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Carcinogenicity

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Description of key information

Combined chronic toxicity and carcinogenicity study, oral (OECD 453), rat: males:

carcinogenicity: NOAEL >= 18000 ppm (equivalent to 741 mg/kg bw/day); females: 4000 ppm (equivalent to 221 mg/kg bw/day)

systemic toxicity (24 month): NOAEL = 4000 ppm (equivalent to 159 and 221 mg/kg bw/day in males and females, respectively)

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Jan 2013 - 19 Oct 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
adopted in 2009
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)
Version / remarks:
adopted in 1987
Qualifier:
according to
Guideline:
EPA OPPTS 870.4300 (Combined Chronic Toxicity / Carcinogenicity)
Version / remarks:
adopted in 1998
Qualifier:
according to
Guideline:
other: MAFF in Japan, notification 12 Nousan n° 8147
Version / remarks:
adopted in 2000
GLP compliance:
yes
Species:
rat
Strain:
other: Wistar Rj:WI (IOPS HAN)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: R. Janvier, Le Genest St Isle, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks
- Weight at study initiation: 206 - 209 g (mean group weight males), 158 - 159 g (mean group weight females)
- Housing: 5 animals of the same sex per cage in suspended, stainless steel and wire mesh cages
- Diet: A04CP1-10 from SAFE (Scientific Animal Food and Engineering, Augy, France,) ad libitum except at designated time periods
- Water: filtered and softened tap water from the municipal water supply, ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): every 4 weeks
- Mixing appropriate amounts with (Type of food): A04CP1-10
- Storage temperature of food: ambient
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The first (F1), the seventh (F7) and the thirteenth formulation (F13) prepared for the study were evaluated for homogeneity using HPLC-UV analysis. Triplicate samples were collected of the lowest and the highest dose (900 and 18000 ppm) from the top, the middle and the bottom of the formulations. The mean values obtained from the homogeneity checks were used as measured concentrations. In addition, for concentration analysis, two samples of formulations F1, F4, F7, F10, F13 and F16 were collected. One sample was analyzed and the other was stored frozen for possible verification purpose. Data indicate homogenous mixture of the test material in the diet. The measured values for the individual homogeneity samples ranged between 91 and 107% of nominal. Additionally, samples were at the targeted concentration (93 – 111% of nominal). One value at 111% was slightly out of in-house target ranges but considered as having no impact on the study results reliabilities. The stability of the test item has been demonstrated in a previous study at concentrations of 120 and 12000 ppm for up to 104 days when stored frozen and then kept for 10 days at ambient temperature (Study completion: 2011; Study number: SA 10372; Doc No: M-568723-03-1). Stability of the high dose 18000 ppm was verified during the current study over a frozen storage period of 167 days followed by 105 days of storage at room temperature. With measured concentrations of 99 and 100% of nominal following storage procedure the test substance was found to be stable.
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
continously (via diet)
Dose / conc.:
900 ppm
Remarks:
equivalent to approximately 35.3 and 51.2 mg/kg bw/day in males and females, respectively
Dose / conc.:
4 000 ppm
Remarks:
equivalent to approximately 159 and 221 mg/kg bw/day in males and females, respectively
Dose / conc.:
18 000 ppm
Remarks:
equivalent to approximately 741 and 1052 mg/kg bw/day in males and females, respectively
No. of animals per sex per dose:
10 (12 month interim sacrifice)
60 (24 month sacrifice; carcinogenicity phase)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results from a previous 90-day dietary study in the rat (2012, Doc number M-443358-02-1), where no toxicity was seen at the highest dose tested of 10000 ppm equating to 608 mg/kg bw/day in males and 723 mg/kg bw/day in females. Consequently, the high dose level chosen on the current study was 18000 ppm, which over the course of the study was expected to equate to a limit dose in females and close to a limit dose in males, but was a dose that was expected to be tolerated. The low dose of 900 ppm was expected to be a dose level where no toxic findings were observed. The mid dose of 4000 ppm, provides a 4.5 fold factor between the high and low dose.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily (including weekends and public holidays) except on a limited number of occasions
- Cage side observations included: mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least daily (clinical signs), at least weekly (detailed physical examination including palpation for masses), ill health

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly during the acclimatization period, weekly for the first 13 weeks of study, approximately every 4 weeks thereafter and prior to necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE:
The weight of food supplied and of that remaining at the end of the food consumption period was recorded for each animal. Food consumption was recorded twice weekly during the first 6 weeks of treatment, then weekly up to Week 13, and once approximately every 4 weeks thereafter.The weekly mean achieved dosage intake in mg/kg bw/day for Weeks 1 to 13, then 1 week per month thereafter was calculated. The monthly and overall mean achieved dosage intake for the 24 months of treatment were derived from the weekly data.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during acclimatization phase, after approximately 12 and 24 months
- Dose groups that were examined:all animals (acclimatization phase), control and high dose groups (12 months), all surviving animals (24 months)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Weeks 15, 24, 25, 51, 78 or 79 and 105; puncture of the retro orbital venous plexus
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: first ten surviving animals
- Parameters examined: hematocrit, hemoglobin concentration, white blood cell count, red blood cell count, platelet count, prothrombin time, white blood cell differential evaluation count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, reticulocyte count, blood smears

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Weeks 15, 24, 25, 51, 78 or 79 and 105; puncture of the retro orbital venous plexus
- Animals fasted: Yes
- How many animals: first ten surviving animals
- Parameters examined: calcium, chloride, inorganic phosphorous, potassium, sodium, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, albumin, creatinine, urea, total cholesterol, glucose (fasting), total bilirubin, total protein, triglycerides

URINALYSIS: Yes
- Time schedule for collection of urine: Weeks 13, 25 or 26, 52, 77 and 104
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters examined: appearance, volume, specific gravity / osmolality / refractive index, pH, sediment (microscopic), protein, glucose, ketones, bilirubin, blood / red blood cells, urobilinogen

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Bioanalytical examination: After approximately 3 months of treatment, as well as at the end of 12 months and during the month before final sacrifice, a blood sample was collected from the sublingual vein of five suitable animals of each sex and all dose groups following anaesthesia with isoflurane. Blood was also collected from 2 control males and 2 control females to determine analytical baseline. Based on the results of a blood kinetic analysis study (2012, Doc number M-427766-02-1), blood was collected at approximately 8.00 am on the day of sampling. Plasma was prepared and determination of the test substance level and main metabolite using HPLC Tandem Mass Spectrometry (HPLC-MS/MS) analysis was conducted.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 1)
On study Days 730 to 745 all surviving animals, respectively, were sacrificed by exsanguination under deep anesthesia (inhalation of isoflurane). An approximately equal number of animals randomly distributed amongst all groups were sampled on each day at the interim sacrifice. Animals were diet fasted overnight prior to sacrifice. All animals, including animals either found dead or killed for humane reasons, were necropsied. The necropsy included the examination of external surfaces, all orifices, all major organs, tissues and body cavities. The organs or tissues checked in table 1 were sampled and/or weighed. All significant macroscopic abnormalities (including masses and their regional lymph nodes when possible) were recorded, sampled and examined microscopically. Two femoral bone marrow smears were prepared from sacrificed animals, one of which was stained with May-Grünwald Giemsa, but not examined as no relevant changes were observed in hematology or bone marrow histology. The second smear was stored unstained. Samples were fixed by immersion in 10% neutral buffered formalin with the exception of the eye and optic nerve, Harderian gland, epididymis and testis that were fixed in Davidson's fixative.

HISTOPATHOLOGY: Yes (see table 1)
The samples listed in table 1 (except exorbital lachrymal gland, larynx/pharynx and nasal cavities) were embedded in paraffin wax. Histological sections, stained
with hematoxylin and eosin, were prepared from all organs and tissue samples.
Histopathology examination of the carcinogenicity phase was performed on all organs and tissues embedded including gross abnormalities in all animals from all groups including decedents. For all unscheduled sacrificed or dead animals on study, the cause of death was determined when it was possible.
Statistics:
In general Bartlett test was performed. If the Bartlett test was not significant (p>0.05), means were compared using the analysis of variance (ANOVA). If the ANOVA was significant (p≤0.05), means of the exposed groups were compared to the mean of the control group using the Dunnett test (2-sided). If the Bartlett test was significant (p≤0.05), group means were compared using the Kruskal-Wallis test. If the Kruskal-Wallis test was significant (p≤0.05), means of the exposed groups were compared to the mean of the control group using the Dunn test (2-sided). If the Bartlett test was significant for body weight and average food consumption/day parameters and selected haematology parameters (red blood cell count, platelet count, white blood cell count, neutrophil count, lymphocyte count, reticulocyte count) data were transformed using the log transformation for body weight and food consumption and the square root transformation for haematology parameters before the same methods were applied as mentioned above. If the Bartlett test was significant (p≤0.05) even after log transformation, statistical analysis was proceeded as mentioned above. For survival analysis Kaplan-Meier estimation procedures were used. Dose relative trends in survival were assessed using log- rank trend test (2-sided). Pairwise comparison was assessed using Tarone tests with Scheffe adjustment for multiple comparisons (1-sided). Histopathological findings were assessed with poly-k (k=3) tests which take into account time to death and the presence of the findings. Firstly, a trend poly-k test (2-sided) was performed regarding all doses. Then, pairwise comparisons were assessed using poly-k test (1-sided) with Sidak adjustment where applicable. The Cochran-Armitage trend test (2-sided) followed by the Fisher’s exact test (1-sided) with Sidak adjustment was applied for selected clinical signs, ophthalmic and macroscopic observations.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
FEMALES
18000 ppm: increased incidence of prolapsed vagina (4/58 animals)
It is specified in the study report that these effects may be treatment-related.

(see table 2)
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
MALES
18000 ppm: decreased body weights compared to controls (7% during the second year), decreased mean cumulative body weight gains compared to controls (4 - 10% during the second year), decreased overall mean cumulative body weight gain at the end of the study compared to controls (10%)

FEMALES
18000 ppm: decreased mean body weight compared to controls (15% at the end of the second year), decreased mean cumulative body weight gains compared to controls (8 - 24% during the second year), decreased overall mean cumulative body weight gain at the end of the study compared to controls (24%)

(see tables 5 and 6)
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
FEMALES
18000 ppm (carcinogenicity phase): increased incidence and severity of diffuse squamous cell hyperplasia in the cervix when compared to controls, increased incidence of focal squamous cell metaplasia in the endometrium when compared to controls, increased incidence of diffuse squamous cell hyperplasia in the vagina when compared to controls, tendency towards a higher severity of corpora lutea depletion in the ovary when compared to controls, increased incidence of focal pars distalis hyperplasia when compared to controls (within the range of the in-house historical control data and therefore considered to be incidental and not treatment-related)

(see tables 9, 10 and 11)
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
FEMALES
18000 ppm (carcinogenicity phase): slightly increased incidence of epithelial tumors in the uterus when compared to controls

(see table 12)
Details on results:
For details on results including tables see the attached document.
Relevance of carcinogenic effects / potential:
Neoplastic histopathological findings were observed at the highest dose level of 18000 ppm. The administration of the test substance at 18000 ppm in the diet for 2 years resulted in an achieved intake of 1052 mg/kg bw/day in females which exceeds the guideline limit dose of 1000 mg/kg bw/day. In addition, based on a reduction of 15% in final mean body weight and an overall 24% reduction of the mean cumulative body weight gain compared to controls, this dose level was considered to be above the Maximum Tolerated Dose (MTD), where a 10% reduction in body weight gain is considered adverse. Therefore, the effects observed in the target organs (uterus, ovary and vagina) were a late onset occurrence, observed at a dose which exceeds both the MTD and the limit dose.
Key result
Dose descriptor:
NOAEL
Remarks:
carcinogenicity
Effect level:
>= 18 000 ppm
Based on:
test mat.
Remarks:
equivalent to 741 mg/kg bw/day
Sex:
male
Basis for effect level:
other: highest dose tested
Key result
Dose descriptor:
NOAEL
Remarks:
carcinogenicity
Effect level:
4 000 ppm
Based on:
test mat.
Remarks:
equivalent to 221 mg/kg bw/day
Sex:
female
Basis for effect level:
histopathology: neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
chronic toxicity 24 month
Effect level:
4 000 ppm
Based on:
test mat.
Remarks:
equivalent to 159 and 221 mg/kg bw/day in males and females, respectively
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
18 000 ppm
System:
female reproductive system
Organ:
ovary
uterus
vagina
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified

For details on results including tables see the attached document.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
221 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex X, 8.5, of Regulation (EC) No 1907/2006.
System:
female reproductive system
Organ:
uterus

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Reliable studies regarding carcinogenicity are available for the test substance.

Rat

The oncogenic potential and the chronic toxicity of the test substance were assessed in a study performed according to OECD Guideline 453 and in compliance with GLP (M-568723-03-1). Groups of 70 male and 70 female Wistar Rj:WI (IOPS HAN) rats were fed diet containing 0, 900, 4000, and 18000 ppm of the test substance for at least 104 weeks. Ten males and 10 females from each group were allocated to the chronic (12 month) phase and were necropsied after 52 weeks of treatment.  The remaining 60 animals/sex/group were allocated to the carcinogenicity (24 month) phase of the study, continued treatment until final sacrifice of the study after at least 105 weeks of treatment. The mean achieved dose levels of the test substance received by the animals over the 12-month period were approximately 41.0, 184 and 854 mg/kg/day in males and 56.7, 246 and 1147 mg/kg/day in females, corresponding to the dietary levels of 900, 4000 and 18000 ppm in both sexes. The mean achieved dose levels of test substance received by the animals overthe 24-month period were approximately 35.3, 159 and 741 mg/kg/day in males and 51.2, 221 and 1052 mg/kg/day in females, corresponding to the dietary levels of 900, 4000 and 18000 ppm in both sexes. During the first 12-months of treatment, no treatment-related clinical signs were observed and the mortality rate was low across all groups. There were no treatment-related effects on the following parameters, at any dose-level tested; food consumption throughout the study, ophthalmic changes at the 12 and 24-month examination, hematology, clinical chemistry or urinalysis parameters after approximately 3, 6, 12, 18 or 24 months of treatment. In addition, no treatment-related organ weight changes were observed at either the 12-month interim or 24-month terminal sacrifice.

No macroscopic or microscopic changes were observed in animals allocated to the12-month interim sacrifice phase and no macroscopic changes were observed at the 24-month terminal sacrifice, which could be attributable to treatment.

In conclusion, adverse treatment-related effects were limited to the high dose of 18000 ppm in both sexes. In males, these effects were limited to an effect on body weight parameters whereas in females, these effects also included non-neoplastic changes in the uterus, ovary and vagina and a slight increase in epithelial uterine tumors after two years of treatment. However, in females, the administration of the test substance at this dose level in the diet for 2 years resulted in an achieved intake of 1052 mg/kgbw/ day which exceeds the guideline limit dose of 1000 mg/kg bw/day in females. Therefore, the effects observed in the target organs (uterus, ovary and vagina) were a late onset occurrence, observed at   a dose which exceeds both the MTD and the limit dose. Thus, under the conditions of this study the NOAEL in terms of carcinogenic potential was the high dose of 18000 ppm for males (equivalent to 741 mg/kg body weight/day) and the mid dose of 4000 ppm for females (equivalent to 221 mg/kgbody weight/day),based on the slight increase in epithelial uterine tumors observed in females at 18000 ppm (equivalent to 1052 mg/kg body weight/day), following a 24-month period of dietary administration with the test substance. The NOAEL over a 24 -month period of dietary administration with the test substance to the rat was 4000 ppm in both sexes (equivalent to 159 mg/kg body weight/day in males and 221mg/ kg body weight/day in females).

 

Mouse

The oncogenic potential of the test substance was assessed in a study performed according to OECD Guideline 451 and in compliance with GLP (M-552413-01-1). Groups of 60 male and 60 female C57BL/6J mice were fed diet containing 0, 260, 1300 or 6500 ppm of the test substance for 52 weeks. After 52 weeks, 10 males and 10 females from each group allocated to the chronic phase of the study were necropsied at the scheduled interim sacrifice to assess chronic toxicity. The remaining 50 animals/ sex/group, allocated to the carcinogenicity phase of the study, continued treatment until the scheduled final sacrifice of the study after at least 78 weeks of treatment. The mean intake of the test substance over 18 months was 0, 32.9, 166 and 825 mg/kg/day in males and 0, 43.1, 215 and 1073 mg/kg/day in females, at 0, 260, 1300 and 6500 ppm, respectively. Up to the highest dose level tested (6500ppm), there were no treatment-related effects on mean body weight parameters, mean food consumption and hematology assessments throughout the study, or on an earlier development or increased incidence of tumors in either sex. The bioanalytical examination showed a dose-related increase in test substance and metabolite mean plasma concentrations, with similar levels observed throughout the study and slightly higher mean values in females compared to males. Thus, under the conditions of this study, the test substance did not induce carcinogenic effects. The NOAEL corresponded to 6500ppm (equivalent to 825 and 1073 mg/kg bw/day in males and females,respectively).

Justification for classification or non-classification

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