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Description of key information

key; M-027741-02-1 (suppl. M-027135-01-1); combined carcinogenicity + chronic (2 years, rat, oral): NOAEL (males) 100 ppm (equivalent to 5.7 mg/kg bw/day) and NOAEL (females) 300 ppm (equivalent to 24.9 mg/kg bw/day)

key; M-026310-01-1 (suppl. M-026038-01-1); carcinogenicity (2 years, mouse, oral): NOAEL (males) 330 ppm (equivalent to 65.6 mg/kg bw/day) and NOAEL (females) 330 ppm (equivalent to 103.6 mg/kg bw/day)

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Referenceopen allclose all

Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jul 1987 - Jul 1989 (M-027741-02-1)

Sep 1988 - Sep 1990 (M-027135-01-1)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
adopted 2018
Deviations:
yes
Remarks:
see 'additional information on materials and methods incl. tables' section
Qualifier:
according to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
adopted 1981
Deviations:
no
Principles of method if other than guideline:
In this RSS, the main study (M-027741-02-1) and a supplementary study (M-027135-01-1) are reported. The supplementary study was performed to support determination of a MTD of the test item in rats. The main study consisted of a control group (0 ppm) and the dose groups 100, 300 and 900 ppm, while the supplementary study consisted of a control group (0 ppm) and a high-dose group (1800 ppm). If not stated otherwise, the procedures in the supplementary study were the same as compared to the main study.
GLP compliance:
yes
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was chosen since it is a recommended species for chronic toxicological/cancerogenicity studies in test guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Winkelmann Experimental Animal Breeder, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 4 - 6 weeks
- Weight at study initiation: 56 - 102 g (males), 59 - 89 g (females)(M027741-02-1); 77 - 111 g (males), 68 - 100 g (females)(M-027135-01-1)
- Housing: animals were individually kept in Type II Makrolon cages on low-dust wood shavings.
- Diet: Altromin 1321 flour (Altromin GmbH & Co KG, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): about 50
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: Jul 1987 To: Jul 1989 (M-027741-02-1) and From: Sep 1988 To: Sep 1990 (M-027135-01-1)
Route of administration:
oral: feed
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: standard diet containing 1% ground nut oil to minimise dust formation during mixing
- Storage temperature of food: deposit samples of each mix taken for re-analysis were kept at approx. +4 °C and from Oct 1987 at -20 °C.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For verification of nominal concentration, homogeneity and stability of the test substance in the diet (Altromin 1321 with 1% peanut oil), the test item was analysed by high performance liquid chromatography and UV detection. Verification of nominal concentration was performed 10-times over the course of the study for all 4 dose levels (100, 300, 900 and 1800 ppm). Mean analytical concentrations found were 98, 295, 897 and 1790 ppm (mean in % of nominal: 98, 98, 100 and 99 for the dose levels 100, 300, 900 and 1800 ppm, respectively). For homogeneity and stability checks, analyses of another study were used (report no. T 1025699). The nominal concentrations of 50 and 1000 ppm were analyzed for homogeneity and stability. To check for homogeneity, 5 feed mixture samples of 100 to 200 g each were obtained. Three of the 5 samples were selected at random and analyzed. The distribution of active ingredient was homogeneous because the relative standard deviation of the measured values did not exceed 10%. The analytical concentrations found were 51 and 1020 ppm at Day 0 and 43 and 880 ppm at Day 7 and 42 and 870 ppm at Day 14 of storage for the nominal concentrations 50 and 1000 ppm, respectively. The active ingredient was stable in the feed mixture over a period of 14 days in consideration of the tolerance range of +- 20% of the nominal concentration.
Duration of treatment / exposure:
up to 24 months (interim section after 12 months)
Frequency of treatment:
continously via the diet
Dose / conc.:
100 ppm
Remarks:
5.7 mg/kg bw/day (males), 7.6 mg/kg bw/day (females)
Dose / conc.:
300 ppm
Remarks:
16.9 mg/kg bw/day (males), 24.9 mg/kg bw/day (females)
Dose / conc.:
900 ppm
Remarks:
51.3 mg/kg bw/day (males), 73.0 mg/kg bw/day (females)
Dose / conc.:
1 800 ppm
Remarks:
102.6 mg/kg bw/day (males), 143.7 mg/kg bw/day (females)
No. of animals per sex per dose:
Terminal sacrifice (24 months): 50
Interim sacrifice (12 months): 10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: dose selection was based on two subchronic feeding studies in which doses of 0, 120, 600 and 3000 ppm and 0, 150, 600 and 2400 ppm were used, respectively.
Positive control:
None.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day (once a day on weekend and public holidays)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week. Body surfaces, body openings, posture, general behavior, respiration and excretory products were inspected.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to treatment (week 0), at weekly intervals thereafter until and including week 14, and at two-week intervals from week 16 until and including week 102. The body weights were also recorded immediately prior to scheduled necropsy after 12 or 24 months to allow calculation of the relative organ weights.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumptions during weeks 2 and 15 and for four-week intervals starting with week 19 were individually determined for all animals.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: water intake was determined in weeks 14, 27, 40, 53, 66, 79 and 92 for each dose and sex group (M-027741-02-1) and in weeks 2, 16, 29, 42, 55, 68, 81 and 94 for each dose and sex group (M-027135-01-1).

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at the start of the study and after 12 months
- Dose groups that were examined: 0, 900 and 1800 ppm (10 per dose and sex)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 6, 12, 18 and 24 months after initiation of the study
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No
- How many animals: 10 randomly selected animals from each group
- Parameters checked: Differential blood count, erythrocyte morphology, erythrocyte count, blood haemoglobin concentration, hematocrit, leukocyte count, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular cell volume (MCV), thrombocyte count, coagulation time, reticulocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 6, 12, 18 and 24 months after initiation of the study
- Animals fasted: No
- How many animals: 10 randomly selected animals from each group
- Parameters checked: activities of alkaline phosphatase, optimized aspartate aminotransferase, optimized alanine aminotransferase, creatine kinase, GLDH, cholinesterase (plasma and erythrocyte); glucose, bilirubin, cholesterol, total protein, urea, albumin, inorganic phosphate, sodium, potassium, calcium, chloride

URINALYSIS: Yes
- Time schedule for collection of urine: The urine samples were collected during sampling periods of about 16 hours (overnight) a few days before blood was withdrawn in each case. About 5 mL drinking water was administered to the animals by stomach tube prior to the collection periods.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- How many animals: 10 randomly selected animals from each group
- Parameters checked: blood, glucose, bilirubin, ketone bodies, pH, protein, urobilinogen, sediment (semiquantitative) and specific gravity, volume, protein (quantitative)

OTHER: Determination of TSH, T3 and T4 (M-027135-01-1)
-Time schedule for collection of blood: 76th week of the study
-How many animals: 10 randomly selected animals from each group
Serum was obtained for determination of the TSH, T3 and T4 levels. For the TSH assay, positive controls were required. Therefore, 8 µg/kg bw doses of the hormone preparation TRH (Ferring Arzneimittel GmbH, Kiel, Germany) were administered i.p. to groups of 10 male and 10 female rats and blood samples were collected after 30 minutes.
Sacrifice and pathology:
SACRIFICE: After 12 months all surviving animals selected for the interim post mortem and at the end of the study after 24 months all surviving animals were sacrificed by exsanguination under deep ether anaesthesia.

GROSS PATHOLOGY: Yes
The animals were then dissected, and their organs/tissues subjected to thorough patho-anatomical evaluation.

HISTOPATHOLOGY: Yes

The following tissues/organs were sampled for histopathological examination: aorta, eyes, eyelids, cecum, colon, duodenum, extraorbital lacrimatory gland, femur, brain, harderian gland, urinary bladder, ureter, urethra, skin, heart, testes, pituitary, ileum, jejunum, larynx, bone marrow (in femur and sternum), head, liver, lung, lymph nodes (mesenteric and mandibular), stomach, mammary glands, spleen, musculature of thigh, epididymis, adrenals, sciatic nerve, optic nerve, kidneys, esophagus, tattooed ear scoops, ovaries, oviduct, pancreas, prostate, rectum, lower intestine, spinal cord (cervical, thoracic, lumbar), seminal vesicles, thyroid gland, cranial salivary gland, sternum, thymus, trachea, uterus, vagina, tongue, zymbal gland
Other examinations:
Organ weights: The following organs of animals sacrificed for the interim post mortem after 12 months or at the end of the study after 24 months were weighed: brain, heart, liver, lung, spleen, kindeys (both), adrenal glands (both), ovaries (both) and testes (both)
Statistics:
The arithmetic group means and standard deviations were calculated from all individual animal results. Except for the cholinesterase data, the test cohort results were subjected to a two-tailed comparison with those of the control cohort using the significance test ("U" test) of H.B. Mann and D.R. Whitney (Ann. Math. Stat. 18, 50 [1947]) and F. Wilcoxon (Biometrics 1(6). 80 [1945]) at significance levels of a = 5 % and a = 1 %.

The survival curves were analyzed using BMDP Routine 1L. The incidences of the individual status variable intensities have been grouped according to the treatment. Intensities deviating from "died intercurrently/sacrificed in moribund condition" - such as "interim necropsy" or "terminal necropsy" - were treated as censored observations, since the animals had not been observed until occurrence an event, i.e. to the point of "natural" death in these cases. The survival curves were subsequently compared using the generalized Wilcoxon test (Breslow test) suggested by N.E. Breslow, Biometrika 51, 579 - 594 (1979), the weight assigned to each event point being proportional to the size of the pertinent group.

The cholinesterase data were separately assessed for each sex by means of single-factorial variance analysis for each time point.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant increased incidences of findings were observed in the groups receiving the test item up to 1800 ppm. Poor general condition, loss of hair and palpable masses were among the most frequently observed clinical signs noted. No exceptional features in the body surfaces and openings, or with respect to the general behavior, posture, respiration and excretory products could be observed at doses to 1800 ppm. No treatment effect was inferred from the incidence, location and chronology of palpable tissue masses at doses to 1800 ppm.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
mortality observed, non-treatment-related
Description (incidence):
M-027741-02-1: Mortality observed was not treatment-related, since incidences were comparable between treatment groups. The cumulative mortalities after the first 12 months of study were 1/60, 1/60, 1/60 and 1/60 (males) and 0/60, 0/60, 3/60 and 1/60 (females) for control, 100, 300 and 900 ppm, respectively. The cumulative mortalities at the end of the study (24 months) were 6/50, 6/50, 6/50 and 6/50 (males) and 13/50, 6/50, 10/50 and 13/50 (females) for control, 100, 300 and 900 ppm, respectively.

M-027135-01-1: Mortality observed was not treatment-related, since incidences were comparable between groups. The cumulative mortalities after the first 12 months of study were 1/60 and 0/60 (males) and 1/60 and 1/60 (females) for control and 1800 ppm, respectively. The cumulative mortalities at the end of the study (24 months) were 10/50 and 5/50 (males) and 13/50 and 10/50 (females) for control and 1800 ppm, respectively.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain was comparable with that of control rats at doses up to and including 300 ppm. At a dose of 900 ppm, body weight gain of male animals was up to 5% slower and that of females up to 8% slower than in the control group. At a dose of 1800 ppm body weight gains in male and female rats were significantly retarded at all times. The weight declined reached a maximum of 12 % (males) or 11 % (females) in week 10. At the close of the study males were 5 % lighter and females were 11 % lighter than controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
not applicable
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
The 1800 ppm females drank 13 % less water per animal compared to the controls. For all other groups no effects were observed.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the control as well as 900 and 1800 ppm group several animals exhibited spontaneous alteration such as focal diffuse clouding of the light-refracting media, foreign boy inclusions or eminences in the cornea. These are common observations made in rats of this breed and age and are not regarded as evidence for an ocultotoxic effect by the test item.
Haematological findings:
no effects observed
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In the 1800 ppm dose group significantly increased aspartate aminotransferase (males and females) and creatine kinase (females) levels were found at the end of the study.
Endocrine findings:
no effects observed
Description (incidence and severity):
No statistically significant differences between the hormone titers of TSH, T3 and T4 of control and treated animals (1800 ppm) were determined.
Urinalysis findings:
no effects observed
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, non-treatment-related
Description (incidence and severity):
M-027741-02-1: After 12 months of study significantly depressed figures for the absolute liver (14 %) and kidney (13 %) weights were observed in the 900 ppm group of female rats. At the end of the study (24 months), these organ weights were still significantly lower compared to control (4 and 5 % for liver and kidney, respectively), but not considered as evidence for organ damage due to the lower deviation between treated and control animals. In male rats only in the high-dose group a decrease in liver weight (13 %) at the interim sacrifice (12 months) was recorded.

M-027135-01-1: After 12 and 24 months of study depressed absolute and relative organ weights in the case of several organs, particular the liver, were found in the 1800 ppm group (males and females) compared to controls. These were considered as a result of the decreased body weights rather than evidence for a specific toxic effect by the test item.
Gross pathological findings:
no effects observed
Description (incidence and severity):
not applicable
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
M-027741-02-1: In the thyroid glands, mineralized particles (MPs) were found in the colloid of isolated, large-lumen follicles in both sexes at the interim and the final section. Interim section: MPs were observed in the thyroid glands of male rats of all dose groups, but incidence increased dose-dependently from 3/10 (control) to 3/10 (100 ppm), 6/10 (300 ppm) and 10/10 (900 ppm). In female rats the effect was less pronounced and MPs were only found in the top dose (3/10), but in no other group. Final section: MPs in thyroid glands were found in 2/50 (control), 12/50 (100 ppm), 31/50 (300 ppm) and 44/50 (900 ppm) male rats and 11/50 (control), 6/50 (100 ppm), 11/50 (300 ppm) and 27/50 (900 ppm) in female rats. The incidence of 12/50 in the 100 ppm treated males was significantly higher compared to the control animals of this study, however, when compared to historical control data from four chronic studies from with the rats being sacrificed between 1987 and 1990, and histopathological samples analysed retrospectively, no differences were found. Therefore 300 ppm is considered as a threshold for this effect in male rats and 900 ppm in female rats.
No findings exhibiting treatment-related distribution were seen in the other organs at doses up to and including 900 ppm.

M-027135-01-1: In the thyroid glands, mineralized particles (MPs) in the colloid of isolated, large-lumen follicles were found more frequently and colloid aggregation sire more rarely in both sexes at the interim and the final section. Interim section: MPs were observed in the thyroid glands of male rats of all dose groups; 5/10 (control) and 10/10 (1800 ppm). In female rats the effect was less pronounced; 2/10 (control) and 5/10 (1800 ppm). Colloid aggregation: 6/10 (control males) and 0/10 (1800 ppm males), 2/10 (control females) and 0/10 (1800 ppm females). Final section: MPs in thyroid glands were found in 12/50 (control) and 46/50 (1800 ppm) male rats and 3/50 (control) and 38/50 (1800 ppm) in female rats. The incidence of colloid aggregation decreased in male rats from 41/50 (control) to 20/50 (1800 ppm) and in female rats from 22/50 (control) to 7/50 (1800 ppm).
Furthermore, enhance porphyrin accumulation in the Harderian glands and an increased incidence of retinal atrophy were found in female rats of the 1800 ppm group.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The total number of tumors observed and the incidence of begnin and malignant neoplasms were comparable in all study groups. Most tumors were observed in the pituitary gland, thyroid, mamma and uterus. Their incidence was independent of the dose. Tumors in the liver only occurred in male rats of the 1800 ppm group (one adenoma, one carcinoma and two cholangiocellular carcinomas). However, comparison with historical control data shows that the incidences of hepatic adenomas and carcinomas do not lie outside the reference ranges for these tumors.
Other effects:
not examined
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
other: maximum tolerated dose, MTD
Effect level:
1 800 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Remarks on result:
other: mineralization in the thyroid follicles, increased levels of aspartate aminotransferase
Key result
Dose descriptor:
NOAEL
Effect level:
100 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: presence of mineralized particles in thyroid glands
Key result
Dose descriptor:
NOAEL
Effect level:
300 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: presence of mineralized particles in thyroid glands
Conclusions:
The studies were performed under GLP conditions and according to OECD TG 453 (adopted 1981). Deviations to the current version (adopted 2018) are minor. Thus the studies are considered reliable and valid. Based on nature, incidence or chronology of neoplasms, or from the numbers of animals exhibiting tumors in treated and control rats, there was no indication of a treatment-related oncogenic effect of the test item. For male rats a NOAEL of 100 ppm (equivalent to 5.7 mg/kg bw/day) and for female rats a NOAEL of 300 ppm (equivalent to 24.9 mg/kg bw/day) were established based on the absence of compound-induced mineralized particles in the colloid of large-lumen follicles of the thyroid gland.
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 Sep 1987 - 28 Sep 1989 (M-026310-01-1);

28 Sep 1988 - 28 Sep 1990 (M-026038-01-1)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
Version / remarks:
adopted 2018
Deviations:
yes
Remarks:
no blood sampling after 3 and 6 months, no prothrombin and activated partial thromboplastin time determined, not all tissues/organs examined (coagulating and lacrimal gland, nose, turbinates, olfactory bulb, seminal vesicles)
Qualifier:
according to guideline
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
Version / remarks:
adopted 1981
Deviations:
no
Principles of method if other than guideline:
In this RSS, the main study (M-026310-01-1) and a supplementary study (M-026038-01-1) are reported. The supplementary study was performed to support determination of a MTD of the test item in mice. The main study consisted of a control group (0 ppm) and the dose groups 100, 330 and 1000 ppm, while the supplementary study consisted of a control group (0 ppm) and a high-dose group (2000 ppm). If not stated otherwise, the procedures in the supplementary study were the same as compared to the main study.
GLP compliance:
yes
Species:
mouse
Strain:
B6C3F1
Details on species / strain selection:
B6C3F1 mice were chosen, since the mouse is considered an acceptable species in carcinogenicity testing.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany (M-026310-01-1); Winkelmann Experimental Animal Breeders, Borchen, Germany (M-026038-01-1)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 5 weeks (M-026310-01-1); 7 - 8 weeks (M026038-01-1)
- Weight at study initiation: 18 - 25 g (males), 14 - 18 g (females)(M-026310-01-1); 19 - 31 g (males), 18 - 24 g (females)(M-026038-01-1)
- Housing: Makrolon type I cages equipped with low-dust wood granulate (Ssniff Spezialdiäten GmbH, Soest, Germany)
- Diet: Altromin 1321 meal (Altromin GmbH & Co KG, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): approx. 50
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 21 Sep 1987 To: 28 Sep 1989 (M-026310-01-1) and From: 28 Sep 1988 To: 28 Sep 1990 (M-026038-01-1)
Route of administration:
oral: feed
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: standard diet containing 1% ground nut oil to minimise dust formation during mixing
- Storage temperature of food: deposit samples of each mix taken for re-analysis were kept at approx. -20 °C.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For verification of nominal concentration, homogeneity and stability of the test substance in the diet (Altromin 1321 with 1% peanut oil), the test item was analysed by high performance liquid chromatography and UV detection. Verification of nominal concentration was performed at least 10-times over the course of the study for all 4 dose levels (100, 330, 1000 and 2000 ppm). Mean analytical concentrations found were 96, 320, 1020 and 1980 ppm (mean in % of nominal: 96, 97, 102 and 99 for the dose levels 100, 330, 1000 and 2000 ppm, respectively). For homogeneity and stability checks, analyses of other studies were used (study nos. T 1025699 and T 7027297). The nominal concentrations of 50, 150, 1000 and 2400 ppm were analyzed for homogeneity and stability. To check for homogeneity, 5 feed mixture samples of 100 to 200 g each were obtained. Three of the 5 samples were selected at random and analyzed. The distribution of active ingredient was homogeneous because the relative standard deviation of the measured values did not exceed 10%. The analytical concentrations found were 50 and 1020 ppm at Day 0 and 43 and 880 ppm at Day 7 and 42 and 870 ppm at Day 14 of storage for the nominal concentrations 50 and 1000 ppm, respectively, and 2570 ppm at Day 0 and 2350 ppm at Day 12 for the nominal concentration of 2400 ppm. The active ingredient was stable in the feed mixture over a period of 14 days in consideration of the tolerance range of +- 20% of the nominal concentration.
Duration of treatment / exposure:
up to 24 months (interim section after 12 months)
Frequency of treatment:
continuously via the diet
Dose / conc.:
100 ppm
Remarks:
20.2 mg/kg bw/day (males), 30.3 mg/kg bw/day (females)
Dose / conc.:
330 ppm
Remarks:
65.6 mg/kg bw/day (males), 103.6 mg/kg bw/day (females)
Dose / conc.:
1 000 ppm
Remarks:
208.2 mg/kg bw/day (males), 274.4 mg/kg bw/day (females)
Dose / conc.:
2 000 ppm
Remarks:
413.5 mg/kg bw/day (males), 423.9 mg/kg bw/day (females)
No. of animals per sex per dose:
Terminal sacrifice (24 months): 50
Interim sacrifice (12 months): 10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: dose selection was performed on the basis of a subchronic feeding study testing 0, 120, 600 and 3000 ppm.
Positive control:
None.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were inspected at least twice daily (once on weekends and public holidays) for clinical signs and abnormalities.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed examination of individual animals was carried out once a week. Body surfaces, orifices, posture, general behaviour, respiration and excretory products were assessed.


BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were determined prior to treatment (week 0) and then weekly thereafter. Each animal was also individually weighed immediately before the interim or final sacrifice to calculate relative organ weights.

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. Individual food intake was recorded once a week in 10 animals per group up to and including week 23 and in 20 animals per group from week 24.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Each group's intake of drinking water was calculated every four weeks.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were taken from the retro-orbital venous plexus after 12 and 24 months of study and additionally from mice of control, 1000 and 2000 ppm groups after 18 months of study
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No
- How many animals: 10 per group
- Parameters examined: differential blood count, erythrocyte morphology and count, haemoglobin concentration, haematocrit, leucocyte count, mean corpuscular haemoglobin (MCH), MCH concentration (MCHC), mean corpuscular cell volume (MCV), platelet count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were taken from the retro-orbital venous plexus after 12 and 24 months of study and additionally from mice of control and top dose group after 18 months of study
- Animals fasted: No
- How many animals: 10 per group
- Parameters examined: alkaline phosphatase, aspartate-aminotransferase and alanine aminotransferase activity in plasma; glucose (whole blood); bilirubin, cholesterol, creatinine, total protein and urea (in plasma).
Sacrifice and pathology:
SACRIFICE: After 12 months all surviving animals selected for the interim post mortem and at the end of the study after 24 months all surviving animals were sacrificed by exsanguination under deep ether anaesthesia.

GROSS PATHOLOGY: Yes
The animals were then dissected, and their organs/tissues subjected to thorough patho-anatomical evaluation.

HISTOPATHOLOGY: Yes

The following organs/tissues were sampled for histopathological examination: aorta, eyes, eyelids, caecum, colon, duodenum, extraorbital gland, femur, gallbladder, brain, Harder's gland, bladder, ureter, urethra, skin, heart, testicle, pituitary, ileum, jejunum, larynx, bone marrow (in femur and sternum), head, liver, lung, lymph nodes (mesenteric and mandibular), stomach, mammary glands, spleen, muscles (thigh), epididymis, adrenal glands, sciatic nerve, optic nerve, kidneys, oesophagus, tattooed auricles, ovaries, oviduct, pancreas, prostate, rectum, residual intestine, spinal cord (spinal column, cervical, thoracic, lumbar), seminal vesicles, thyroid, salivary glands, sternum, thymus (if present), trachea, uterus, vagina, tongue, cymbocephalic glands
Other examinations:
Organ weights: The following organs of animals sacrificed for the interim post mortem after 12 months or at the end of the study after 24 months were weighed: brain, liver, lung, spleen, kindeys (both), adrenal glands (both), ovaries (both) and testes (both)
Statistics:
From the results for each animal of body weight measurement, food intake determination, medical laboratory tests, and measurement of organ weights were calculated arithmetic group means and standard deviations. Comparison of the results for treated animals with those for the control group was carried out using the significance test (U test, two-sided) after MANN, H.B. and WHITNEY, D.R. (Ann. Math. Stat. 18, 50 (1947) and after WILCOXON, F. (Biometrics Vol. 1, no. 6, 80 (1945) at the significance level a = 5% and a = 1%.

Evaluation of the survival curves was carried out using the BMDP-Routine 1L. The incidences of individual characteristics of the status variables were grouped by dosage. Characteristics other than "animals which died or were sacrificed moribund during the study" such as, for example, "interim post mortem" or "final post mortem" were treated as censored observations as in these cases the animals were not observed until the event occurred, i.e. until "natural death". Subsequent comparison of survival curves was carried out separately for each sex using the generalised WILCOXON-test (BRESLOW-Test: BRESLOW, N.E. (1970): A generalized Kruskal-Wallis test for comparing k samples subject to unequal pattern of censorship, Biometrika 51, 579-594), weighted per time of occurrence in proportion to the size of the group in question. The given probability of error a was 0.05
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Most observed effects are considered non-treatment-related since incidences are similar between groups. Main findings in individual animals were poor general condition, rough coat, loss of hair, increased circumference and palpable masses. However, in the 2000 ppm group a "squeaking and twittering" type of vocalization was perceived in male and female mice from the inception of the study onwards. No abnormalities were recorded as regards body surfaces and orifices, general behaviour, posture, respiration, excretory products in any of the animals. The incidence, localisation and time of appearance of palpable tissue masses provided no evidence of any treatment-related effects in the dose range investigated.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
mortality observed, non-treatment-related
Description (incidence):
M-026310-01-1: Mortality observed was not treatment-related, since incidences were comparable between treatment groups. The cumulative mortalities after the first 12 months of study were 0/60, 0/60, 1/60 and 0/60 (males) and 2/60, 2/60, 0/60 and 0/60 (females) for control, 100, 330 and 1000 ppm, respectively. The cumulative mortalities at the end of the study (24 months) were 3/50, 6/50, 3/50 and 8/50 (males) and 7/50, 9/50, 10/50 and 9/50 (females) for control, 100, 330 and 1000 ppm, respectively.

M-026038-01-1: The cumulative mortalities after the first 12 months of study were 0/60 and 7/60 (males) and 4/60 and 7/60 (females) for control and 2000 ppm, respectively. The cumulative mortalities at the end of the study (24 months) were 6/50 and 17/50 (males) and 19/50 and 14/50 (females) for control and 2000 ppm, respectively. Of the animals that died or were sacrificed in moribund condition before the commencement of terminal necropsy, 1 control female, and 7 males and 4 females of the 2000 ppm group died in connection with blood withdrawal; 2 males of the 2000 ppm group died following tattooing; 1 control female and 2 males and 4 females of the 2000 ppm group after being caught in the automatic feeders. These incidences are attributed to indirect test substance effects such as general debilitation and a major decrease in body weight.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain was comparable with that of control mice at doses up to and including 330 ppm. At a dose of 1000 ppm, body weights of male animals were up to 10% lower and those of females up to 5% lower. In the 2000 ppm group, body weights were statistically significant lower from the first week of study onwards compared to control. In male mice the highest extent of deviation was 29 % and in female mice up to 26 %.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Over the course of the study, food consumption was not affected at dose levels of up to and including 1000 ppm (males) and 330 ppm (females). A slight decrease in food intake was found in female mice of the 1000 ppm group (-10% per animal per day compared to control). At a dose level of 2000 ppm, male mice consumed slightly (10 %) and female mice markedly (35 %) less food than controls. In relation to body weight, no difference in food intake was found between treated and control males, while the food intake per kg body weight in treated females was 24 % below the control figure.
Food efficiency:
not specified
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Over the course of the study, water consumption was not affected at dose levels of up to and including 1000 ppm (males) and 330 ppm (females). At a dose of 1000 ppm water intake of females was slightly decreased (10% per animal per day and 10% per kg bw per day) compared to control animals. Doses of 2000 ppm test item led to a decrease in the water intake per animal (29 % in males and 38 % in females), as well as per kg body weight (11 % in males and 27 % in females) in both sexes.
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At doses of up to 2000 ppm there was no sign of adverse treatment-induced effects on blood or haemopoietic organs. In the 2000 ppm males and females, depressed leukocyte counts were found compared to control animals at the interim (statistically significant difference) and terminal section.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In males and females of the 2000 ppm group elevated alkaline phosphatase activities were found in the plasma at interim and terminal sacrifice, while cholesterol levels were significantly decreased compared to control. The levels of aspartate and alanine aminotransferase activities as well as levels of other substrates determined (glucose, protein, creatinine and urea) either did not deviate from the control figures or only within the range of variation appropriate for mice of this age.
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 applicable
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
-1000 ppm: At the interim sacrifice (12 months) for males and females, decreased absolute weights of liver and kidneys (males only) were recorded, but regarded as a consequence of the lower body weight, since changes were relatively slight. At the final sacrifice (24 months), too, the mean weight of liver in males was lower compared to control animals, possibly due to there being more liver tumours (carcinomas) in the 0 ppm group. Furthermore, a decreased absolute and relative weight of the liver was found in the females treated with 1000 ppm test item, but this was regarded to be of no toxicological relevance, since the differences compared with control group were marginal (max. 3%).

-2000 ppm: Absolute weights of lung, liver, kidney and adrenals at both necropsy time points (adrenal weights in males only at interim section), absolute weights of brain and ovary in females after 24 months and relative weights of liver and spleen in females after 12 and 24 months as well as relative liver weight in males after 12 months were statistically significant lower than the control figures. However, these differences may be explained by the markedly reduced body weights of treated animals compared to the controls.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic findings in mice that died or were sacrificed moribund or as scheduled provided no indications of treatment-specific organ changes. Necropsy findings in the interim and final sacrificed animals were infrequent and generally minor such as discolorations and changes in size and shape of several organs, cystic and enlarged ovaries, collapsed lungs, hair loss in skin and nodes in several organs.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The range of non-tumour histopathology data in treated animals up to 1000 ppm was generally similar to that in control animals with minor intergroup variations. At the 2000 ppm dose level a slight periacinary liver cell hypertophy was observed in 5/50 males, whereas this lesion was not found in animals of the other groups. Also an increased incidence of mineralization in the brain (thalamus) was found in both sexes. In the kidneys a loss of vacuolation in the tubules was observed in males of the 2000 ppm group (1/50 vs 25/50 in the controls). Additionally, greatly reduced incidences of tubular basophilia (in males) and of lymphocytosis in the renal pelvis (males and females) were observed at the 2000 ppm level. These effects might be a cause of reduced food and thus reduced protein intake.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The spectrum of neoplastic lesions in control animals was consistent with that expected in ageing mice. The more common tumours were those of the Harderian gland in both sexes, those of the liver and lung in males and those of the haemolyphoreticular system and uterus in females. The spectrum and incidence of neoplasia in animals treated with the test item was generally similar to that in the control group with minor intergroup variations. There was no evidence of a treatment-related increase in the incidence of tumour-bearing animals or of specific tumour types suggestive of any carcinogenic response attributable to test article administration.
Other effects:
not examined
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
other: maximum tolerable dose, MTD
Effect level:
> 1 000 - < 2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: No carcinogenic potential is evidenced from the incidence, nature, location or chronological occurrence of tumors following administration of the test item up to and including 2000 ppm.
Key result
Dose descriptor:
NOAEL
Effect level:
330 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Conclusions:
The study was performed under GLP conditions and according to OECD TG 451(adopted 1981). Deviations to the current version (adopted 2018) are considered minor. Thus the study is considered reliable and valid. Based on type, incidence and organ distribution of neoplastic lesions in treated and control mice, there was no indication of a treatment-related oncogenic effect of the test item. A NOAEL of 330 ppm (equal to 65.6 mg/kg bw/day in male mice and 103.6 mg/kg bw/day in females mice) was established based on the absence of compound-induced toxicological responses.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
5.7 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Two reliable guideline studies are available, conducted with rat and mouse, which both fulfill the criteria of key studies. The emphasis is set on the rat study, since this is the preferred rodent species as recommended by current guidelines.

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

No data available.

Justification for classification or non-classification

Two reliable guideline studies are available, which both provide no evidence for the test substance to bear any carcinogenic potential; accordingly, no classification is warranted according to CLP Regulation (EC) No. 1272/2008.

Additional information

To assess the potency of the test item for carcinogenicity, two studies are available, one performed in the rat and one performed in the mouse:

 

A 2-year combined chronic toxicity and carcinogenicity study (M-027741-02-1 and suppl. M-027135-01-1) was performed in Wistar rats. The study was performed under GLP condition and in accordance with OECD TG 453 (adopted 1981). Deviations to the current version of the guideline (adopted 2018) are considered minor and not to be expected to have an impact on reliability of this study. The test substance was administered via diet to 50 animals per sex and dose at levels of 100, 300, 900 and 1800 ppm, corresponding to 5.7, 16.9, 51.3 and 102.6 mg/kg bw/day (males) and 7.6, 24.9, 73.0 and 143.7 mg/kg bw/day (females). Control animals received plain diet. Additionally, 10 animals per sex and dose were used for interim sacrifice after 1 year. The top dose group (1800 ppm) was treated in a supplementary study (M-027135-01-1) under the same conditions as compared to the main study. The aim of this additional study was to determine the maximum tolerated dose (MTD) for the present substance.

Analytical verification of test item stability, homogeneity and concentration in the diet was performed.The test item was stable in the food for 14 days and the homogeneity was confirmed, since the standard deviations of analysed samples obtained from different areas of mixing bottles did not exceed 10%. The achieved analytical concentration were within a range of 98 to 100% of respective nominal concentrations.

No treatment-related effects on clinical signs, mortality, food consumption, haematology, organ weights and gross pathological findings were observed. Body weight gain was not affected at doses up to 300 ppm, but slightly lower (5% in males, 8% in females) at the 900 ppm level and significantly retarded (12% in males, 11% in females) at 1800 ppm, compared to the controls. Also in the 1800 ppm group, significantly increased aspartate aminotransferase (both sexes) and creatine kinase (creatine kinase) levels were found at the terminal sacrifice. The test item also induced formation of mineralized particles (MPs) in the thyroid glands in both sexes. This effect was dose-dependently and observed at the interim and final sacrifice with a NOAEL of 100 ppm in male rats and 300 ppm in female rats, based on comparison of occurrence of MPs in treated animals compared to historical control data. No treatment-related neoplastic findings were observed.

Taking together in this study a NOAEL of 100 ppm (equivalent to 5.7 mg/kg bw/day) for male and 300 ppm (equivalent to 24.9 mg/kg bw/day) for female Wistar rats was established based on the absence of adverse test item-induced effects in the thyroid glands (mineralized particles).

 

 

Additionally, a 2-year carcinogenicity study (M-026310-01-1 and suppl. M-026038-01-1) was performed in B6C3F1 mice. The study was performed under GLP conditions and in accordance with OECD TG 451 (adopted 1981). Deviations to the current version of the guideline (adopted 2018) are only minor and not considered to have an impact on the outcome of this study. The test item was administered via diet to 50 animals per sex and dose at nominal concentrations of 100, 330, 1000, and 2000 ppm, corresponding to 20.2, 65.6, 208.2, and 413.5 mg/kg bw/day (males) and 30.3, 103.6, 274.4, and 423.9 mg/kg bw/day (females). Control animals received plain diet only. Additionally, 10 animals per sex and dose were used for interim sacrifice after 1 year of dosing. The top dose group (2000 ppm) was treated in a supplementary MTD finding study (M-026038-01-1) under the same conditions as compared to the main study.

Analytical verification of test item stability, homogeneity and concentration in the diet was performed. The test item was stable in the food for 14 days and the homogeneity was confirmed, since the relative standard deviation of the analyzed samples obtained from different locations within the mixing bottle did not exceed 10%. The achieved analytical concentrations were within a range of 96 - 102 % of nominal concentrations.

No treatment-related effects on mortality, organ weights, gross or histopathological findings were observed. Also most clinical signs observed, such as poor general condition, rough coat, loss of hair or increased circumference and palpable masses were considered to be non-treatment-related since incidences were similar between groups. However, at 2000 ppm a squeaking and twittering type of vocalization was perceived in male and female mice from the inception of the study onwards. Further treatment-related effects noted were a decrease of body weight and food consumption in male and female mice. Body weight gain was comparable to control mice at doses of up to and including 330 ppm (both sexes), but was slightly lower at 1000 ppm (-10% for males and -5% for females) and markedly decreased at a dose level of 2000 ppm (-29% for males and -26% for females). This effect was accompanied by a slight decrease in food consumption in the 1000 ppm group females (-10%), which was even enhanced at 2000 ppm (-35% for females and -10% for males). No effect on food consumption was observed in male mice treated with up to and including 1000 ppm test item and in female mice treated with up to and including 330 ppm test item. Haematological analysis revealed a treatment-dependent decrease of leukocyte counts in the 2000 ppm dose group (both sexes), but only in individual cases differences in e.g. erythrocyte count, haematocrit, mean cell haemoglobin or platelet count were observed between 1000 and 2000 ppm dose groups and the controls. Also in the 2000 ppm group, elevated levels of alkaline phosphatase were found in the plasma, while cholesterol levels were decreased compared to control. No treatment-related neoplastic findings were observed. The more common tumors were those of the Harderian gland in both sexes, those in the liver and lung in males and those of the haemolyphoreticular system and uterus in females. However, the spectrum and incidence iof neoplasia in animals treated with the test item was generally similar to that in control mice with only minor intergroup variations.

Hence, the NOAEL obtained in this study is 330 ppm (equivalent to 65.6 mg/kg bw/day in males and 103.6 mg/kg bw/day in females) for B6C3F1 mice, based on the absence of test item-induced general toxicity noted as a decrease in body weight at 1000 ppm.

 

 

In summary the test item was tested in two different rodent species (rat and mouse) for its carcinogenic potential. Both studies are considered reliable and valid and the test item was not found to possess carcinogenic properties.