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Carcinogenicity

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Description of key information

Not carcinogenic. Oral, Key study: OECD 453, EPA OPP 83-5, EU Method B.33, Toxicity NOAEL male 1.14 mg/kg bw, female 1.3 mg/kg bw in rats. Gerspach 1998a
Not carcinogenic. Oral, Supporting study: OECD 451, EPA OPP 83-2, EU Method B.32, NOAEL male 0.36 mg/kg bw, female 1.2 mg/kg bw in mice. Gerspach 1998b

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:
18 January 1996 to 17 February 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed to GLP and in line with the standardised guidelines OECD 453, EPA OPP 83-5, EU Method B. 33 and Japanese MAFF with no deviations thought to impact the reliability of the presented results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPP 83-5 (Combined Chronic Toxicity / Carcinogenicity)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Guidance on Toxicology Study data for Application of Agricultural Chemical Registration, 28 January 1985.
Deviations:
not specified
GLP compliance:
yes
Species:
rat
Strain:
other: Tif: RAIf (Spraque-Dawley derived)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 5 weeks old.
- Weight at study initiation: Males 191.5 to 294.1 g; Females 153.7 to 223.1 g.
- Housing: Housed in groups of 5 by sex in 1800 cm² cages with soft wood bedding.
- Diet: Pelleted, certified standard diet, provided ad libitum. All batches were assayed for composition and contaminant levels by the manufacture.
- Water: Tap water, provided ad libitum. The results of the routine chemical examination of water showed that contaminant levels were within the acceptable limits and were not considered to have affected the conduct of the study or the interpretation of the data.
- Acclimation period: 18 days for males and 17 for females.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 ºC
- Humidity (%): 55 ± 10%
- Air changes (per hr): 16 to 20 air charges/ hour
- Photoperiod (hrs dark / hrs light): 12 hours light per day.

IN-LIFE DATES: From: 18 January 1996 To: 17 February 1998.
Route of administration:
oral: feed
Vehicle:
water
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet: Diets were prepared at about monthly intervals.
- Mixing appropriate amounts with: The test material was weighed on a calibrated balance. The pulverized food was then homogenously mixed with the appropriate concentrations of the test material and about 25% water was added before pelleting to ensure the necessary pellet quality. The pellets were subsequently air dried. The test material was used without adjustment for purity.
- Storage temperature of food: Food was stored in stainless steel containers at room temperature in a separate area.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Pellet samples (about 200 g each) were collected during the pelleting process (for homogeneity analyses at beginning, middle, and end of pelleting process) and dispatched deep frozen for analysis.

Method: Samples were first extracted with water and methanol and stored at -18 ºC until analysis. Appropriate aliquots of the extracts were either directly diluted with mobile phase or first evaporated to dryness and then the residue dissolved in mobile phase before injection, depending on the nominal concentration.
The determination was performed by HPLC using two columns switching and UV detection.

Results: The test material was found to be chemically stable at 22 ± 2 ºC in diet, at all concentrations used in the study. The test material was found to be homogeneously distributed in the diet at all concentrations used in the study.
Procedural recovery ranged between 75 and 107%.
The measured concentrations were found to be in agreement with the nominal concentrations. Nominal concentrations were used for the evaluation of the results.
Duration of treatment / exposure:
24 months
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 10, 30, 100 and 300 mg/kg food
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 9.92, 29.6, 99.0 and 296.0 mg/kg food
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0.39, 1.14, 3.76 and 11.4 mg/kg bw/day (males)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0.44, 1.30, 4.43 and 13.0 mg/kg bw/day (females)
Basis:
actual ingested
No. of animals per sex per dose:
Eighty per sex per dose, divided into the following groups:
> Group 1 consisted of fifty rats per sex per dose for carcinogenicity observations.
> Group 2 consisted of ten rats per sex per dose, used as a satellite group for haematology observations.
> Group 3 consisted of ten rats per sex per dose, used for blood chemistry and urine parameters.
> Group 4 consisted of ten rats per sex per dose, used for interim sacrifice at month 12.
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels were based on the results of the following previously conducted study:
Conditions: 3 month dietary toxicity study in rats, conducted at 20, 100, 300, 1000 and 4000 mg/kg food in 10 males and 10 females per dose group. The calculated average of daily doses were 1.21, 6.12, 18.8, 62.3, and 243 mg/kg bw in males, and 1.42, 7.07, 20.6, 69.3 and 282mg/kg bw in females, respectively.
Results: Body weight gains were decreased by 41% (males) and 20% (females) at 4000 mg/kg and 17% (males) and 12% (females) at 1000 mg/kg. In addition, Red blood cell parameters were affected at these upper dose levels. The liver was a target organ as indicated by the incidence of changes in males and females at feeding levels of 1000 and 4000 mg/kg. The liver lesions were characterized by minimal or moderate necrosis of single hepatocytes in animals at 1000 and 4000 mg/kg, areas of modest or marked necrosis in three males at 4000 mg/kg which died during the study; minimal to marked inflammatory cell infiltration in males at 1000 mg/kg and in males and females at 4000 mg/kg, minimal to moderate pigmentation (most likely hemosiderin) in the Kupffer cell of the liver in males and females at 4000 pmg/kg and in the hepatocytes of three males which died; and minimal to marked cytoplasmic vacuolization in females at 1000 mg/kg and in males and females at 4000 mg/kg. Additionally, in males at 4000 mg/kg, minimal to moderate hypertrophy of hepatocytes and minimal to moderate hypertrophy of hepatocytes and minimal to moderate extramedullary hematopoiesis were observed.
Thus, body weight gain data, laboratory investigations, and histopathology in the liver showed that the maximum tolerated dose (MTD) criteria were clearly exceeded at 4000 and 1000 mg/kg.
- Rationale for selecting satellite groups: Individuals assigned to satellite groups were done so at random.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily, twice on weekdays and once at weekends.
- Cage side observations included: Mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly for the first 3 months and then weekly thereafter.
- Bodyweight gain: Determined as the difference in body weight between that and the previous measurement.

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

WATER CONSUMPTION: Yes
- Time schedule for examinations: Weekly for the first 3 months and then monthly thereafter.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Beginning of treatment and then at 6, 12, 18 and 24 months.
- Dose groups that were examined: Both males and females from group 1 of the 300 mg/kg dose groups and the control were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected from the orbital plexus on weeks 13, 27, 53, 78 and 105.
- Anaesthetic used for blood collection: Yes, light ether anaesthesia.
- Animals fasted: Yes, overnight before sampling.
- How many animals: Twenty animals per sex per dose.
- Parameters checked in Table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected from the orbital plexus on weeks 13, 27, 53, 78 and 105.
- Animals fasted: Yes, overnight before sampling.
- How many animals: Ten animals per sex per dose.
- Parameters checked in Table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected overnight weeks 13, 27, 53, 78 and 105.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, both food and water were withheld overnight before sampling.
- How many animals: Ten animals per sex per dose.
- Parameters checked in Table 3 were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, see Table 4.
HISTOPATHOLOGY: Yes, see Table 5.
Statistics:
For each time point and parameter an univariate statistical analysis was performed. Nonparametric methods (Lehmann, 1975) were applied, to allow for non normal as well as normal data distribution.
Each treated group was compared to the control group either by Lepage's (Lepage, 1971) or Wilcoxon's two-sample test and tested for increasing or decreasing trends from control up to the respective dose group by Jonckheere's test for ordered alternatives (Jonckheere, 1954). The Lepage test is a combination of Wilcoxon and Ansari-Bradley statistics, i.e. a combined test for location and dispersion. The Lepage test has a good power against the more general alternative that the distributions differ not only in location but also in dispersion. The Jonckheere test is sensitive to dose related monotonic trends.
Survival analysis was performed by the regression model (partial likelihood) introduced by Cox (Cox, 1972) in order to compare survival time of treated animals (experimental group 1) with control animals.
All microscopic findings of animals of the carcinogenicity subgroup were subjected to a statistical analysis performed by a computer program.. Neoplastic lesions were analyzed by Peto's mortality-prevalence test (Peto et al., 1980), non-neoplastic lesions by Cochran-Armitage's linear trend test (Armitage, 1955). One-sided tests for positive trend using the group numbers (1,2,...,5) as scoring coefficents were performed. In all cases where an effect was found (p<=0.05), further analyses were carried out in order to determine the highest non-significant dose group, by deleting the highest dose group and re-performing the above analysis until a non-significant result was obtained. This is a so-called "closed testing procedure" which guarantees the observance of the nominal significance level of 5%. The p-values were computed using standard normal approximations.
Statistical significance, however, does not necessarily imply biological relevance.
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment related effects.
Mortality:
no mortality observed
Description (incidence):
No treatment related effects.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment related effects.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment related effects.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No treatment related effects.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment related effects.
Haematological findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant effects.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment related effects.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment related effects.
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No toxicologically relevant effects.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related effects.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver effects in both males and females at 300 mg/kg.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No toxicologically relevant effects.
Details on results:
CLINICAL SIGNS AND MORTALITY
The incidence and distribution of clinical signs recorded during the study gave no indication of a treatment-related effect on the appearance and behaviour of the animals.
Considering animals of experimental group 1 (carcinogenicity group) only there were no adverse effects on survival as a results of treatment. The number and percentage of animals surviving to terminal necropsy are presented in Table 6.

BODY WEIGHT AND WEIGHT GAIN
Throughout the study, the body weight development of the treated animals was comparable to that of the control group. Cumulative mean body weight gain can be seen in Table 7. Statistical significances that were obtained for female groups during the initial study period, were considered to reflect the range of biological variability.

FOOD CONSUMPTION AND COMPOUND INTAKE
The mean food consumption of the treated animals was comparable to that of the control group. Cumulative food consumptions (CFC) and time weighted average (TWA) can be seen in Table 8.
No experimental relevance was given to the few isolated statistical significances which were considered to reflect the values' range of normal biological variability.
Daily male compound intake was calculated to be 0.39, 1.14, 3.76 and 11.4 mg/kg bw/day, for the nominal treatment groups 10, 30, 100 and 300 mg/kg.
Daily female compound intake was calculated to be 0.44, 1.30, 4.43 and 13.0 mg/kg bw/day, for the nominal treatment groups 10, 30, 100 and 300 mg/kg.

WATER CONSUMPTION
No experimental relevance was given to the few isolated statistical significances which were considered to reflect the values' range of normal biological variability.

OPHTHALMOSCOPIC EXAMINATION
The examinations of the eyes (lids and surrounds, conjunctiva, pupillary reflex, cornea, sclera, anterior chamber, lens, vitreous, and fundus) at pre-test and months 6, 12, 18, and 24 did not indicate an effect of the test material on the eyes.

The incidences of the findings in cornea, lens, vitreous, or fundus were not dose dependently distributed and occurred in comparable numbers in the control group. The corneal findings were overwhelmingly considered to reflect artifacts due to the mydriasis procedure. Occurrence of turbid lens is considered an age-dependent process. The fundus findings at 18 and 24 months were consequent to the examiners impaired sight onto the fundus due to turbid lens of the animal.

HAEMATOLOGY
No treatment-related changes were identified.
Myeloid leukemia was observed in one male (no. 292) of the 100 mg/kg group and contributed to the high mean values for white blood cell count, neutrophil count and large unstained cell count in this group at week 53. No toxicological relevance is attached to this finding which occasionally occurs spontaneously in this colony of rats.
A number of differences between the means achieved a level of statistical significance or showed a significant positive or negative trend. However, none of these differences was considered to be related to treatment as they were not consistent across time, did not form a dose relationship and/or were of insufficient magnitude to be of toxicological relevance.
In males, these differences included lower mean values for erythrocyte counts at weeks 13, 27, 53 and 78 for the 30 mg/kg dose group and at weeks 27 and 53 for 100 mg/kg; a lower hematocrit value at week 53 for 100 mg/kg; higher mean cell volume and mean corpuscular hemoglobin at week 53 for 30 mg/kg; a higher red cell volume distribution width for 30 mg/kg at week 78; a higher mean corpuscular hemoglobin concentration at week 27 for 300 mg/kg; higher hemoglobin concentration distribution width at weeks 13 and 53 for 30 mg/kg; higher reticulocyte counts for 30 mg/kg at weeks 53 and 78 and for 100 mg/kg at weeks 53 and 105; higher absolute and relative neutrophil counts for 100 mg/kg at week 53; lower absolute basophil count at week 13 for 300 mg/kg, and a higher platelet count for 100 mg/kg at week 78. Furthermore, there were a few changes in relative values of differential white blood cell counts (basophils, lymphocytes, monocytes, large unstained cells) without effects on the absolute values, which therefore, were considered to be without toxicological relevance.
In females, these differences included lower mean values for erythrocytes, hemoglobin and hematocrit at week 105 for 100 and 300 mg/kg; higher hemoglobin concentration and hematocrit values at week 13 for 10 mg/kg; a lower hemoglobin concentration distribution width for 10 mg/kg at week 78; higher absolute and relative neutrophil counts for 100 mg/kg and higher dispersion of neutrophil counts (individual values) for 300 mg/kg at week 27, and a lower mean prothrombin activity at week 27 for 100 mg/kg. Furthermore, there were a few changes in relative values of differential white blood cell counts (neutrophils, lymphocytes, monocytes) without effects on the absolute values, which therefore, were considered to be without toxicological relevance.

CLINICAL CHEMISTRY
No treatment-related changes to blood chemistry parameters were apparent.
A small number of differences between the means achieved a level of statistical significance or showed significant positive or negative trends. However, these differences were neither related to the dose administered, nor to the duration of treatment, and/or the change was of insufficient magnitude to be toxicologically relevant.
In males, these differences included a lower dispersion of glucose values at week 13 for 10 mg/kg, a lower mean value for glucose at week 53 for 100 mg/kg; a higher bilirubin value at week 13 for 30 mg/kg; a lower albumin value at week 27 for 100 mg/kg and a higher albumin value at week 13 for 300 mg/kg; a higher cholesterol value at week 13 and a higher dispersion of cholesterol values at week 105 for 30 mg/kg; lower sodium values at week 13 for 30 and 100 mg/kg and at week 27 for 30, 100 and 300 mg/kg; a higher potassium value at week 53 for 30 mg/kg; lower chloride values at week 27 for 30, 100 and 300 mg/kg, and at week 78 for 300 mg/kg higher phosphate values at weeks 53 and 105 for 300 mg/kg, and a higher mean value for alkaline phosphatase at week 78 for 300 mg/kg.
In females, these differences included lower values for glucose at week 13 for 30, 100 and 300 mg/kg and at week 53 for 10 and 30 mg/kg; a higher globulin value for 30 mg/kg at week 78; a lower albumin to globulin ratio at week 13 for 30 mg/kg; a lower value for cholesterol and a higher value for sodium at week 53 for 10 mg/kg; a lower triglyceride value for 300 mg/kg at week 105; higher potassium values at weeks 53 and 78 for 300 mg/kg; a higher calcium value for 30 mg/kg at week 78; higher phosphate values for 30 mg/kg at week 105 and for 300 mg/kg at weeks 27 and 105, and a higher alkaline phosphatase value for 300 mg/kg at week 78.

URINALYSIS
The quantitative and qualitative tests performed on urine samples collected at intervals during the study revealed no evidence of a treatment related effect on urine parameters investigated.
A small number of differences between the means achieved a level of statistical significance or showed significant positive or negative trends. However, these differences showed no dose relationship and/or the change was of insufficient magnitude to be toxicologically relevant. In males, these differences included lower mean values for relative density at week 53 in 100 and 300 mg/kg; higher pH values at week 13 for 30, 100 and 300 mg/kg; lower protein content at weeks 53 and 78 for 10 mg/kg, and higher bilirubin at week 13 for 100 mg/kg. Differences in females were limited to a higher urine volume at week 105 and higher pH values at weeks 13 and 105 for 100 mg/kg.

ORGAN WEIGHTS
The carcass weights and the organ weights of the treated animals were comparable to those of the control group at both the 12 and 24 month necropsy.
No toxicological relevance was attributed to the testes to body weight ratios (300 mg/kg) and the ovaries to body weight ratios (30 and 100 mg/kg) which reached statistical significances at the 12 month necropsy. The absolute mean testes weight value was less than control, and lay well within the range of reference values for our rat strain. The increase in testes weight ratio was very slight, approximately 3%, and there was no statistical significance at terminal necropsy. Additionally, there were no corroborative histological findings indicative of a treatment related effect. The absolute mean ovary weights in groups 30 and 100 mg/kg lay well within the historical reference range of our rat strain. There were no other corroborative findings, no consistent trend over the whole dose range and also no effect after two years of treatment.

GROSS PATHOLOGY
Positive results for the terminal necropsy can be seen in Table 9.
> Interim sacrifice (12 months)
At the 12 months interim sacrifice, all animals of experimental group 4 were examined macroscopically. A variety of macroscopic findings was observed in this study. They occurred in comparable numbers in all experimental groups and were similar to those occurring spontaneously in our colony of rats. Thus, no experimental relevance is attributed to these findings. In particular, masses on the body surface (chest wall, abdominal wall) microscopically reflecting neoplasias of the mammary gland, all were incidentally distributed among the dose groups.
> Final sacrifice (24 months) (including intercurrent deaths)
At the terminal sacrifice, all animals were examined macroscopically, including animals which died intercurrently.
No treatment related findings were observed during examination at final sacrifice. A variety of findings, as listed in Table 9, occurred in slightly increased incidences in treated animals. The histological counterparts of these gross lesions, occurring normally in aged rats, were not indicative of an effect due to the treatment. Therefore, these findings are considered devoid of toxicological relevance. They included cysts in the liver of males, corresponding histologically with biliary cyst, spongiosis and peliosis hepatis and cholangioma, enlarged spleens, corresponding with extramedullary hematopoiesis, congestion and/or infiltration by systemic neoplasia, cysts in the kidneys of males, corresponding histologically with simple cysts and an abscess, fluid in the tunica albuginea of testes in males, corresponding histologically with interstitial edema of testes, and deformation of the brain in females, corresponding histologically with hydrocephalus and/or pressure atrophy.
All other findings occurred in comparable numbers in all dose groups and were similar to those occurring spontaneously in our colony of rats. Thus no experimental relevance is attributed to these findings. In particular, masses on the body surface (head, back, chest wall, abdominal wall, inguinal region), all reported under 'skin/subcutis', microscopically reflecting neoplasias of the skin, subcutaneous tissue and the mammary gland, are all incidentally distributed among the dose groups.

HISTOPATHOLOGY: NON-NEOPLASTIC
> Interim sacrifice (12 months)
Microscopic findings are presented in Table 10.
Liver: Histopathology at 100 and 300 mg/kg confirmed the liver as a target organ. In animals sacrificed at 12 months, there was a minimal increase in incidence and/or severity of liver necrosis in males at 100 and 300 mg/kg, when compared to controls. In females, liver necrosis was seen only at 100 and 300 mg/kg, not in controls. Single cell necrosis (necrosis of single hepatocytes or minute clusters of hepatocytes) was seen only in 300 mg/kg females. There was an increase in severity of cholangiofibrosis in females at 300 mg/kg. Eight of 10 high dose females had an average severity grade of 2.3 compared with an average grade of 1.4 from 5 of 10 controls. The incidence appeared to be slightly increased in females at 100 and 300 mg/kg (8 of 10), although 5 of 10 controls were also noted with cholangiofibrosis. Neither the incidence nor the severity was affected by treatment in males; there was an apparent decrease in high dose (3 of 10) compared with the controls (6 of 10).
Additionally, a variety of other changes were found in the animals scheduled for interim sacrifice. They commonly occur in our colony of rats of this age, and, neither their incidences nor their distribution and morphologic appearance gave any indication of a treatment related association.
> Final sacrifice (24 months) (including intercurrent deaths)
Microscopic findings are presented in Table 11.
Liver: The following treatment related findings were observed: Compared to the controls, statistically significant higher numbers of females with fatty change were observed in the 300 mg/kg treatment group. The severity of this lesion was mainly minimal to slight and the distribution diffused. Compared to the control animals, the severity was slightly increased. Furthermore, slightly increased incidences of minimally to slightly increased mitotic activity of hepatocytes were recorded in high dose females. Compared to the controls, the severity was slightly increased.
In contrast to the 12-month data, the findings for cholangiofibrosis were generally comparable at final sacrifice. The incidences of this common finding in aged rats were greater than 70% in all female groups, although a significant positive trend was noted. There were no differences in average grade, 2.1 - 2.2, for all groups. In males, the incidences were greater than 50% in all groups and there appeared to show a slight decrease in average grade at 300 mg/kg (high dose 2.1 versus control 2.5). Therefore, cholangiofibrosis is considered of no toxicological relevance after 24 months of treatment. Slightly increased incidences of chronic congestion of the liver in high dose females proved to be significant on statistical examination. This lesion occurred in animals found dead or killed moribund and was in relation with their deteriorated general condition. Due to its low incidence of only 3/50 animals affected, this finding is considered devoid of toxicological relevance.

HISTOPATHOLOGY: NEOPLASTIC
> Interim sacrifice (12 months)
There was no evidence of an increased occurrence of neoplastic lesions. Microscopic findings are presented in Table 10.
> Final sacrifice (24 months) (including intercurrent deaths)
Microscopic findings are presented in Table 11.
No treatment induced neoplastic changes were observed after two years of treatment.
Systemic neoplasias: 2/50 males in the high dose group presented with malignant lymphoma. These incidences proved to be significant on statistical examination. This finding occasionally occurs in control animals of our strain of rats and is within the range of the historical control data as shown at the end of this section. Furthermore, up to 5.7% malignant lymphomas were observed in other colonies of old control Sprague-Dawley rats. Therefore, it is considered an incidental finding of no toxicological relevance.
Oral cavity: 2/50 males in the high dose group presented with squamous cell carcinoma of the oral cavity versus none in the control group. This incidence was slightly above the historical control values of our strain of rats shown at the end of this section. Oral squamous cell carcinomas may occur consequent to chronic inflammatory changes and foreign bodies in the oral cavity. Since inflammation and/or foreign bodies of the oral cavity were observed in several control and treated animals from this study, the oral squamous cell carcinomas observed in these few animals are considered incidental findings devoid of toxicological relevance.

Additionally, a variety of other non-neoplastic and neoplastic changes such as hemosiderosis and extramedullary hematopoiesis in the spleen, biliary cysts, hepatocellular and cholangiocellular tumors in the liver, cysts, chronic tubular lesion and and chronic nephropathy in the kidneys, foam cells and bronchiolo-alveolar adenoma in the lung, myocardial inflammation with fibrosis, atrophy of the thymus, interstitial edema and tubular atrophy of the testes, inflammatory changes in the prostate gland, hyperplasia, adenoma and carcinoma of the adrenal cortex and medulla, c-cell and follicular hyperplasia and tumors of the thyroid gland, hyperplasia and adenoma of acinar and islet cells of the pancreas, several types of tumors of the skin and subcutaneous tissue, fibroadenoma and adenocarcinoma of the mammary gland, degenerative changes in the eyes and Harderian glands, and schwannoma, hemangioma and hemangiosarcoma in different tissues was found in animals of the oncogenicity group in this study. These and the other changes not mentioned here commonly occur in our colony of rats, and, neither their incidences nor their distribution and morphologic appearance gave any indication of a treatment-related association.

HISTORICAL CONTROL DATA
Historical data can be seen in Tables 12 and 13.
Relevance of carcinogenic effects / potential:
Under the conditions of the test there was no evidence of treatment related carcinogenic effects.
Dose descriptor:
NOEL
Effect level:
1.14 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: liver necrosis
Remarks on result:
other: Effect type: toxicity (migrated information)
Dose descriptor:
NOEL
Effect level:
1.3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: liver necrosis
Remarks on result:
other: Effect type: toxicity (migrated information)

Table 6. Mortality Data

Nominal Test Concentrations (mg/kg)

Number of Animals Surviving to Termination

Males

Females

Control (0)

26 (52%)

32 (64%)

10

33 (33%)

35 (70%)

30

31 (62%)

36 (72%)

100

29 (29%)

28 (56%)

300

25 (25%)

30 (60%)

 

Table 7. Cumulative Body Weight Gain (g/animal)

Sex

Week

Nominal Test Concentration (mg/kg)

0

10

30

100

300

g

g

%

g

%

g

%

g

%

Male

12

22.9

225.0

- 2.1

232.1

+ 1.0

233.6

+ 1.6

224.9

- 2.2

50

433.4

431.6

- 0.4

444.6

+ 2.6

435.6

+ 0.5

430.9

- 0.6

130

476.0

468.2

- 1.6

482.1

+ 1.3

493.3

+ 3.6

496.9

+ 4.4

Females

12

98.67

101.5

+ 2.9

103.2

+ 4.6

106.8

+ 8.2

105.9

+ 7.3

50

192.6

191.2

- 0.7

198.0

+ 2.8

201.8

+ 4.8

194.8

+ 1.1

130

264.8

274.0

+ 3.5

264.6

- 0.1

260.9

- 1.5

261.3

- 1.3

 

Table 8. Cumulative Food Consumption (CFC) and Time Weighted Average (TWA) per Week

CFC (g/animal)

TWA (g/week)

Nominal Test Concentrations (mg/kg) (% of control)

0

10

30

100

300

CFC males

16856.0

16962.0 (100.6)

16981.6 (100.7)

16809.7 (99.7)

16722.6 (99.2)

TWA males

163.7

164.7 (100.6)

164.9(100.7)

163.2 (99.7)

162.4 (99.2)

CFC females

11900.0

11573.4 (97.3)

11726.4 (98.5)

11874.5 (99.8)

11488.2 (96.5)

TWA females

115.5

112.4 (97.3)

113.8 (98.5)

115.3 (99.8)

111.5 (96.5)

Table 9. Gross Pathology, Incidence of Findings at Final Sacrifice

Observations

Males

Females

Nominal test concentrations (mg/kg)

0

10

30

100

300

0

10

30

100

300

No. of animals examined

50

50

50

50

50

50

50

50

50

50

Liver: cyst

1

1

4

3

4

7

1

6

8

4

Spleen: large

1

4

3

3

4

3

0

5

1

3

Kidneys: cyst

0

4

3

2

4

2

1

0

1

1

Testes tunica albug: Contents fluid

8

9

10

10

14

-

-

-

-

-

Brain: deformation

2

1

2

2

1

1

1

6

4

4

 

Table 10. Histopathology, Incidence of Findings at Interim Sacrifice

Observations

Males

Females

Nominal test concentrations (mg/kg)

0

10

30

100

300

0

10

30

100

300

No. of animals examined

10

10

10

10

10

10

10

10

10

10

Liver Necrosis:

Minimal

1

0

0

2

1

0

0

0

1

3

Slight

1

1

3

2

2

0

0

0

1

0

Moderate

0

0

0

0

1

0

0

0

0

0

Total

2

1

3

4

4

0

0

0

2

3

Liver Single Cell Necrosis:

Minimal

0

0

0

0

0

0

0

0

0

1

Slight

0

0

0

0

0

0

0

0

0

3

Total

0

0

0

0

0

0

0

0

0

4

Liver Cholangiofibrosis:

Minimal

0

0

0

0

0

3

4

2

4

1

Slight

6

6

6

3

3

2

2

2

3

4

Moderate

0

0

0

1

0

0

0

1

1

3

Total

6

6

6

4

3

5

6

5

8

8

Table 11. Histopathology, Incidence of Findings at Terminal Sacrifice

Observations

Males

Females

Nominal test concentrations (mg/kg)

0

10

30

100

300

0

10

30

100

300

No. of animals examined

50

50

50

50

50

50

50

50

50

50

Treatment Related Increased Incidence

Liver: Fatty change

Minimal

0

0

0

0

0

10

11

11

6

13

Slight

9

12

9

10

8

1

4

0

4

11

Moderate

1

0

2

0

4

1

1

2

2

3

Severe

0

0

1

0

0

0

0

0

1

0

Total

10

12

12

10

12

12

16

13

13

27

Liver: Increased mitotic activity

Minimal

0

0

0

0

0

1

0

1

1

3

Slight

0

0

0

1

0

0

0

0

0

3

Total

0

0

0

1

0

1

0

1

1

6

Differences in Incidences Not Associated with Treatment

Liver: Cholangiofibrosis

35

37

36

33

28

37

41

44

42

45

Liver: Congestion chronic

0

0

0

0

0

0

0

1

0

3

Systemic Neoplasias: Lymphoma malignate

0

0

1

1

2

0

0

0

1

0

Oral Cavity: Carcinoma squamous

0

0

0

0

2

1

0

0

1

0

 

Table 12. Historical Data, Incidence of Malignant Lymphoma (Systemic Neoplasia) in Untreated Male Rats

Date of 1stDose*

No. of Animals with Systemic Neoplasia **

No. of Organs Examined

February 1992

0

50

June 1992

1

50

August 1992

1

50

August 1992

2

50

May 1993

0

50

May 1993

1

50

June 1993

0

50

January 1994

1

60

January 1994

0

60

October 1994

0

50

August 1995

0

50

Total

6 (1.05%)

570

SD

1.36%

 

Range High

2/50 (4.00%)

 

Range Low

0/60 (0.00%)

 

* All studies were performed for 24 months.

** Malignant lymphoma (M-LM-3) in the lymphoreticular tissue (LRT).

 

Table 13. Historical Data, Incidence of Squamous Cell Carinoma, in the Mouth or Nasopharynx of Untreated Male Rats

Date of 1stDose*

No. of Animals with Squamous Carinoma in the Mouth, Oral Cavity (MOU SQ-CE-CC-3)

No. of Animals with Squamous Carinoma in the Nasopharynx, Nasal Cavity (PHA SQ-CE-CC-3)

No. of Organs Examined

February 1992

0

0

50

June 1992

1

0

50

August 1992

0

0

50

August 1992

0

0

50

May 1993

0

0

50

May 1993

0

0

50

June 1993

1

0

50

January 1994

1

0

60

January 1994

0

0

60

October 1994

0

0

50

August 1995

0

0

50

Total

3 (0.53%)

0 (0.00%)

570

SD

0.89%

0.00%

 

Range High

1/50 (2.00%)

0/50 (0.00%)

 

Range Low

0/60 (0.00%)

0/60 (0.00%

 

* All studies were performed for 24 months.

Conclusions:
Under the conditions of the test, the test material was tolerated without occurrence of overt toxicity up to the maximum dose level of 300 mg/kg. All In-life end points (clinical signs, behaviour, body weight development, food consumption, and laboratory data) and mortalities of animals of the treated groups were similar to those of the control group. An overall slightly decreased water consumption was measured for the high dose females only. Organ weights and macroscopic examination at necropsy were without indication of effects related to treatment.
Histopathology revealed the liver as a target organ. Observations at the maximum dose, 300 mg/kg after 12 months of treatment were a slightly increased incidence and/or severity of liver necrosis in males and females, increased incidence of hepatocellular single cell necrosis in females, and increased incidence and/or severity of cholangiofibrosis in females. Observations after 24 months were, overall slightly reduced water consumption in females, increased incidence and slightly increased severity of fatty change in females, and slightly increased incidence and severity of mitotic activity of hepatocytes in females.
Observations at 100 mg/kg after 12 months were, slightly increased incidence and/or severity of liver necrosis in males and females, and increased incidence and/or severity of cholangiofibrosis in females.
There was no evidence for the occurrence of treatment related neoplastic lesions at any dose level in this study.
The findings observed in the liver (necrosis, fatty change, and higher mitotic activity) at 100 and/or 300 ppm, however, clearly indicate these dose levels to have met or exceeded the maximum tolerated dose (MTD) requirement. Therefore the NOEL and NOAEL was determined to be 30 mg/kg in both males and females, corresponded to 1.14 mg/kg bw in males and to 1.30 mg/kg bw in females.
Executive summary:

The carcinogenic potential of the test material was determined in a 24 month study with both male and female rats, conducted to the standardised guidelines OECD 453, EPA OPP 83-5, EU Method B. 33 and Japanese MAFF.

The test material was administered oral to Sprague-Dawley derived rats at concentrations of 0, 10, 30, 100, and 300 mg/kg in their diet. A total of eighty rats per sex per dose were exposed, consisting of fifty animals per sex for evaluation of carcinogenic potential, and thirty split into satellite groups for the evaluation of haematological parameters, blood chemistry, urine parameters, and the interim sacrifice at month 12. Clinical signs, body weight, food and water consumption, opthalmoscopic effects, and mortality were monitored throughout the study for all animals. Haematological investigations, blood chemistry and urine parameters were examined on weeks 13, 27, 53, 78 and 105. At sacrifices, animals were examined macroscopically and organ weights were recorded. Organs and tissues were collected and prepared for histopathological evaluation. Organs and tissues from all animals in the carcinogenicity group were examined microscopically.

Under the conditions of the test, the test material was tolerated without occurrence of overt toxicity up to the maximum dose level of 300 mg/kg. All In-life end points (clinical signs, behaviour, body weight development, food consumption, and laboratory data) and mortalities of animals of the treated groups were similar to those of the control group. An overall slightly decreased water consumption was measured for the high dose females only. Organ weights and macroscopic examination at necropsy were without indication of effects related to treatment. Histopathology revealed the liver as a target organ. Observations at the maximum dose, 300 mg/kg after 12 months of treatment were a slightly increased incidence and/or severity of liver necrosis in males and females, increased incidence of hepatocellular single cell necrosis in females, and increased incidence and/or severity of cholangiofibrosis in females. Observations after 24 months were, overall slightly reduced water consumption in females, increased incidence and slightly increased severity of fatty change in females, and slightly increased incidence and severity of mitotic activity of hepatocytes in females. Observations at 100 mg/kg after 12 months were, slightly increased incidence and/or severity of liver necrosis in males and females, and increased incidence and/or severity of cholangiofibrosis in females. There was no evidence for the occurrence of treatment related neoplastic lesions at any dose level in this study.

The findings observed in the liver (necrosis, fatty change, and higher mitotic activity) at 100 and/or 300 ppm, however, clearly indicate these dose levels to have met or exceeded the maximum tolerated dose (MTD) requirement. Therefore the NOEL and NOAEL was determined to be 30 mg/kg in both males and females, corresponded to 1.14 mg/kg bw/day in males and to 1.30 mg/kg bw/day in females. The study is considered to be reliable, relevant and adequate for risk assessment purposes.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1.14 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Both available studies were conducted under GLP and in accordance with standardised guidelines. Both studies were assigned a reliability score of 1 according to the criteria of Klimisch (1997) and were considered to be suitable as an accurate reflection of the test material. The quality of the database is high.

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In the absence of any carcinogenic effects observed, test material does not meet classification criteria under Regulation (EC) No. 1272/2008 or EU Directive 67/548/EEC for carcinogenicity.

Additional information

The carcinogenic potential of the test material was determined in a 24 month study with both male and female rats, conducted to the standardised guidelines OECD 453, EPA OPP 83-5, EU Method B. 33 and Japanese MAFF. The test material was administered oral to Sprague-Dawley derived rats at concentrations of 0, 10, 30, 100, and 300 mg/kg in their diet. A total of eighty rats per sex per dose were exposed, consisting of fifty animals per sex for evaluation of carcinogenic potential, and thirty split into satellite groups for the evaluation of haematological parameters, blood chemistry, urine parameters, and the interim sacrifice at month 12. Clinical signs, body weight, food and water consumption, opthalmoscopic effects, and mortality were monitored throughout the study for all animals. Haematological investigations, blood chemistry and urine parameters were examined on weeks 13, 27, 53, 78 and 105. At sacrifices, animals were examined macroscopically and organ weights were recorded. Organs and tissues were collected and prepared for histopathological evaluation. Organs and tissues from all animals in the carcinogenicity group were examined microscopically.

Under the conditions of the test, the test material was tolerated without occurrence of overt toxicity up to the maximum dose level of 300 mg/kg. All In-life end points (clinical signs, behaviour, body weight development, food consumption, and laboratory data) and mortalities of animals of the treated groups were similar to those of the control group. An overall slightly decreased water consumption was measured for the high dose females only. Organ weights and macroscopic examination at necropsy were without indication of effects related to treatment. Histopathology revealed the liver as a target organ. Observations at the maximum dose, 300 mg/kg after 12 months of treatment were a slightly increased incidence and/or severity of liver necrosis in males and females, increased incidence of hepatocellular single cell necrosis in females, and increased incidence and/or severity of cholangiofibrosis in females. Observations after 24 months were, overall slightly reduced water consumption in females, increased incidence and slightly increased severity of fatty change in females, and slightly increased incidence and severity of mitotic activity of hepatocytes in females. Observations at 100 mg/kg after 12 months were, slightly increased incidence and/or severity of liver necrosis in males and females, and increased incidence and/or severity of cholangiofibrosis in females. There was no evidence for the occurrence of treatment related neoplastic lesions at any dose level in this study. The findings observed in the liver (necrosis, fatty change, and higher mitotic activity) at 100 and/or 300 ppm, however, clearly indicate these dose levels to have met or exceeded the maximum tolerated dose (MTD) requirement. Therefore the NOEL and NOAEL was determined to be 30 mg/kg in both males and females, corresponded to 1.14 mg/kg bw in males and to 1.30 mg/kg bw in females.

The carcinogenic potential of the test material was determined in an 18 month study with both male and female mice, conducted to the standardised guidelines OECD 451, EPA OPP 83-2, EU Method B. 32 and Japanese MAFF.The test material was administered oral to albino mice at concentrations of 0, 1, 3, 10, and 60 mg/kg in their diet. Fifty animals per sex and group were utilized for evaluation of carcinogenic potential and ten animals per sex and group were utilized for evaluation of haematological parameters. Clinical signs, body weight, food consumption, and mortality were monitored throughout the study for all animals. Haematological investigations were performed at 12 and 18 months. At sacrifices, animals were examined macroscopically and organ weights were recorded. Organs and tissues were collected and prepared for histopathological evaluation. Organs and tissues from all animals in the carcinogenicity group were examined microscopically.

Under the conditions of the treatment was tolerated without occurrence of overt toxicity. Females treated with the high dose level (60 mg/kg) had minimally lower overall food consumption and consequent decreased food consumption ratio values. Haematological findings concerning red blood cells (at 60 and 10 mg/kg) and white blood cells (at 60 mg/kg) parameters were confined to male animals. The liver was a target organ, in both sexes, absolute and relative weights were increased at the high dose level. In addition, an increased number of macroscopically enlarged livers were seen in the males of this dose group. Microscopically, increased incidence and/or severity of single cell necrosis of hepatocytes and hyperplasia of Kupffer cells were observed in males and females dosed at 60 mg/kg. A slight increase in incidence and severity, of questionable toxicological significance, was also noted in males dosed at 10 mg/kg. There was no evidence of treatment related carcinogenic effects. The NOEL after 18 months of treatment was 3 mg/kg for males and 10 mg/kg for females, equivalent to an average daily intake of 0.36 or 1.20 mg/kg bw/day for males and females, respectively.


Justification for selection of carcinogenicity via oral route endpoint:
Robust study summary selected was performed on the preferred species in a chronic study. Although adverse effects were seen no increased incidence of tumor formation was noted indicating that butafenacil was not carcinogenic in the rat. This finding is true for both key and supporting study. The NOAEL level for the key study was reported under Repeated dose toxicity (oral).

Carcinogenicity: via oral route (target organ): digestive: liver