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Carcinogenicity

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Description of key information

The carcinogenicity of benoxacor has been evaluated in guideline compliant GLP studies in both the rat and mouse. These studies indicated that prolonged oral exposure to the material at dose levels that produce systemic toxicity caused an increased incidence of forestomach tumours in rats and mice. The rodent forestomach has no human equivalent and tumours confined only to this tissue are of questionable human significance. There is insufficient evidence to conclude that benoxacor is likely to be a human carcinogen.

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:
15 February 1990 - 3 March 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF 4200
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPP 83-5 (Combined Chronic Toxicity / Carcinogenicity)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Species:
rat
Strain:
other: Crl:CD (SD) BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, Michigan, USA
- Age at study initiation: approximately 6-7 weeks
- Weight at study initiation: mean weight: 124 g (males), 114 g (females)
- Fasting period before study: none
- Housing: 5 per sex per cage in suspended cages with wire mesh floors
- Diet: SDS Rat and Mouse No.1 modified maintenance diet (powdered) ad libitum, except overnight prior to blood and urine collection
- Water: tap water ad libitum, except overnight prior to urine collection
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature 21±2°C
- Humidity: 55±10%
- Air changes (per hr): not reported
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 15 February 1991 To: 3 March 1992
Route of administration:
oral: feed
Vehicle:
other: diet
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): a pre-mix was prepared by grinding the test substance directly into SDS Rat and Mouse No.1 modified maintenance diet. The required concentrations were then prepared by direct dilution of the pre-mix with untreated diet. Homogeneity was achieved by mixing for a minimum of 7 minutes in a double cone blender.
- Storage temperature of food: room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the start of treatment, the proposed diet mixing procedures were checked by chemical analysis of diet trials to confirm homogeneity, concentration and stability. During the study, analysis was performed at approximately 6-week intervals. Only diets with achieved concentrations within ±10% of nominal levels were used in the study, with the exception of the 10 ppm dose level at week 57 that was +11% of nominal.
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
Continuous
Post exposure period:
not applicable
Remarks:
Doses / Concentrations:
0, 10, 50, 500 and 1000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 0.4, 2.0, 20.6 and 41.0 mg/kg/day (males); 0, 0.6, 2.8, 28.2 and 59.0 mg/kg/day (females)
Basis:
actual ingested
No. of animals per sex per dose:
70 (20 rats/sex/group allocated to the satellite groups of which 10 sex/group were killed after 52 weeks of treatment)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: based on the results of a 13 week toxicity study
- Rationale for selecting satellite groups: the satellite groups (20 rats/sex/group) were intended for blood sampling at intervals and 10 rats/sex/group were used for interim kill investigations after 52 weeks of treatment
- Post-exposure recovery period in satellite groups: not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily for any signs of behavioural changes, reaction to treatment or ill health

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly. A detailed palpation noted the appearance, location and dimension, of all new palpable masses

BODY WEIGHT: Yes
- Time schedule for examinations: at allocation to the study, on the day of commencement of treatment and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined weekly and food intake per rat (g food/rat/week): Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, weekly

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: Yes
- Time schedule for examinations: monitored daily by visual appraisal of the water bottles. Water consumption was measured, by weight, over daily periods during weeks 12, 25, 51 and 103 for all cages of satellite group animals

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to treatment, at 52 weeks and at study termination
- Dose groups that were examined: all animals. Prior to examination, the pupils of all animals were dilated using a Tropicamide ophthalmic solution.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during weeks 13, 26, 52, 78 and 104 weeks
- Anaesthetic used for blood collection: Yes (light ether anaesthesia)
- Animals fasted: Yes
- How many animals: 10 males and 10 females per satellite group
- Parameters checked: packed cell volume, haemoglobin, red cell count, mean corpuscular volume, mean cell haemoglobin concentration, total white cell count, platelet count, thrombotest, reticulocyte count, differential WBC count, cell morphology (if abnormal cells were observed on any stained slide). Venous blood smears were prepared from all rats killed during the study; these slides were fixed and stained but not examined.
- An additional (repeat) investigation of haematological parameters was performed in week 30, due to a number of samples collected in week 26 being clotted.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during weeks 13, 26, 52, 78 and 104 weeks
- Animals fasted: Yes
- How many animals: 10 males and 10 females per satellite group
- Parameters checked: total protein, albumin, globulin, urea nitrogen, creatinine, sodium, potassium, calcium, inorganic phosphorus, chloride, total cholesterol, alkaline phosphatase, total bilirubin, glucose, creatine phosphokinase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase and uric acid.

URINALYSIS: Yes
- Time schedule for collection of urine: during weeks 13, 26, 52, 78 and 104 weeks
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: appearance, colour, volume, pH, specific gravity, protein, total reducing substances, glucose, ketones, bile pigments, urobilinogen, haem pigments. Sediment was examined microscopically for the presence of epithelial cells, polymorphonuclear leucocytes, mononuclear leucocytes, erythrocytes, organisms, renal tubule casts, sperm and any abnormal constituents.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; 10 rats/sex in the satellite groups were killed for interim study. Remaining animals in the Satellite groups were retained for laboratory investigations in week 78 and at termination. After 104 weeks of treatment, all surviving rats were killed.
- Animals killed by carbon dioxide asphyxiation
- Tissues/organs examined: all superficial tissues including the urogenital orifices and tail, each pinna, eye and external auditory meatus. The external nares, buccal cavity and tongue, brain, pituitary gland and cranial nerves; all subcutaneous tissues including regional lymph nodes, mammary and thyroid/parathyroid glands; thoracic viscera particularly thymus, lymph nodes and heart. Stomach and caeum and sections of duodenum, jejunum, ileum, colon and oesophagus were incised and examined. Pleural surfaces of the lungs were examined. Liver and kidneys were incised and examined. Any abnormalities in the appearance and/or size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded. Any lesion suggestive of neoplasia was noted.

ORGAN WEIGHTS: Yes
- The following organs were weighed: adrenals, brain, heart, kidneys, liver, ovaries, pituitary, spleen, testes and thyroid

HISTOPATHOLOGY: Yes
- Any abnormal tissue, adrenals, alimentary tract (oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon and rectum, aorta, brain (medullary, cerebellar and cortical sections), eyes, femur (with joint), heart, kidneys, liver, lungs (all lobes and mainstem bronchi), lymph nodes (cervical and mesenteric), mammary gland, ovaries, pancreas, pituitary, prostate, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal column (cervical, thoracic and lumbar levels), spleen, sternum (for bone and marrow), testes with epididymides, thymus, thyroid with parathyroid, tongue, trachea, urinary bladder, uterus (corpus and cervix) and vagina.
Statistics:
All analyses were carried out separately for males and females. Data relating to food and water consumption were analysed on a cage basis; other parameters used the individual animal as the basic experimental unit. The following statistical tests were used as appropriate for food and water consumption, bodyweight, organ weight and clinical pathology data: Bartlett's test; the Kruskal-Wallis analysis of ranks; analyses of variance using Student's t-test and the William's test; Shirley's test. Where appropriate, analysis of covariance was used in place of analysis of variance.
Mortality was analysed using Logrank methods. The incidence of tumours was analysed by Fisher's exact test and if necessary according to Peto et al.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
BODY WEIGHT AND WEIGHT GAIN
- Bodyweight gain was reduced in both sexes at 500 and 1000 ppm compared to control animals. This was dose-related, statistically significant and had been evident throughout the treatment period. After week 52, males at 50 ppm also showed reduced bodyweight gain. Overall weight gain was reduced by 5% and 14% for males and females at 500 ppm and 11% and 23% for males and females at 1000 ppm. There were no effects of treatment on weight gain at 10 ppm.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- There was a marginal reduction in cumulative food intake for animals fed 500 and 1000 ppm, statistically significant in males. Food consumption at 10 and 50 ppm was unaffected by treatment.

FOOD EFFICIENCY
- Marginally reduced overall at 500 and 1000 ppm, although consistent trends in calculated food consumption ratios were not apparent on a weekly basis. Food utilisation at 10 and 50 ppm was unaffected by treatment.

ORGAN WEIGHTS
- Marginally increased liver weight at week 52 in males at 1000 ppm. No intergroup differences in organ weights were evident at week 104.

GROSS PATHOLOGY
- At 52 weeks, an increased incidence of reduced adipose tissue was seen in males (3/10) and females (1/10) treated with 1000 ppm and males only treated at 500 ppm (2/10). There were no incidences in the control animals at the interim kill. A similar finding was observed in a slightly greater number of 1000 ppm treated rats at week 104 compared to control animals.
- In animals killed in extremis, at interim or terminal kill, there was an increased incidence of excrescence/s observed in the forestomach of animals fed 500 or 1000 ppm compared to control.
- Raised area/s of the forestomach were seen in greater number in 1000 ppm treated rats.
- A nodular appearance of the limiting ridge was observed on 1/34 male rats fed 500 ppm at termination and 3/35 male rats fed 1000 ppm, compared to 0/29 control group male rats.

MICROSCOPIC PATHOLOGY
- Increased incidences of hyperplastic lesions of the non-glandular epithelium of the stomach was increased in both sexes at 1000 ppm and in females at 50 or 500 ppm.
- Increased centrilobular hepatocyte enlargement and centrilobular enlargement with vacuolation of hepatocytes were seen in males fed 50, 500 and 1000 ppm. An increased incidence of ballooned hepatocytes was seen in males at 500 and 1000 ppm.
- Some age-related findings were increased in rats at 1000 ppm eg in the heart and lungs (males), and ovaries (females).
- There was a treatment-related increased incidence of squamous cell tumours in the stomach (non-glandular region), in both sexes at 1000 ppm 4/60 males and 5/60 females compared to 0/60 males and 1/60 females in the control group). The incidence did not acheive statistical significance but a positive trend was evident in the Peto test.
- There was a slight increase in the incidence of benign liver tumours in rats at 1000 ppm (0, 0, 2, 2 and 2 for males and 0, 2, 0, 2 and 3 in females for 0, 10, 50, 500 and 1000 ppm groups respectively). The findings was not statistically significant, showed no positive trend and was within the laboratory historical control range. It was therefore considered to be of no toxicological significance.

ADDITIONAL HISTOPATHOLOGY ON INTERIM KILL ANIMALS: The only additional findings detected in the sections of stomach examined were from two male rats in the 500 ppm group, as follows:
one rat; stomach (non-glandular) - ulcer with associated hyperplasia and hyperkeratosis
one rat; stomach (non-glandular) - minimal hyperkeratosis (limiting ridge)
All other stomach sections examined showed no abnormalities.
These additional findings were considered to be of uncertain relationship to treatment and the overall conclusion and outcome of the study remains unaltered.


Relevance of carcinogenic effects / potential:
There was an increased incidence of squamous cell papillomas at 1000 ppm. This increase did not achieve statistical significance.
Dose descriptor:
NOEL
Effect level:
500 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: Increased incidence of forestomach squamous cell papillomas at 1000 ppm
Remarks on result:
other: Effect type: carcinogenicity (migrated information)

Intergroup comparison of mean bodyweight gain (g/rat)

 

Dietary Concentration of CGA154281 (ppm)

Males

Females

 Weeks

0

10

50

500

1000

0

10

50

500

1000

0-13

359

362

356

324**

317**

120

120

120

109**

100**

0-26

454

461

452

405**

399**

154

156

155

140

128

0-51

597

607

590

543**

524**

230

236

232

208**

188**

1-104

672

687

648

636

598*

354

340

325

306*

274**

* Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided) (Williams’ test)

 

Intergroup comparison of mean food consumption (g/rat)

 

Dietary Concentration of CGA154281 (ppm)

Males

Females

Week

0

10

50

500

1000

0

10

50

500

1000

1-13

2495

2542

2516

2363**

2302**

1681

1712

1693

1580**

1583**

1-26

4890

4934

4904

4627**

4520**

3291

3378

3332

3118**

3112**

1-52

9718

9791

9752

9270*

9051**

6616

6866

6724

6367**

6314**

1-104

19977

19865

19667

19043*

18430**

14044

14718

14136

13590

13560

* Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided) (Williams’ test)

 

Group mean food utilisation for the first 26 weeks of treatment

 

Dietary Concentration of CGA154281 (ppm)

Males

Females

Week

0

10

50

500

1000

0

10

50

500

1000

1-25

10.4

10.3

10.5

11.0

11.0

20.6

20.7

20.5

21.5

23.4

 

Mean Dose Received (mg/kg/day), weeks 1 -104

CGA154281 (ppm)

10

50

500

1000

Males

0.4

2.0

20.6

41.0

Females

0.6

2.8

28.2

59.0

 

Intergroup comparison of liver weight (g), adjusted for bodyweight, week 52

Dietary Concentration of CGA154281 (ppm)

Males

Females

0

10

50

500

1000

0

10

50

500

1000

28.1

26.7

30.2

31.7

32.5*

14.3

13.0

12.8

14.8

14.7

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided) (Williams’ test)

 

Conclusions:
Dietary exposure of rats to benoxacor at up to 1000 ppm for 2 years produced no statistically or toxicologically significant changes in the incidence of neoplastic findings. There was a slightly increased incidence of forestomach squamous cell papillomas at 1000 ppm. The NOEL for carcinogenicity was 500 ppm (20.6 and 28.2 mg/kg/day for males and females respectively).
Executive summary:

Groups of 70 male and 70 female Crl:CD (SD) BR rats were fed diet containing 0 (control), 10, 50, 500 or 1000 ppm benoxacor. Mortality, clinical appearance and behaviour, body weights, body weight changes, food consumption, ophthalmology, haematology, blood and urine biochemistry, organ weights, gross pathology and histopathology were assessed. Ten animals per group per sex were killed after 52 weeks of treatment for interim examination.

At 1000 ppm there was a slightly inreased incidence of benign squamous cell papillomas in the forestomach of both male and female rats which did not achieve statistical significance.

Based on these findings, the NOEL for carcinogenicity was 500 ppm benoxacor (20.6 and 28.2 mg/kg/day for males and females respectively).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
20.6 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Two reliable, chronic carcinogenicity studies in rats and mice are available.

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Two key studies have been identified: a 2-year combined chronic toxicity and carcinogenicity study in the rat (Ryle et al, 1993, 1994) and an 80-week study in the mouse (Ryle et al, 1993).

Rats were fed diet containing 0 (control), 10, 50, 500 or 1000 ppm benoxacor (0, 0.4, 2.0, 20.6 and 41.0 mg/kg/day in males; 0, 0.6, 2.8, 28.2 and 59.0 mg/kg/day in females) for up to 104 weeks. Dose related reductions in body weight gain and food consumption were seen at 1000, 500 and 50 ppm. There was a slightly increased incidence of forestomach (non-glandular) squamous cell papillomas at 1000 ppm. The NOEL for carcinogenicity was 500 ppm (20.6 and 28.2 mg/kg/day for males and females respectively).

In the mouse treatment-related neoplastic and non-neoplastic findings were seen in the non-glandular stomach following dietary administration of 0 (control), 10, 30, 600 or 1200 ppm benoxacor for up to 80 weeks. There was a statistically significantly increased incidence of squamous cell tumours (papillomas and carcinomas) and papillomatous hyperplasia in both sexes at the 600 and 1200 ppm treatment levels. The incidence of malignant tumours was low (maximum of 6% in males at 1200 ppm) and there was no evidence of a treatment-related effect on the incidence of any other tumour type, nor on the total number of tumour bearing mice per group when tumours of the non-glandular stomach were excluded. Benoxacor has been examined in a number of genotoxicity assays both in vitro and in vivo and found to be non-genotoxic. Any tumours seen in these long-term studies are therefore considered to have arisen through a non-genotoxic mechanism. Tumours were only seen at systemically toxic dose levels and were accompanied by hyperplastic changes in the forestomach epithelium, consistent with a proliferative response to irritation/cytotoxicity. Induction was dependent on prolonged high dose administration of the test material. It is also important to note that the forestomach, primarily a storage organ where food can be held for many hours, is not present in humans. An increase in tumours in this organ alone is of questionable relevance to humans.


Justification for selection of carcinogenicity via oral route endpoint:
Reliable, GLP- and guideline-conform study on the preferred rodent species demonstrating an induction of tumour formation by a non-mutagenic mechanism and at doses exhibiting systemic toxicity. In addition, tumours were only found in the forestomach of treated animals.

Justification for classification or non-classification

The available studies are considered adequate and reliable for the purposes of classification and labelling.

Although benoxacor produced an increased incidence of forestomach tumours in rats and mice following prolonged oral exposures these are considered to be as a consequence of a non-genotoxic mechanism and generally occurred at dose levels that also produced systemic toxicity. There was no evidence for tumours in other tissues, malignant tumours were only seen at low incidence in the mouse and there is evidence of a threshold for tumour induction. As humans have no equivalent to the rodent forestomach this tumour type is of questionable, if any, significance and it is proposed that no classification for carcinogenicity is warranted.