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Carcinogenicity

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Description of key information

NOAEL was determined to be 3.46 mg/kg bw/day (males) and 4.21 mg/kg bw/day (females) in Crl:CD rats; study conducted in accordance with EPA OPP 83-5; Trutter (1991)

The test material was determined to be carcinogenic at 40 mg/kg bw/day in Crl:CD rats; study conducted in accordance with EPA OPP 83-2; Goldenthal (1993)

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:
12th February 1988 to 16th April 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPP 83-5 (Combined Chronic Toxicity / Carcinogenicity)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OTS 798.3320 (Combined Chronic Toxicity / Carcinogenicity)
Deviations:
no
GLP compliance:
yes
Species:
rat
Strain:
other: Crl:CD BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Kingston, New York, USA
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 200.7 g ± 12.61 (males); 150.5 g ± 10.58 (females)
- Housing: individually in stainless steel cages
- Diet: Purina® Certified Rodent Laboratory Chow® #5002 ad libitum
- Water: ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature: 71 ± 1.6 ºF
- Humidity: 49 ± 10.4 %
- Photoperiod: 12 hours light/12 hours dark
Route of administration:
oral: feed
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The required amount of test material for each dose level was mixed with the vehicle and added to 5 kg of basal diet and the premix mixed for 20 minutes. The premix was added to the remaining basal diet and the final formulation mixed for 1 minute per kg of diet.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were collected from the top, middle and bottom levels of each concentration, including controls, of test diet for analysis. During the study, samples of diets mixed for weeks 1, 2, 3 and 4 and once every 4 weeks thereafter were analysed for verification of dose level concentrations.
Duration of treatment / exposure:
Two years (terminal sacrifice of males started at 103 weeks; terminal sacrifice of females occurred at 104 weeks)
Frequency of treatment:
Daily
Post exposure period:
None
Dose / conc.:
50 mg/kg diet
Dose / conc.:
80 mg/kg diet
Dose / conc.:
400 mg/kg diet
Dose / conc.:
800 mg/kg diet
No. of animals per sex per dose:
Sixty
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: based on results of a preliminary subchronic toxicity study and an older chronic study which suggested that the maximum tolerated dose was less than 1000 mg/kg diet. 800 mg/kg was therefore set as the highest dose with 400 mg/kg chosen on the basis that it was half this value.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: prior to treatment and then weekly for weeks 1 to 16 and once every 4 weeks for the rest of the study

FOOD CONSUMPTION AND COMPOUND INTAKE/FOOD EFFICIENCY: Yes
- Time schedule for examinations: prior to treatment and then weekly for weeks 1 to 16 and once every 4 weeks for the rest of the study

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to treatment and prior to sacrifice
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 26, 52, 78 and 104 (males) or 105 (females)
- Anaesthetic used for blood collection: Yes (ketamine)
- Animals fasted: Yes
- How many animals: first 10 surviving animals in each group
- Parameters checked: leucocyte count, corrected leucocyte count, erythrocyte count, haemoglobin, absolute reticulocyte count, haematocrit count, platelet count, leucocyte differential cell morphology and reticulocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: weeks 26, 52, 78 and 104 (males) or 105 (females)
- Animals fasted: Yes
- How many animals: first 10 surviving animals in each group
- Parameters checked: sodium, potassium, chloride, total protein, aspartate aminotransferase, total bilirubin, blood urea nitrogen, glucose, albumin, globulin, albumin/globulin ratio, calcium, alanine aminotransferase, total cholesterol, creatine kinase, inorganic phosphorus and serum iron.

URINALYSIS: Yes
- Time schedule for collection of urine: weeks 26, 52, 78 and 104 (males) or 105 (females)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: appearance, pH, specific gravity, glucose, ketones, microscopic examination of sediment, volume, bilirubin, occult blood, urobilinogen and protein.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Performed on all animals that died or were sacrificed in a moribund condition. Ten animals/sex/group were sacrificed and necropsied following 52 weeks of treatment. All rats surviving to termination were sacrificed and necropsied after 103 (males) or 104 (females) weeks. Necropsies included examination of the following: external surface of the body, all orifices, cranial cavity, carcass, external surfaces of the brain, nasal cavity and paranasal sinuses, thoracic, abdominal and pelvic cavities and their viscera and cervical tissues and organs
- From all animals sacrificed at interim or terminal kills, the following organs were weighed and relative ratios determined: liver, kidneys, brain, testes with epididymides

HISTOPATHOLOGY: Yes
- Performed after 52 weeks and after study termination. During the second year of the study, an additional interim evaluation was performed of six high-dose animals to evaluate the significance of abdominal masses found at necropsy.
- The following tissues were examined microscopically: lesions and tumours, cervical spinal cord, mid-thoracic spinal cord, lumbar spinal cord, brain with brainstem, pituitary, thyroid, thymus, lung, heart, salivary glands, spleen, kidneys, adrenals, pancreas, testes with epididymides, trachea, liver, prostate, seminal vesicles, ovaries, uterus with vagina and cervix, mammary gland (females only), oesophagus, stomach, duodenum, jejunum, ileum, colon, cecum, rectum, urinary bladder, skin, mesenteric lymph node, mandibular lymph node, sciatic nerve, sternum with bone marrow, skeletal muscle, eyes and aorta.
- In addition, gross lesions from all animals, as well as lungs, kidneys and small intestine from all animals (except the control and high-dose groups) were examined microscopically.
Statistics:
Statistical evaluation applied to survival, body weight, food consumption, haematology, clinical chemistry, urine volume and organ weight data to determine differences between treatment and control groups. One way analysis of variance at the 5 % two-tailed probability level or at the 5% one-tailed probability level where more appropriate.
Clinical signs:
no effects observed
Description (incidence and severity):
Daily/weekly clinical examinations revealed no test material-related effects; the various clinical signs observed are considered normal for rats of this age/strain.
Mortality:
mortality observed, treatment-related
Description (incidence):
Survival in the 50 and 80 mg/kg males and in all female groups was similar to, or higher than, in the controls. By week 104, there was a statistically significant linear trend of reduced survival among males exposed to dietary concentrations of 400 and 800 mg/kg, although the difference to survival in the other groups was not significant (see Table 1).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no effect on body weight of males or females in the 50 and 80 mg/kg groups. There was a small, but statistically significant reduction in body weight of 400 mg/kg males from week 24 onwards and a moderate decrease in body weight for 800 mg/kg males and females from week 24 (see Table 2 and Figure 1).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was similar in the 50 and 80 mg/kg groups, with minimal to moderate statistically significant dose-related decreases at 400 and 800 mg/kg. Decreases in food consumption ranged from 2-5 and 12-17 % in the 400 and 800 mg/kg animals, respectively, over the study duration.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Daily/weekly ophthalmoscopic observations revealed no test material-related effects; the various ophthalmic lesions observed are considered normal for rats of this age/strain.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The principal haematological finding consisted of a significant increase in reticulocyte count in the 800 mg/kg males at week 104. This was considered to be a regenerative mechanism in response to a (non-significant) decline in red blood cell mass. The majority of the decrease was contributed by just 3 males. Other statistically significant findings were considered to have no biological or toxicological relevance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The serum chemistry data revealed a significant decrease in the total protein of the 400 and 800 mg/kg males and 800 mg/kg females at week 26, accompanied by a significant decrease in total serum calcium in these groups as well as decreased globulin and increased albumin/globulin ratios in the 800 mg/kg males. Declines in aspartate and alanine aminotransferases were noted at weeks 26 and 52 in the 400 and 800 mg/kg animals and may have been linked to a reduction in food intake/body weight. Other significant differences were noted, but isolated in occurrence and not considered to be of toxicological relevance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis results were generally unremarkable.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Evaluation of data from the 53 week interim sacrifice and at terminal sacrifice indicates that there were no treatment related effects on absolute weight of any organs. At the 53 week interim sacrifice there were some increased organ to body weight ratios in all treatment groups, although these were associated with the decreased body weights observed at this time.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Necropsy of unscheduled deaths through the first 64 weeks of study showed no apparent test material-related gross tissue changes. Beginning at week 65, several 800 mg/kg animals (principally male) exhibited abdominal masses, mostly associated with the small intestine (principally the jejunum). By study termination, the incidence of this finding was 15-25 % in 400 and 800 mg/kg males and 800 mg/kg females. Abdominal masses were also observed in a few individuals from other test groups, including the controls. There were no other treatment related tissue alterations.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathological examination revealed that there were no compound related non-neoplastic effects.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
During the second year of treatment there was an increased frequency of undifferentiated sarcomas in the jejunum of 400 and 800 mg/kg males and in 800 mg/kg females, necropsied after unscheduled deaths and after terminal sacrifice. In addition, single cases of this rare tumour type were also observed in females in the 50, 80 and 400 mg/kg groups, although the incidence was only statistically significant for 400 and 800 mg/kg males and 800 mg/kg females. Electron microscopy findings indicated that dietary exposure to the test material was linked with proliferation of malignant mesenchymal tumours in various stages of differentiation. The incidence of the jejunal sarcomas is summarised in Table 3.
Relevance of carcinogenic effects / potential:
There was a significantly increased incidence of undifferentiated sarcomas in the jejunum of 400 and 800 mg/kg males and in 800 mg/kg females (22, 40 and 21 %, respectively). There was also a single occurrence of this rare tumour type in females in the 50 and 80 mg/kg groups (equal to 2 %), although this was not statistically significant.
Key result
Dose descriptor:
NOAEL
Remarks:
carcinogenicity
Effect level:
80 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
400 mg/kg diet
System:
gastrointestinal tract
Organ:
jejunum
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified

Table 1: Mortality

 Response  0 mg/kg  50 mg/kg  80 mg/kg  400 mg/kg  800 mg/kg
 Males
 Found dead  15  11  23  22  32
 Sacrificed moribund  9  5  2  10  3
 Accidental death  2  0  0  0  0
 Total  26  16  25  32  35
 Percent adjusted survival  50  68  50  36  30
 Females
 Found dead  13  15  10  14  16
 Sacrificed moribund  17  14  11  14  13
 Accidental death  0  1  0  2  0
 Total  30  30  21  30  29
 Percent adjusted survival  40  41  58  42  42

Table 2: Percent body weight changes (as compared to the controls)

 Weeks  50 mg/kg  80 mg/kg  400 mg/kg  800 mg/kg
 Males            
 At randomisation  1  0  -1  -1
 0  0  -1  -2  -2
 24  0  -1  -5  -17
 52  1  0  -4  -14
 76  1  -1  -5  -13
 103/104  1  -2  -6  -12
 Females            
 At randomisation  1  1  0  1
 0  1  1  0  0
 24  0  1  -4  -16
 52  0  3  -2  -19
 76  2  5  0  -20
 103 -104  2  6  1  -10

Table 3: Percent incidence of jejunal sarcomas

   % incidence of jejunal sarcoma
 Concentration (mg/kg)  Male  Female
 0  0  0
 50  0  2
 80  0  2
 400  22  2
 800  40  21
Conclusions:
Under the conditions of the test, clinical observations, ophthalmic examination and clinical urinalysis showed no findings that could be correlated with exposure to the test material. There was also no treatment-related mortality in females of any exposure group. Although mortality was higher in 400 and 800 mg/kg males in the later weeks of the study, the difference in survival to the other groups was not significant (although there was a significant linear trend). Mean body weight of the 400 and 800 mg/kg males and the 800 mg/kg females was significantly reduced from week 26. Differences from the control ranged from <10-20 %. There was also a corresponding decrease in food consumption. Significant changes in blood chemistry and decreases in organ to body weight ratios during the first year of the study, reflected the poor nutritional status of animals in the 400 and 800 mg/kg groups. Principal findings in the haematology data consisted of significant increases in reticulocyte counts in the 800 mg/kg males and a corresponding decrease in red cell mass.

Histopathology indicated no non-neoplastic changes. However, there was a significantly increased incidence of undifferentiated sarcomas in the jejunum of 400 and 800 mg/kg males and in 800 mg/kg females. There was also a single occurrence of this rare tumour type in females in the 50 and 80 mg/kg groups (equal to 2 %), although this was not statistically significant.

Based on the absence of statistically significant effects at dietary concentrations of up to 80 mg/kg in this study, the NOEL is estimated to be 80 mg/kg diet. This is equivalent to 4 mg/kg bw/day.
Executive summary:

The test material was administered to rats for 104 weeks at doses of 0, 50, 80, 400 and 800 mg/kg diet with 60 animals/sex/group. These dose levels corresponded to an overall estimated test material consumption for males of 2, 4, 19 and 39 mg/kg/day and for females of 3, 5, 24 and 49 mg/kg/day, respectively. An interim sacrifice was performed during week 53. Terminal sacrifice of males began at 103 weeks; terminal sacrifice of females occurred at 104 weeks.

Survival was similar to, or higher than, the control except for 400 and 800 mg/kg males where an increasing number of unscheduled deaths occurred during the later weeks of the study. A positive linear trend for mortality was seen in males although overall mortality rates were not significantly increased. In-life findings included minimal to moderate decreases in body weights observed consistently in 400 mg/kg males and 800 mg/kg males and females while disappearing in 400 mg/kg females over time. Food consumption measurements were also increased in these groups.

Principal haematology findings consisted of significant increases at week 104 in mean percent and absolute reticulocyte counts in 800 mg/kg males accompanied by a nonsignificant decline in red cell mass (erythrocyte count, haemoglobin, haematocrit) and increase in the nucleated red blood cell count. Serum chemistry data showed significant decreases in the total serum protein and calcium values of 400 mg/kg males and 400 and 800 mg/kg males and females at week 26 as well as decreased globulin and increased albumin/globulin ratio values for 800 mg/kg males. Non-significant declines in total protein and globulin persisted in the 800 mg/kg males through week 104. Decreasing tendencies in aspartate aminotransferase and alanine aminotransferase activities were noted for females at weeks 26 and 52 and aspartate aminotransferase activities of the 400 and 800 mg/kg females were significantly decreased at week 52. Other findings related to dose-related decreases in body weight included statistically significant increases in relative organ weight values (organ-to-terminal-body weights) at week 53 for liver in the 400 and 800 mg/kg females and kidney and brain in the 800 mg/kg males and females.

Beginning at week 65, necropsy of unscheduled deaths showed an increasing frequency of 800 mg/kg animals (principally males) with abdominal masses. In most instances the masses were associated with the small intestine (principally the jejunum). By study termination, the 400 and 800 mg/kg males and 800 mg/kg females showed a high frequency of this finding with their overall incidences ranging from 15 to 25 %. Histopathology revealed the presence of undifferentiated sarcomas in the jejunum of 22 and 40 % of the 400 and 800 mg/kg males and in 2, 2, 2 and 21 % of the 50, 80, 400 and 800 mg/kg females, respectively. Frequencies of occurrence for this rare tumour were significantly increased for the 400 mg/kg males and the 800 mg/kg males and females with a significant positive trend for both sexes. Electron microscopic findings suggested that exposure to the test material was related to a proliferation of malignant mesenchymal tumours in various stages of differentiation. There were no other remarkable histopathologic findings in this study.

There were no non-neoplastic microscopic tissue alterations in either males or females at the dose levels tested. Neoplastic effects were limited to the presence of the undifferentiated sarcoma tumour. The occurrence of this type of tumour was significantly increased for 400 mg/kg females and 800 mg/kg males and females. Body weight data suggested that the maximum tolerated dose was exceeded at 800 mg/kg in both sexes based on body weight decreases of 10-20 % beginning early in the study and continuing throughout the study duration. Body weight decreases for males and females were 14 and 19 % after one year on the study, respectively.

Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13th November 1990 to 10th November 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPP 83-2 (Carcinogenicity)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OTS 798.3300 (Carcinogenicity)
Deviations:
no
GLP compliance:
yes
Species:
rat
Strain:
other: Crl: CD
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Kingston, New York, USA
- Age at study initiation: six weeks
- Weight at study initiation: 139-179 g
- Housing: individually in wire-mesh cages
- Diet: Certified Rodent Chow® #5002 ad libitum
- Water: ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 73 ± 1.2 ºF
- Humidity: 52 ± 8.2 %
- Photoperiod: 12 hours light/12 hours dark
Route of administration:
oral: feed
Vehicle:
not specified
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- A premix for each concentration was prepared by mixing an appropriate amount of test material with a small amount of diet in a mixer for 5 minutes. The premix was then blended with an appropriate additional amount of diet in a twin shell blender for 10 minutes
- Storage temperature of food: room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- For determination of homogeneity: ten 50 g test material:diet mixture samples were collected from evenly distributed portions of the test diet while being dispensed from the blender after a blending interval of ten minutes.
- For determination of test material concentration: at the time the test material:diet mixtures were prepared for study weeks 1-4 and every four weeks thereafter (except week 28), duplicate composite samples were collected and analysed for determination of test material concentration.

-Analytical method:
Sample preparation: 50 mL acetonitrile added to 5 g of sample in duplicate, samples were shaken for 60 minutes and then centrifuged at 2000 rpm for 5 minutes. A 4 mL extract was evaporated to dryness and then eluted with 10 % acetone/hexane.
Method: GC with FID detection
Column: 3' x 2 mm ID, glass column packed with 1.5 % OV-17 on gas chrom Q
Temperature: Column - 200 ºC for 12 min, then 35 ºC/minute to a hold (at 250 ºC) for 4 minutes followed by a return to initial temperature and then a hold for 2 minutes; Injection port - 250 ºC; Detector - 280 ºC
Gas flow: N2 30 mL/min, air 300 mL/min, H2 30 mL/min
Calculations: the peak heights of the samples were calculated from the calibration curve computed from the peak height of the standards.
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
Daily
Dose / conc.:
800 mg/kg diet
No. of animals per sex per dose:
Sixty male
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: available data from previous studies
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily for signs of overt toxicity at the same time as the moribundity/mortality checks. Detailed observations on appearance and condition, behaviour and activity, excretory functions, respiration, orifices, eyes and palpable masses at least once weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: pretest, weekly at weeks 1 to 16 and once every two weeks thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: weekly at weeks 1 to 16 and once every two weeks thereafter.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Ten animals from each group were sacrificed at 12 months. All animals received a thorough external examination and the contents of the abdominal, thoracic and cranial cavities were examined in situ and after removal and dissection. The small intestine was opened and examined for gross lesions and masses.
- The following organs were weighed: brain (with stem), kidneys, liver and testis.

HISTOPATHOLOGY: Yes
- The following tissues were collected: adrenals, aorta, bone (femur), bone marrow (femur), bone marrow smear, brain, eye including optic nerve, oesophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, testis with epididymides, heart, kidneys, liver, lung with mainstream bronchi, mediastinal, mesenteric and regional lymph nodes, pancreas, pituitary, prostate and seminal vesicle, salivary glands, sciatic nerve, spinal cord, spleen, thymic region, thyroid/parathyroid, trachea, urinary bladder, tissue masses and gross lesions.
- Representative samples of the duodenum, jejunum and ileum were microscopically examined. Sections were also prepared of gross lesions of the small intestine and all tissue masses.
Statistics:
Body weight, food consumption and absolute/relative organ weight data were analysed by one-way analysis of variance and Bartlett's test for homogeneity of variance. The treatment and control data were compared using an appropriate t-statistic and the significance of difference determined using Dunnett's multiple comparison tables or pairwise comparisons with a Bonferroni correction. Survival and tumour incidence data were analysed using the Kaplan-Meier method.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no treatment related clinical findings, although, at antemortem observation, there was a greater incidence of palpable masses of the abdomen in the treated group.
Mortality:
mortality observed, treatment-related
Description (incidence):
During the first year of the study survival rates in the control and 800 mg/kg groups were very similar. After two years, survival was significantly reduced in the 800 mg/kg treatment group as compared to survival in the control (33/60 deaths vs. 21/60 deaths in the control, representing a 20% reduction in survival at two years). Signs observed in animals prior to death were typical of those associated with morbidity and age (see Table 1 and Figure 1).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Following 104 weeks of exposure, there was a statistically significant reduction in body weight of rats in the 800 mg/kg group (up to 18 % lower than in the control). Most of this weight reduction occurred within the first 6 weeks of exposure.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Following 104 weeks of exposure, there was a statistically significant reduction in body weight of rats in the 800 mg/kg group. There was also a corresponding decrease in food consumption, mostly occurring in the first week of the study. As the study progressed, food consumption rates were more comparable.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the interim sacrifice, there was a statistically significant increase in all organ/body weight ratios in the 800 mg/kg group. At the terminal sacrifice, there were significant increases in the absolute weight of the testis, brain/body ratio, testis/body ratio and testis brain weight ratio; and decreases in the absolute weight of the kidney and kidney/brain weight ratio. These changes were probably due to a decreased body weight of the 800 mg/kg animals.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At the 12 month interim sacrifıce, there were no treatment related macroscopic findings. In animals which died between 12 and 24 months and in those at terminal sacrifıce, there was a high incidence of treatment related thickening/nodes/masses in the jejunum/duodenum/abdomen. Jejunal masses were observed in 19 animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopic examination revealed no treatment related changes at the 12 month interim sacrifice. In the 800 mg/kg animals that died between 12 and 24 months and in those sacrificed at 24 months, all microscopic changes were considered to be unrelated to the treatment.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the 800 mg/kg animals that died between 12 and 24 months and in those sacrificed at 24 months, there was an increased incidence of treatment related undifferentiated sarcomas of the duodenum and jejunum (principally in the latter). One additional undifferentiated sarcoma was found in the abdominal soft tissue, although this was considered to have arisen from the duodenum or jejunum. A further tumour, a mucinous adencarcinoma was seen the jejunum of a single 800 mg/kg animal, although this was considered to be unrelated to the administration of the test article. The presence of all other microscopic changes and/or tumours were considered to be unrelated to the treatment. The incidence of the jejunal/duodenal sarcomas is summarised in Table 2.
Relevance of carcinogenic effects / potential:
In the second year of the study, jejunal/duodenal/abdominal masses, nodes and thickening were observed. Microscopic examination revealed a significantly increased incidence of undifferentiated sarcomas of the duodenum and jejunum, primarily of the latter. It can therefore be concluded that administration of the test material at a dietary concentration of 800 mg/kg is carcinogenic to Crl CD rats.
Key result
Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
800 mg/kg diet
System:
gastrointestinal tract
Organ:
jejunum

Table 1: Summary of survival

 Study week  0 mg/kg  800 mg/kg
 1 to 12  60  60
 13 to 34  60  59
 35 to 52  60  58
 53  60  57
 54 (scheduled sacrifice)  50  47
 55  50  47
 56  49  47
 57 to 66  48  47
 67 to 69  47  47
 70 to 73  47  46
 74 to 75  47  45
 76  47  44
 77  47  44
 78  46  44
 79  46  44
 80  46  44
 81  45  43
 82  45  41
 83  45  40
 84  45  40
 85  44  40
 86  43  40
 87  42  38
 88  41  38
 89  40  37
 90  40  35
 91  40  35
 92  40  35
 93  38  32
 94  37  30
 95  35  27
 96  35  26
 97  35  26
 98  35  24
 99  35  23
 100  33  22
 101  33  22
 102  33  20
 103  32  19
 104  30  17
 105  29  17

Table 2: Percent incidence of jejunal/duodenal sarcomas

   Number of sarcomas observed   
   0 mg/kg  800 mg/kg
 Jejunum  0/49  23/47
 Duodenum  0/50  2/47
 Abdominal soft tissue  0/0  1.1
Conclusions:
Under the conditions of the test, survival was reduced in the second year of the study. Body weight was significantly reduced in the first two months of exposure; thereafter weight gain was comparable to that in the control. There was a corresponding decrease in food consumption. Organ weight changes were primarily linked to decreased body weight.

In the second year of the study, jejunal/duodenal/abdominal masses, nodes and thickening were observed. Microscopic examination revealed a significantly increased incidence of undifferentiated sarcomas of the duodenum and jejunum, primarily of the latter. It can therefore be concluded that administration of the test material at a dietary concentration of 800 mg/kg is carcinogenic to Crl CD rats. This is equivalent to 40 mg/kg bw/day.
Executive summary:

The test material was administered to male rats in the diet at a concentration of 800 mg/kg for two years with an appropriate control group. Each rat was observed twice daily for mortality, moribundity and overt toxicity and further detailed observations were conducted weekly. Individual body weights and food consumption were recorded weekly for weeks 1-16 and once every two weeks thereafter. An interim sacrifice of ten animals from each group was conducted following one year of treatment and after two years of treatment, all surviving animals were sacrificed. A thorough post-mortem was conducted on all animals. A complete set of all major tissues and organs was harvested and selected organs were weighed. Microscopic examination of specific tissues was performed.

Increased mortality was observed in the treated group during the second year of the study. A moderate reduction in body weight was seen in the treated group compared to the controls. Food consumption was reduced in the treated group throughout the study. Large jejunal masses were seen in the treated group during the second year of the study; these masses were diagnosed as undifferentiated sarcomas and resulted in the death of many of the treated animals. Under the conditions of the test, the test material is considered to be carcinogenic to male rats at 800 mg/kg diet.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
3.46 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Two GLP studies with a Klimisch score of 1, five GLP studies with a Klimisch score of 2 and one non-GLP study with a Klimisch score of 2 are available; therefore the quality of the database is high. Study with lowest NOAEL has been selected as key.
System:
gastrointestinal tract
Organ:
jejunum

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 accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does require classification with respect to carcinogenicity as Category 2 (H351: Suspected of causing cancer).

Additional information

In chronic feeding studies, dietary administration of propargite did not lead to the development of tumours in Wistar rats, CD-1 mice or dogs at dietary concentrations of up to 2000, 1000 and 900 mg/kg, respectively; the only treatment-related effects in these species (if any; none on dogs) typically being on body weight and/or food consumption and/or absolute organ weights/relative organ:body weight ratios at these high concentrations. However, in a chronic feeding study with the Sprague-Dawley (CD) rat, there was a significantly increased incidence of undifferentiated sarcomas in the jejunum of males receiving 400 and 800 mg/kg and in females administered 800 mg/kg (22, 40 and 21 %, respectively) (both doses at or above the MTD). There was also a single occurrence of this rare tumour type in 50 and 80 mg/kg females (equal to 2 %), although this was not statistically significant. In a second study with Sprague-Dawley (CD) rats, exposure for two years to a dietary concentration of 800 mg/kg led to a significantly increased incidence of undifferentiated sarcomas of the duodenum and jejunum, primarily of the latter (23/47 800 mg/kg rats compared to 0/49 control rats).

These data indicate that the carcinogenic effect is both species (rats, but not mice or dogs) and strain (Sprague-Dawley CD but not Wistar rats) specific.

In a range of mutagenicity studies, including an in vivo transgenic rat (F344 rats) assay which included jejunal/duodenal, tissue propargite was not genotoxic.

In vivo metabolism studies have shown that, while propargite is more extensively metabolised in mice than rats, the qualitative nature of the metabolites is similar in both species. In a subsequent in vitro comparative ADME study conducted in 2015 in rat (Sprague Dawley and Fisher 344), mouse, dog and human hepatocytes, the most extensive metabolism of [14C]Propargite (based on the lowest percentage of total chromatogram radioactivity in the peak attributed to [14C]Propargite) was observed following 1 h incubation with rat hepatocytes (both Sprague-Dawley and Fischer 344; ca. 4 % [14C]Propargite remaining). The rat appeared to metabolise propargite faster than the mouse, dog or human. There were no major differences observed in the metabolite profiles between the two strains of rat. In vitro, with the exception of the two rat strains, which produced a greater proportion of radioactivity as metabolites with a retention time <15.1 minutes (particularly M2 and M7), the metabolite profiles obtained across the species were qualitatively very similar (particularly at the 1 hour incubation time point). In all species M10 was either the predominant or one of the predominant metabolite fractions formed. Metabolite fraction M2 and M7 were major metabolite fractions in both strains of rat, but not in the other species investigated (mouse, dog and human).

Since the difference in the carcinogenic response between the rat and mouse cannot be explained by genotoxicity, studies were conducted to investigate cell proliferation as a mechanism for the carcinogenicity seen in Sprague-Dawley rats. The in vitro comparative ADME study included hepatocytes from both F344 and Sprague Dawley rats, and shows the strain used in the Big Blue transgenic rat genotoxicity assay (F344 derived) produced a similar metabolic profile to the Sprague Dawley rat in which jejunal tumours were observed. This confirms the relevance of the non-mutagenic result from the Big Blue transgenic rat assay to the Sprague Dawley jejunum.

There was a statistically and biologically significant increase in cell proliferation in jejunal smooth muscle of male and female Sprague-Dawley CD rats following one week exposure to propargite technical, at a dietary concentration of 800 mg/kg. In this study, cell proliferation was not increased in rats and mice receiving dietary concentrations of 40/80 and 1000 mg/kg, respectively. In a second study, a dietary concentration of 400 mg/kg led to a statistically and biologically significant increase in cell proliferation of the jejunum in male and female Sprague-Dawley rats, following one week exposure. Following 20 months exposure of Sprague-Dawley CD rats to a dietary concentration of 800 mg/kg, there was a biologically significant increase in jejunal cell proliferation in males (not females), but this increase was not statistically significant and occurred towards the end of the animal's natural life-span. There was no cell proliferative response following exposure of male rats to 800 mg/kg diet for 4, 8 and 12 months. Administration of a dietary concentration of 900 mg/kg to Wistar rats for one week caused no significant increase in jejunal cell proliferation in either males or females.

The cell proliferative response in Sprague-Dawley rats, correlates well with the formation of undifferentiated jejunal sarcomas seen in this strain/species in long-term feeding studies. This cell proliferative response (and accompanying lack of mutagenicity) is consistent with a carcinogenic substance having a non-genotoxic, mitogenic secondary mode of action. Moreover, the absence of cell proliferation and subsequent formation of jejunal tumours in mice and Wistar rats, indicates that the carcinogenic effect of propargite is highly species and strain specific. Furthermore, the marked cell proliferation observed in Sprague Dawley rats following short-term exposure (1-2 weeks), as compared to the absence of proliferation seen after long-term exposure (4-16 months), indicates that the proliferative response is transitory in nature.

Although the mechanism of the jejunal tumours is still not precisely known, it is now possible to conclude that it is non-genotoxic, and thus highly likely to have a threshold (Slikker et al., 2004). Carcinogenesis is a multistage process which is well documented (e.g. Foulds, 1958; Weinberg, 1989; Navin et al., 2011). Within the multistep model, initiators tend by definition to be genotoxins. Non-genotoxic carcinogens tend to enhance (“promote”) the progression of cells to tumorigenic states, through tumour “promotion”, as opposed to “initiation” (Jones et al., 1996; Lima & Van der Lann, 2000; Hernandez et al., 2009). There is typically a strong correlation between hyperplastic foci induction and tumour incidence which suggest proliferation plays a pivotal role in carcinogenesis (e.g. Horn et al., 1996, Jones et al., 1996; Mally & Chipman). In 1- and 4-week, and 20 month oral mechanistic studies (Goldenthal, 1999; Eldridge, 1994; Goldenthal, 1998) investigating jejunal cell proliferation (site of tumourigenesis in rats) following exposure to propargite, increased proliferation of jejunal cells was observed in CD rats of both sexes (but not in CD-1 mice) when treated at exposure levels used in previous carcinogenicity studies where jenunal tumours were reported (i.e. 800 ppm and 1000 ppm for rat and mouse respectively). Jejunal proliferation was significantly elevated in all studies following one week of exposure to propargite and is good evidence that propargite is likely to have tumour promotor potential (affecting mechanisms relating to cellular proliferation or survivability past their intended life span), rather than acting as a tumour initiator as supported by the overall negative genotoxicity conclusion.

There was an apparent increase in mammary tumours observed in female rats in the 1966 (Ober, 1966) carcinogenicity assay with a lab-specific strain of Wistar rat. If the mammary tumours observed in the 1966 study cannot be discounted on the basis of different tumour classification schemes employed in 1966, or as a result of normal (high) biological variation, it can be considered that they are not-genotoxic in origin (given the genotoxicity data available), and would have a threshold below which they would not occur. Time to onset of the mammary tumours was not decreased, and they were not observed in a second more recent and GLP compliant rat carcinogenicity bioassay in a different strain. In addition their naturally high prevalence in some strains does not make them a sensitive indicator of direct carcinogenic potential. Mammary tumours tend to be extremely common in geriatric rats (fibroadenomas in particular), and are therefore not a reliable indicator of direct carcinogenic potential of a test substance. This is because any increase in mammary tumour incidence only represents an enhancement of the existing physiological processes underlying the already naturally high background incidence. Any increase in incidence is hence unlikely to represent any independent mechanism of tumorigenesis caused directly by the chemical.

More detail consideration is contained in the attached document >>2015-06-12 1405586.UK0 - 2729 Propargite MRL Evaluation Report.doc<<.

On the basis that the observed tumours were threshold related, the lowest NOEL for cell proliferation (female Sprague Dawley rat, one week exposure) and hence for subsequent carcinogenicity was 40 mg/kg diet (2 mg/kg bw/day). Since human dietary exposure will be many orders of magnitude lower than this, it can be concluded that propargite presents a negligible risk to human health under normal use conditions.