weight.

Summarized results can be found in Attachment 1 in the attached background material.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- 50 ppm: No difference between the control and treatment groups.
- 500 ppm: areas of erosion in the non-glandular stomach (2/10 males, 2/10 females vs 0/10 males and 0/10 females in the control group) and 1000 ppm (4/10 males, 2/10 females), diffusely red mesenteric lymph nodes (1/10 females vs 0/10 females in the control group), mottled mesenteric lymph nodes (7/10 males, 5/10 females vs 0/10 males and 0/10 females in the control group).
- 1000 ppm: areas of erosion in the non-glandular stomach (4/10 males, 2/10 females vs 0/10 males and 0/10 females in the control group), diffusely red mesenteric lymph nodes (3/10 males, 3/10 females; vs 0/10 males and 0/10 females in the control group), mottled mesenteric lymph nodes (6/10 males, 6/10 females vs 0/10 males and 0/10 females in the control group).

Summarized results can be found in Attachment 1 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):
- 50 ppm: No difference between the control and treatment groups.
- 500 ppm: mucosal hyperplasia of the non-glandular stomach (2/10 males, 3/10 females vs 0/10 males and 0/10 females in the control group), focal areas of ulceration in the stomach (1/10 females vs 0/10 females in the control group), inflammation or oedema of the non-glandular stomach (2/10 males, 3/10 females vs 0/10 males and 0/10 females in the control group), the mesenteric lymph nodes were frequently congested but otherwise normal (4/10 males, 5/10 females vs 0/10 males and 0/10 females in the control group).
- 1000 ppm: mucosal hyperplasia of the non-glandular stomach (3/10 males, 4/10 females; vs 0/10 males and 0/10 females in the control group), focal areas of ulceration in the stomach (3/10 females vs 0/10 females in the control group), inflammation or oedema of the non-glandular stomach (3/10 males, 4/10 females vs 0/10 males and 0/10 females in the control group) , the mesenteric lymph nodes were frequently congested but otherwise normal (7/10 males, 8/10 females vs 0/10 males and 0/10 females in the control group).

Summarized results can be found in Attachment 1 in the attached background material.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
NOAEL
Effect level:
50 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 3.5 mg/kg bw/day for male rats and 4 mg/kg bw/day for female rats.
Key result
Dose descriptor:
LOAEL
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
gross pathology
haematology
histopathology: non-neoplastic
Remarks on result:
other: corresponding to an actual ingested dose of 38 mg/kg bw/day for male and female rats.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 ppm
System:
other: hepatobiliary, haematopoietic
Organ:
blood
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
The present study was conducted to assess the sub-chronic toxicity of the test substance on rats when given for a time frame of approximately 13 weeks. The study was similar to the OECD guideline 408 and was performed under GLP conditions. The test substance was administered via dietary exposure to groups of 10 male and female rats at dose levels of 50, 500 and 1000 ppm corresponding to approximately 3.5, 38 and 67 mg/kg bw/day for males and 4, 38 and 80 mg/kg bw/day for females. Under the conditions of the test, the test substance caused changes in haematology and clinical biochemistry parameters as well as reduced body weight, food consumption, gross pathology and histopathology changes at the mid-and high dose level. Therefore, a NOAEL could was set at 50 ppm, corresponding to 3.5 and 4 mg/kg bw/day for male and female rats, respectively. The LOAEL was set at 500 ppm for males and females (corresponding to 38 mg/kg bw/day).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
0.84 mg/kg bw/day
Study duration:
chronic
Species:
dog
Quality of whole database:
The quality of the database is good comprising several studies in rodent species (rat and mouse) and non-rodent species (dog). All studies were conducted similar to OECD guidelines and under GLP conditions. All studies are considered of reliable quality and validity.
System:
other: haematopoietic and hepatobiliary
Organ:
blood
liver
spleen

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Mar - 13 Aug 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
adopted in 1981
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Froxfield Farms U.K. Ltd., Petersfield, Hampshire, England
- Age at study initiation: 11 - 13 weeks
- Weight at study initiation: group means were 2.599 - 2.736 kg (males) and 2.688 - 2.763 kg (females)
- Fasting period before study: no
- Housing: individually
- Diet: SQC rabbit diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 - 21
- Humidity (%): 42 - 55
- Air changes (per hr): approx. 19
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 25 Mar 1992 To: 13 Aug 1992
Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: dorsal region, 12 x 8 cm
- % coverage: approx. 10% of the total body surface
- Type of wrap if used: impervious bandage consisting of gauze covered with "Elastoplast" elastic adhesive dressing backed with impervious "Sleek" plaster.
- Time intervals for shavings or clippings: 24 h before the first application of the test substance and clipping was repeated as necessary during the experiment period.

REMOVAL OF TEST SUBSTANCE
- Washing: warm water and then gently blotted dry
- Time after start of exposure: 6 h

TEST MATERIAL
- Constant dose level: yes (based on recent body weight)
- For solids, paste formed: no, but the test substance was moistened with distilled water (0.6, 1.8 and 6 mL for the dose groups of 100, 300 and 1000 mg/kg bw/day, respectively).

USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
6 h/day for 21 (males) or 22 (females) consecutive days
Frequency of treatment:
21 (males) or 22 (females) consecutive days
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Fasting period before blood sampling for clinical biochemistry: yes, overnight
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality and morbidity on working days, once on weekends and public holidays, once daily for signs of ill health, behavioral changes or toxicosis.

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION: Yes
- Time schedule for examinations: immediately prior to the first daily application of the test substance and subsequently daily. The observations were scored according to a modified Draize scoring system.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to dosing and subsequently at weekly intervals during the study.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to termination (Week 3)
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes (overnight)
- How many animals: all animals of all dose groups
- Parameters checked: packed cell volume (PCV), haemoglobin concentration (Hb), erythrocyte count (RBC), total leucocyte count (WBC), differential leucocyte count (WBC, differentiating among neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B) and monocytes (M)), platelet count, mean cell volume (MCV), mean cell haemoglobin concentration (MCHC), Thrombotest, cell morphology.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to termination (Week 3)
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes (overnight)
- How many animals: all animals of all dose groups
- Parameters checked: alkaline phosphatase activity (AP), alanine amino-transferase activity (ALT), aspartate amino-transferase activity (AST), urea concentration, creatinine concentration, glucose concentration, total bilirubin concentration, globulin concentration (Glob), albumin concentration (Alb), albumin/globulin ratio (A/G), total cholesterol concentration (Chol), total protein concentration, sodium (Na), potassium (K), chloride (Cl), calcium (Ca) concentrations, inorganic phosphorus concentration (P).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were sacrificed at study termination and a gross pathological examination was performed.

Organs weighed: adrenals, liver, kidneys, testes with epididymides/ovaries

HISTOPATHOLOGY: Yes
- Tissues examined: abnormal tissue, skin (treated and untreated), kidneys, liver
- Fixative: 10% buffered formalin
- Embedding media: Paraffin wax
- Section thickness: 4 µm
- Staining: haematoxylin and eosin
- Animals examined: All tissues of all animals in the control and high-dose group, the skin was examined in all treatment and control groups.
Statistics:
All analyses were carried out separately for males and females. The following tests were used for food and water consumption, bodyweight, relative 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 appropriate methods. 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 a Student’s ‘t’ test and Williams’ test for a dose-related response, although only the one thought most appropriate for the response pattern observed has been reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the ‘t’ test and Williams’ test (Shirleys’ test). Where appropriate for organ weight data, analysis of covariance was used in place of analysis of variance.
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed.

Summarized results can be found in Attachment 1 in the attached background material.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
- 100 mg/kg bw/day: Weekly mean irritation indices of 0 (Week 1), 0.9 (Week 2) and 1.5 (Week 2) for males and 0 (Week 1), 0.5 (Week 2) and 1.0 (Week 2) for females.
- 300 mg/kg bw/day: Weekly mean irritation indices of <0.1 (Week 1), 1.2 (Week 2) and 2.1 (Week 2) for males and <0.1 (Week 1), 0.6 (Week 2) and 1.3 (Week 2) for females.
- 1000 mg/kg bw/day: Weekly mean irritation indices of <0.1 (Week 1), 1.5 (Week 2) and 2.6 (Week 2) for males and <0.1 (Week 1), 1.1 (Week 2) and 2.3 (Week 2) for females.

Slight or well-defined erythema with or without slight or well-defined oedema, were noted with greater frequency in the mid- and high-dose group. Reactions were observed initially in isolated incidences on Day 7 but over the following two weeks developed in the great majority of rabbits. These reactions were frequently accompanied by residual (brown) staining from the test substance and/or desquamation of the stratum corneum. The control group showed no signs of dermal irritation.

Summarized results can be found in Attachment 1 in the attached background material.
Mortality:
no mortality observed
Description (incidence):
There were no mortalities.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- 100 and 300 mg/kg bw/day: No effects on body weight were observed between the control and treatment groups.
- 1000 mg/kg bw/day: Statistically significantly reduced bodyweight gain was observed in females dosed compared to the control group throughout the treatment period. Cumulative body weight gain in females was 40.5% decreased compared to the control group.

Summarized results can be found in Attachment 1 in the attached background material.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 100 and 300 mg/kg bw/day: No effects on food consumption were observed between the control and treatment groups.
- 1000 mg/kg bw/day: : Reduction in mean food consumption was measured for females throughout the study. These changes were statistically significant (p 0.05) in Week 1 and 2.

Summarized results can be found in Attachment 1 in the attached background material.
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:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were observed between the control and treatment groups. The only statistically significant differences were slightly increased RBC (females, at all dose levels, up to +14.9%, but not dose-related) and slightly increased MCHC (females, 1000 mg/kg bw/day, +3.1%). As these were minor changes and not dose related, they were not considered treatment related or adverse.

Summarized results can be found in Attachment 1 in the attached background material.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 100 and 300 mg/kg bw/day: No effects on clinical chemistry were observed between the control and treatment groups.
- 1000 mg/kg bw/day: Liver enzymes AST (+361%), ALT (+356%) and cholesterol (+88%) levels were statistically significantly increased in females, AP was statistically significantly increased (males +59.4%, females +18%), all compared to the control group.

Summarized results can be found in Attachment 1 in the attached background material.
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
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 applicbale
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No dose-related effects were observed.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No dose-related effects were observed.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Minimal generalised/focal acanthosis of epidermis at the treated skin sites of all male and female rabbits were considered treatment-related. Other treatment-related changes were not observed between control and treatment groups.

Summarized results can be found in Attachment 1 in the attached background material.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed up to this dose level.
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Remarks:
dermal irritation
Effect level:
< 100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: dermal irritation was observed in all treatment groups.
Key result
Critical effects observed:
no

The LOAEL per area was 2.7 mg/cm². It was calculated using the lowest body weight (2.6 kg), the lowest dose group (100 mg/kg bw/day) and the exposure area of 12 x 8 cm.

Conclusions:
The study was conducted according to OECD guideline 410 and under GLP. Under the conditions of the test, the test substance caused dermal irritation at all dose levels when applied dermally for three weeks to the intact skin of male and female rabbits (100, 300 and 1000 mg/kg bw/day). Treatment-related disturbances in body weight, food consumption and various biochemical parameters were observed for rabbits receiving the test substance at 1000 mg/kg bw/day. Thus, no NOAEL was set for local skin effects and the NOAEL for systemic effects was 300 mg/kg bw/day for males and females.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Experimental exposure time per week (hours/week):
42
Species:
rabbit
Quality of whole database:
The quality of the database is good comprising one study in rabbits, similar to OECD TG 410 and under GLP conditions. The study is considered of reliable quality and validity, fulfilling the criteria of a key study. Thus, it is suitable for assessment of the present endpoint.

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Mar - 13 Aug 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
adopted in 1981
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Froxfield Farms U.K. Ltd., Petersfield, Hampshire, England
- Age at study initiation: 11 - 13 weeks
- Weight at study initiation: group means were 2.599 - 2.736 kg (males) and 2.688 - 2.763 kg (females)
- Fasting period before study: no
- Housing: individually
- Diet: SQC rabbit diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 - 21
- Humidity (%): 42 - 55
- Air changes (per hr): approx. 19
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 25 Mar 1992 To: 13 Aug 1992
Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: dorsal region, 12 x 8 cm
- % coverage: approx. 10% of the total body surface
- Type of wrap if used: impervious bandage consisting of gauze covered with "Elastoplast" elastic adhesive dressing backed with impervious "Sleek" plaster.
- Time intervals for shavings or clippings: 24 h before the first application of the test substance and clipping was repeated as necessary during the experiment period.

REMOVAL OF TEST SUBSTANCE
- Washing: warm water and then gently blotted dry
- Time after start of exposure: 6 h

TEST MATERIAL
- Constant dose level: yes (based on recent body weight)
- For solids, paste formed: no, but the test substance was moistened with distilled water (0.6, 1.8 and 6 mL for the dose groups of 100, 300 and 1000 mg/kg bw/day, respectively).

USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
6 h/day for 21 (males) or 22 (females) consecutive days
Frequency of treatment:
21 (males) or 22 (females) consecutive days
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Fasting period before blood sampling for clinical biochemistry: yes, overnight
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality and morbidity on working days, once on weekends and public holidays, once daily for signs of ill health, behavioral changes or toxicosis.

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION: Yes
- Time schedule for examinations: immediately prior to the first daily application of the test substance and subsequently daily. The observations were scored according to a modified Draize scoring system.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to dosing and subsequently at weekly intervals during the study.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to termination (Week 3)
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes (overnight)
- How many animals: all animals of all dose groups
- Parameters checked: packed cell volume (PCV), haemoglobin concentration (Hb), erythrocyte count (RBC), total leucocyte count (WBC), differential leucocyte count (WBC, differentiating among neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B) and monocytes (M)), platelet count, mean cell volume (MCV), mean cell haemoglobin concentration (MCHC), Thrombotest, cell morphology.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to termination (Week 3)
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes (overnight)
- How many animals: all animals of all dose groups
- Parameters checked: alkaline phosphatase activity (AP), alanine amino-transferase activity (ALT), aspartate amino-transferase activity (AST), urea concentration, creatinine concentration, glucose concentration, total bilirubin concentration, globulin concentration (Glob), albumin concentration (Alb), albumin/globulin ratio (A/G), total cholesterol concentration (Chol), total protein concentration, sodium (Na), potassium (K), chloride (Cl), calcium (Ca) concentrations, inorganic phosphorus concentration (P).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were sacrificed at study termination and a gross pathological examination was performed.

Organs weighed: adrenals, liver, kidneys, testes with epididymides/ovaries

HISTOPATHOLOGY: Yes
- Tissues examined: abnormal tissue, skin (treated and untreated), kidneys, liver
- Fixative: 10% buffered formalin
- Embedding media: Paraffin wax
- Section thickness: 4 µm
- Staining: haematoxylin and eosin
- Animals examined: All tissues of all animals in the control and high-dose group, the skin was examined in all treatment and control groups.
Statistics:
All analyses were carried out separately for males and females. The following tests were used for food and water consumption, bodyweight, relative 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 appropriate methods. 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 a Student’s ‘t’ test and Williams’ test for a dose-related response, although only the one thought most appropriate for the response pattern observed has been reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the ‘t’ test and Williams’ test (Shirleys’ test). Where appropriate for organ weight data, analysis of covariance was used in place of analysis of variance.
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed.

Summarized results can be found in Attachment 1 in the attached background material.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
- 100 mg/kg bw/day: Weekly mean irritation indices of 0 (Week 1), 0.9 (Week 2) and 1.5 (Week 2) for males and 0 (Week 1), 0.5 (Week 2) and 1.0 (Week 2) for females.
- 300 mg/kg bw/day: Weekly mean irritation indices of <0.1 (Week 1), 1.2 (Week 2) and 2.1 (Week 2) for males and <0.1 (Week 1), 0.6 (Week 2) and 1.3 (Week 2) for females.
- 1000 mg/kg bw/day: Weekly mean irritation indices of <0.1 (Week 1), 1.5 (Week 2) and 2.6 (Week 2) for males and <0.1 (Week 1), 1.1 (Week 2) and 2.3 (Week 2) for females.

Slight or well-defined erythema with or without slight or well-defined oedema, were noted with greater frequency in the mid- and high-dose group. Reactions were observed initially in isolated incidences on Day 7 but over the following two weeks developed in the great majority of rabbits. These reactions were frequently accompanied by residual (brown) staining from the test substance and/or desquamation of the stratum corneum. The control group showed no signs of dermal irritation.

Summarized results can be found in Attachment 1 in the attached background material.
Mortality:
no mortality observed
Description (incidence):
There were no mortalities.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- 100 and 300 mg/kg bw/day: No effects on body weight were observed between the control and treatment groups.
- 1000 mg/kg bw/day: Statistically significantly reduced bodyweight gain was observed in females dosed compared to the control group throughout the treatment period. Cumulative body weight gain in females was 40.5% decreased compared to the control group.

Summarized results can be found in Attachment 1 in the attached background material.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 100 and 300 mg/kg bw/day: No effects on food consumption were observed between the control and treatment groups.
- 1000 mg/kg bw/day: : Reduction in mean food consumption was measured for females throughout the study. These changes were statistically significant (p 0.05) in Week 1 and 2.

Summarized results can be found in Attachment 1 in the attached background material.
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:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were observed between the control and treatment groups. The only statistically significant differences were slightly increased RBC (females, at all dose levels, up to +14.9%, but not dose-related) and slightly increased MCHC (females, 1000 mg/kg bw/day, +3.1%). As these were minor changes and not dose related, they were not considered treatment related or adverse.

Summarized results can be found in Attachment 1 in the attached background material.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 100 and 300 mg/kg bw/day: No effects on clinical chemistry were observed between the control and treatment groups.
- 1000 mg/kg bw/day: Liver enzymes AST (+361%), ALT (+356%) and cholesterol (+88%) levels were statistically significantly increased in females, AP was statistically significantly increased (males +59.4%, females +18%), all compared to the control group.

Summarized results can be found in Attachment 1 in the attached background material.
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
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 applicbale
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No dose-related effects were observed.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No dose-related effects were observed.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Minimal generalised/focal acanthosis of epidermis at the treated skin sites of all male and female rabbits were considered treatment-related. Other treatment-related changes were not observed between control and treatment groups.

Summarized results can be found in Attachment 1 in the attached background material.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed up to this dose level.
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Remarks:
dermal irritation
Effect level:
< 100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: dermal irritation was observed in all treatment groups.
Key result
Critical effects observed:
no

The LOAEL per area was 2.7 mg/cm². It was calculated using the lowest body weight (2.6 kg), the lowest dose group (100 mg/kg bw/day) and the exposure area of 12 x 8 cm.

Conclusions:
The study was conducted according to OECD guideline 410 and under GLP. Under the conditions of the test, the test substance caused dermal irritation at all dose levels when applied dermally for three weeks to the intact skin of male and female rabbits (100, 300 and 1000 mg/kg bw/day). Treatment-related disturbances in body weight, food consumption and various biochemical parameters were observed for rabbits receiving the test substance at 1000 mg/kg bw/day. Thus, no NOAEL was set for local skin effects and the NOAEL for systemic effects was 300 mg/kg bw/day for males and females.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
2.7 mg/cm²
Study duration:
subacute
Species:
rabbit
Quality of whole database:
The quality of the database is good comprising one study in rabbits, according to OECD TG 410 and under GLP conditions. The study is considered of reliable quality and validity, fulfilling the criteria of a key study. Thus, it is suitable for assessment of the present endpoint.

Mode of Action Analysis / Human Relevance Framework

Please refer to the respective endpoint summary. 

Additional information

Repeated dose toxicity: oral administration

Data on repeated dose toxicity are available after exposure for 28 days, 90 days, 52 weeks (dog) and 104 weeks (rat) to characterize the potency of the test substance to induce toxicity after short-term and long-term exposure.

 

Mice

A subacute (Report number: HLA-6111-127) key study was available that served as a dose range finding study for the key carcinogenicity study (Report number: 798-223). Both studies were conducted similar to OECD guidelines and under GLP conditions.

 

In the subacute oral toxicity study (Report number: HLA-6111-127), the test substance was administered at 300, 600 and 1200 ppm for 4 weeks to groups of 10 animals/sex and dose (corresponding to approx. 51-58, 101-115 and 177-226 mg/kg bw/day in males and to approx. 59-66, 111-127 and 221-281 mg/kg bw/day in females, respectively). There were no mortalities during the study. Body weights were significantly lower for males in the 300 and 1200 ppm groups during Week 3 and 4 and for males in the 600 ppm group for Week 4. Females showed no differences. Food consumptions were significantly lower for all treated animals throughout the study period. RBC count, Hb and Hct were slightly lower in all treated males (up to -8.6%). In females RBC count was decreased in the 600 ppm group (-6.2%) while platelet count was increased at 600 and 1200 ppm (up to +27.6%). The only statistically significant alteration in clinical chemistry was the decreased glucose value in females dosed at 1200 ppm. Absolute organ weights alterations were not dose-response-related and regarded as a result of the lower terminal body weight. There were no treatment-related macroscopic or microscopic observations noted.

No NOAEL was set for males, the LOAEL for males was set at 300 ppm, corresponding to 51 mg/kg bw/day. In females, 300 ppm, corresponding to 59 mg/kg bw/day was the NOAEL.

 

 

 

Rats

A subchronic (Report number: HLA 6111-110) and one combined chronic/carcinogenicity studies (Report number: HLA 6111-113) were available, which are considered as key studies. All studies were conducted similar to OECD guidelines and under GLP conditions.

 

Description of the studies with rats

In a sub-chronic study (Report number: HLA 6111-110), the test substance was administered via diet to three groups, each consisting of ten male and ten female Sprague-Dawley rats. The test substance was administered for 13 weeks at dose levels 50, 500 and 1000 ppm, corresponding to 0, 3.5, 38 and 67 mg/kg bw/day for males and 0, 4, 38 and 80 mg/kg bw/day for females. The control group of ten males and ten females received plain diet. No treatment-related deaths or antemortem observations were noted. Body weights, cumulative body weight gains and food consumptions were significantly lower in animals dosed at 500 or 1000 ppm. Changes in clinical pathology occurred at 500, 1000 ppm or both and affected parameters typical for haemolysis, e.g. lower RBC count, Hb and Hc and higher MCV and MHC in both sexes. Besides that, higher leukocyte count, urea nitrogen and chloride and lower albumin in females and lower total protein and glucose in both sexes were noted. Corresponding to the lower terminal body weight animals dosed at 500 or 1000 ppm had lower absolute organ weights. Pathological examinations revealed the nonglandular stomach as target organ. However, since the observed mucosal hyperplasia may be caused by the irritating properties of the substance and the nonglandular stomach is not present in humans, the relevance for human hazard assessment may be questionable. Oral administration of the test substance to rats for a period of 13 weeks at a dose level up to 1000 ppm resulted in treatment-related adverse effects in animals treated with 500 and 1000 ppm. The NOAEL was set at 3.5-4 mg/kg bw/day.

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 (corresponding to approx. 1.46, 7.31 and 14.66 mg/kg bw/day in males and to approx. 1.80, 8.86 and 18.57 mg/kg bw/day in females, respectively). As also observed in the subchronic repeated dose studies, the test substance caused general systemic 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 haemolysis, evident by changes in the number of erythrocytes, haemoglobin and haematocrit concentration, as well as increase 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 were observed, the latter including extramedullary haematopoieses in the liver and spleen, suggesting a test material induced effect on haematopoiesis. 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. For the description of the carcinogenic effects, please refer to the respective Endpoint summary.

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.

 

Conclusion for all studies in rats

 

In all of the repeated dose toxicity studies with rats, the test substance caused decreased body weight gain and food intake. Further, changes in blood parameters suggesting haemolysis were observed. These were consistent decreased mean red blood cells (RBC), haemoglobin (Hb) and haematocrit (Hct) concentrations, increased mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC). The haematological changes occured at an actual ingested dose of 38 mg/kg bw/day (males and females) in the 13-week repeated dose oral toxicity study and at an actual ingested dose of 7.3 mg/kg bw/day (males) and 8.9 mg/kg bw/day (females) in the combined chronic/carcinogenicity study.

Based on the subchronic and combined chronic/carcinogenicity rat studies, the test substance affected several target organs with the following treatment-related tissue changes: erosive lesions of the non-glandular epithelium in the stomach, changes in mesenteric lymph nodes, extramedullary haematopoiesis (EMH) in the spleen and liver. These histopathological findings were accompanied by gross pathological findings (stomach, mesenteric lymph nodes, liver, kidneys). In the 104-weeks dietary combined chronic/carcinogenicity study, increased incidences of steatosis/fatty infiltration were found in the pancreas of mid- and high-dosed animals compared to the control group. This finding cannot be specifically attributed to treatment but could also be age-related or a secondary effect to the presence of acinar atrophy (Covance 6111 -113).

The principal effect caused by the test substance is haemolysis indicated by a decrease in red blood cells and haemoglobin and by an increase in the mean corpuscular volume and mean corpuscular haemoglobin concentration is accompanied by subsequent effects on spleen and liver, seen as extramedullary haematopoiesis.

The carcinogenicity of the test item is discussed in the corresponding section. The ED properties of the substance are discussed in the respective robust study summaries. 

In total, the lowest oral NOAEL in rats was 30 ppm (corresponding to 1.46 mg/kg bw/day for males and 1.8 mg/kg bw/day for females) from the 2 years combined chronic/carcinogenicity study.

 

Dogs

Three oral repeated dose toxicity studies in dogs are available, covering different study durations including a 28-days supporting study (Report number: HLA 6111-109), a 90-days key study (Report number: HLA  6111-121) and a 52-weeks (Report number: HLA 6111-112) study. All three studies were conducted similar to the respective OECD guidelines and under GLP conditions.

 

Description of studies with dogs

 

In a sub-acute supporting study (Report number: HLA 6111-109), two beagle dogs per sex and dose were administered daily doses of 125, 500 and 2000/1500 ppm (corresponding to approx.4, 12 and 27-21 mg/kg bw test substance for females and 4, 16 and 26-15 mg/kg bw/day) test substance for males in the diet for a period of four weeks. Due to mortality, the dose level of the high dosed group was reduced from 2000 ppm to 1500 ppm during week 3 and 4. 

Clinical observations noted included ptyalism and convulsion. One female of the highest dose group died during week 2 and one male of the same group was moribund and sacrificed during week 4. Body weights in both sexes were decreased at all doses. During week 4, food consumptions in males/females at all doses were lower than those of controls. Treatment-related changes in blood parameters appeared to be lower erythrocyte count, haemoglobin and haematocrit in males given 12 and 27-21 mg/kg bw/day as well as slightly lower platelet count in some dogs. One male given 27-21 mg/kg bw/day had higher ALT, AST and AP. Slightly lower absolute lymphocyte and slightly higher total bilirubin and urea nitrogen occurred in some dogs. Corresponding to the lower body weights, surviving animals given the highest dose level had lower absolute organ weights. The liver of the surviving male dosed at 27-21 mg/kg bw/day had hepatocellular degeneration with sinusoidal cell proliferation and pigmentation. Based on reduced body weight and food consumption and alteration in clinical blood parameters in the 16/12 mg/kg bw/day dose group, the NOAEL was set at 4 mg/kg bw/day for both sexes.

 

In the sub-chronic study, the test substance was administered via diet to groups of 4 male and 4 female Beagle dogs for a period of 13 weeks at dietary concentrations of 75, 250 and 500 ppm, corresponding to 1.94 -2.58, 6.17-7.85, 10.55-14.69 mg/kg bw/day for males and 0.00, 2.14-2.55, 6.67-8.01, 11.75-13.42 mg/kg bw/day for females (Report number: HLA  6111-121). Control dogs received plain diet. No mortalities occurred. Clinical signs of toxicological relevance consisted of food-like vomitus, reduced food consumption, and reduced body weights (up to 18% and 21% lower as compared to control animals for males and females of the 500 ppm group in week 13).

There were several statistically significant differences for clinical pathology variables between control and treated dogs. In addition, there were some consistent differences that were not statistically significant. The differences considered to represent treatment-related effects were lower RBC count and higher MCV and MCH in males and females given 75, 250, or 500 ppm; lower haemoglobin and haematocrit in females given 500 ppm, higher platelet count in males given 250 or 500 ppm; lower total protein and albumin in males and females given 75, 250, or 500 ppm; and higher cholesterol in males and females given 250 or 500 ppm. Other differences were considered normal biological variation, not related to the test material, and not toxicologically meaningful. Organ weight analyses, ophthalmological examinations and gross pathology revealed no treatment-related effects. Microscopic changes (chronic inflammatory cells, and hepatocellular degeneration with granulomatous inflammation) were found in the livers of several animals. These changes were thought to be variations of a process caused by low-grade bacterial infection. The treatment appeared to facilitate development and progression of the changes, but the changes were not considered to be toxicological effects. The NOAEL was set at 2 mg/kg bw/day.

 

In the chronic study, six dogs per sex and dose level received daily doses of 0, 30, 90 or 250 ppm test substance in diet, corresponding to 0, 0.84, 2.61, 7.35 mg/kg bw/day for males and 0, 0.90, 2.54, 7.23 mg/kg bw/day for females for 52 consecutive weeks (Report number: HLA 6111-112).

One female at 90 ppm was sacrificed during week 25, due to clonic convulsions with pulmonary congestion. Pathology examinations revealed meningoencephalitis. Another female of the high-dose group was sacrificed during week 26 due to clonic seizures with opisthotonus. Pathology examinations revealed hydrocephalus. However, both deaths were not considered treatment-related. No treatment-related effects were noted in any of the other dogs surviving until termination. There were also no observations that indicated an effect on the neurological system. There were no statistically significant differences in body weights of males and females at any dose level. Food consumption of females at 250 ppm was significantly lower than controls during week 20. However, there were no consistent effects on food consumptions in both sexes throughout the study. Ophthalmoscopic examinations revealed no lesions in any animal. RBC values were reduced in high dose males. Higher MCV and MCH values were also observed in treated animals. However, since there were similar differences noted before initiation of treatment, no conclusion could be drawn from these findings. Clinical pathology examination revealed lower protein and higher cholesterol levels in males at 90 or 250 ppm, and in females at 250 ppm. Albumin values were lower in both sexes at 250 ppm. These effects were considered not treatment-related. Absolute and relative liver weights were significantly increased in males at 90 and 250 ppm. Relative liver weights were also significantly increased in high-dose females, as were liver-to-brain ratios in high-dose males. The changes in liver weights, in conjunction with the altered protein, albumin, and cholesterol levels suggest a test material-related effect on liver function and size. As the liver has an important role in the metabolism of the test substance, these changes may be considered as an adaptation to treatment. There were no gross pathological and histopathological adverse findings at necropsy. However, based on the observed magnitude of ≥ 20% liver weight increase compared to control, this effect may be considered adverse (ECETOC Technical Report No. 85, 2002). Based on the increased liver weights in mid- and high-dose males, and on changes in clinical chemistry parameters in the same groups, as well as for high-dose females, the NOAEL for dogs after 52-week dietary administration of the test substance is 0.84 mg/kg bw/day for males, and 2.54 mg/kg bw/day for females.

 

 

Conclusion for all studies in dogs

 

Dogs were noted with reduced body weight and food consumption in the subchronic key study. Decreased RBC, increased MCV and MCH were noted in all key studies. In the subchronic study, also decreased Hct, Hb and decreased MCHC were noted. In the subchronic and chronic study, the following clinical chemistry parameters were altered: total protein and albumin were decreased while cholesterol was increased. Hepatocellular degeneration was seen in the subacute and subchronic study, in the 52-week study, the absolute liver weight was increased but no histopathological changes were observed. The ED properties of the substance are discussed in the respective robust study summaries. 

 

In total, the lowest most relevant oral NOAEL was 30 ppm for males (corresponding to 0.84 mg/kg bw/day) in the 52-week dog study. For females, the lowest NOAEL was 75 ppm (corresponding to approx. 2 mg/kg bw/day) in the subchronic study.

 

 

Repeated dose toxicity: dermal administration

A 21-day percutaneous study on rabbits (Report number: UCB 421/920767) was conducted according to OECD 410 and under GLP conditions. The test substance caused dermal irritation at all dose levels when applied dermally for three weeks to the intact skin of male and female rabbits (100, 300 and 1000 mg/kg bw/day). Treatment-related disturbances in body weight, food consumption and various biochemical parameters were observed for rabbits receiving the test substance at 1000 mg/kg bw/day. No treatment-related changes were observed between the control and treatment groups regarding haematological changes. The only statistically significant differences were slightly increased RBC and MCHC values in females of the highest dose group. As these were minor changes and not dose related, they were not considered treatment related or adverse. Thus, no NOAEL was set for local skin effects and the NOAEL for systemic effects was 300 mg/kg bw/day for males and females.

 

Conclusion

Overall, the lowest most relevant oral NOAEL was 30 ppm (corresponding to 0.84 mg/kg bw/day for males and 0.9 mg/kg bw/day for females) from the 52-weeks dog study. The lowest dermal LOAEL was 100 mg/kg bw/day in regards to local effects and the lowest dermal NOAEL was 300 mg/kg bw/day for systemic effect in rabbits.

Further, it was concluded that the test substance affected the target organs liver (all animals), non-glandular stomach (rat and mice) and spleen (rat and mouse). The dog is considered to be the most sensitive species. An assessment on endocrine disruption was performed during the CoRAP substance evaluation of Thiram. The outcome of this evaluation in 2018 was that Thiram has no ED concern. 

Effects on target organs or haematology parameters were observed at dose levels below 10 mg/kg bw/day in the combined carcinogenicity/chronic toxicity study in the rat as well as in the subchronic and chronic dog studies. However, according to the Regulation (EC) No 1272/2008, the reference value of 10 mg/kg bw/day for classification as Specific Target Organ Toxicity after Repeated Exposure (STOT-RE), Category 1 is referring to a subchronic study in rats. The corresponding guidance values for a study period of 52 weeks are <= 2.5 mg/kg bw/day for STOT RE, Category 1, and > 2.5 - <= 25 mg/kg bw/day for STOT RE, Category 2. Moreover, it is stated that the effects observed should be “consistent and significant adverse change[s] in clinical biochemistry, haematology, or urinalysis parameters” (p. 469). Even though the changes are consistent over study periods and species, the effect at doses below 10 mg/kg bw/day cannot be seen as significantly adverse. The guideline states as an example of a significant adverse effect a reduction of haemoglobin of at least 10% compared to the control group (p. 470). A reduction of this extent has not been observed in any of the studies with the test substance. Therefore, significant adverse effects are only seen above doses of 10 mg/kg bw/day in the sub-chronic studies and above doses of 2.5 mg/kg bw/day in chronic studies over 52 weeks. Therefore, classification as Specific target Organ Toxicity after Repeated Exposure (STOT RE), Category 1 is not warranted and the classification criteria for Specific Target Organ Toxicity after Repeated Exposure (STOT RE), Category 2, H373 (Regulation (EC) No 1272/2008) are met which is in line with the harmonised classification according to Annex VI of CLP Regulation (Index No.: 006-005-00-4).

 

References:

 

Covance 6111 -113, 104-week dietary combined chronic toxicity and Carcinogenicity study with thiram in rats, Supplementary Information on Pancreas Steatosis/Fatty Infiltration, 2016

Justification for classification or non-classification

Based on the observations of systemic toxic effects (significant changes in the spleen, stomach and liver) in the repeated dose toxicity studies at relevant dose levels, classification criteria for Specific Target Organ Toxicity after Repeated Exposure (STOT RE), Category 2, H373 (Regulation (EC) No 1272/2008) are met. This is in line with the harmonised classification of the test substance (Annex VI of Regulation (EC) 1272/2008, index number 066-012-00-4).

Based on local effects observed in the dermal 21-d repeated dose toxicity study in rabbits (UCB 421/920767) the test substance is classified as skin irritating Category 2 (H315) and included in Annex VI of Regulation (EC) No 1272/2008 (Index No. 006-005-00-4).