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Carcinogenicity

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Description of key information

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, test procedure in accordance with official methods, meets generally accepted scientific principles, acceptable for assessment.
Reason / purpose:
reference to other study
Qualifier:
no guideline available
Guideline:
other: NTP
GLP compliance:
yes
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
Supplier: Charles River Breeding Laboratories under a contract to the Carcinogenesis Program
Details on rats: F344/N, age: 6-7 weeks
Details on mice: B6C3F1 (C57BL/6N, female X C3H/HeN MTV-, male), age: 8-9 weeks
Feed: NIH 07 rat and mouse ration (Zeigle Bros., Gardners, PA) ad libitum
Water: automatic watering system (Edstrom Industries, Waterford, WI)
Cage: polycarbonated (Lab Products, Garfield, NJ)
Housing: 5 per cage
Bedding: beta-chips heat treated harwood chips (Northeastern Products Corp., Warrensburg, NY)
Conditions: Temperature: 21-23 °C; Humidity: 30-50%; fluorescent light 12h/day, 15 room air changes/h

Breeding stock for the foundation colonies at the production facility originated at the National Institutes of Health Repository.
Animals shipped for study were progeny of defined microflora-associated parents that were transferred from isolators to barrier maintained rooms.Rats were shipped to the study laboratory at 4-5 weeks of age and mice, at 5-6 weeks of age.
The rats were quarantined at the study facility for 2 weeks and the mice, for 3 weeks. Thereafter, a complete necropsy was performed on five animals of each sex and species to assess their health status.
The rats were placed on study at 6-7 weeks of age and the mice, at 8-9 weeks of age. The health of the animals was monitored during the course of the studies according to the protocols of the NTP Sentinel Animal Program (Appendix F).
Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
corn oil
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
2 year (103 weeks)
Frequency of treatment:
5 days a week
Post exposure period:
no data
Remarks:
Doses / Concentrations:
0
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
375 mg/kg bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
750 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
50 males and 50 females
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
Body weights: Mean body weights of high dose male rats were 5%-10% lower than those of vehicle controls after week 25 and 11%-16% lower after week 52 (Table 10 and Figure 3). Mean body weights of high dose female rats were 5%-10% lower than those of vehicle controls after week 47 and 11%-19% lower after week 70.
Survival: The survival of the high dose group of both males (after week 33) and females (after week 14) was significantly lower than that of the vehicle controls. By week 97, all of the males receiving 750 mg/kg had died.
Sacrifice and pathology:
Incidences of rats with neoplastic or nonneoplastic lesions of the kidney, glandular stomach, bone, circulatory system, parathyroids, colon, cecum, and testis.
Necropsy and histologic examination performed on all animals; the following tissues were examined:
adrenal glands, aorta, brain, cecum, colon, costochondral junction, duodenum, esophagus, eyes, femur including marrow, gallbladder (mice), gross
lesions, heart, ileum, jejunum, kidneys, larynx including oral cavity, liver, lungs and bronchi, mammary gland, mandibular and mesenteric lymph nodes,nasal cavity and turbinates, pancreas, parathyroids, pituitary gland, preputial or clitoral gland (after 6/1182 l, prostate/testes/seminal vesicles/epidiymis/tunica vaginalis/ scrotal sac or ovaries/uterus, rectum, salivary glands, sciatic nerve, skin, spinal cord, spleen, stomach, thigh muscle, thymus, thyroid gland, tissue masses, trachea, urinary bladder, and Zymbal gland (after 6/1/82)
Statistics:
Analysis of Tumor Incidence: Three statistical methods are used to analyze tumor incidence data: life table tests, incidental tumor analysis, and Fisher exact/Cochran-Armitage trend analyses. Tests of significance include pairwise comparisons of high dose and low dose groups with vehicle controls and tests for overall doseresponse trends. For studies in which administration of the study compound has little effect on survival, the results of the three alternative analyses will generally be similar. When differing results are obtained by the three methods, the final interpretation of the data will depend on the extent to which the tumor under consideration is regarded as being the cause of death. Continuity-corrected tests are used in the analysis of tumor incidence, and reported P values are one-sided. The procedures described below also were used to evaluate selected nonneoplastic lesions.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Mortality:
mortality observed, treatment-related
Description (incidence):
see details on results
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Histopathological findings: neoplastic:
no effects observed
Details on results:
Pathology:
Kidney: The administration of C.l. Acid Orange 3 was associated with a spectrum of nonneoplastic lesions in male and female rats (Table 12). These included an increased incidence and/or severity of nephropathy, hyperplasia of the pelvic epithelium, papillary necrosis, inflammation, and pigmentation.
Six transitional cell carcinomas were observed in female rats given 750 mg/kg C.I. Acid Orange 3. These neoplasms originated from the transitional epithelium of the renal pelvis and exhibited cellular atypia and local invasion of the submucosa. The incidence of this rare tumor in the high dose group was significantly greater than that in the vehicle control group. Renal transitional cell carcinomas have not been observed in 1,697 historical corn oil vehicle control female F344/N rats. No renal neoplasms were observed in dosed male rats; however, one vehicle control male was found to have a tubular cell adenocarcinoma.

Glandular Stomach, Bone, Circulatory System, and Parathyroids: Mineralization, erosion of the epithelium, and ulcers occurred in the glandular stomach of some dosed rats (Table 13). Mineralization of the aorta also occurred in some dosed rats. These lesions are probably related to uremia caused by kidney failure. Parathyroid hyperplasia (to a degree evident by light microscopic examination) was increased in high dose male rats. Fibrous dysplasia of bones was also increased in high dose male and female rats and is considered to be secondary to the renal disease and parathyroid hyperplasia (renal secondary hyperparathyroidism).

Colon and Cecum: Chronic and suppurative inflammation of the colon and cecum were observed in a number of dosed male and female rats. Whether these changes are directly related
to the administration of C.I. Acid Orange 3 or are related to uremia from kidney failure is uncertain.

Testis: Interstitial cell hyperplasia was observed at increased incidences in dosed male rats (vehicle control, 1/50; low dose, 8/50; high dose, 10/48; P<0.05); the incidences of interstitial cell tumors in the dosed groups were significantly lower than that in the vehicle controls (47/50; 34/50; 22/48; P < 0.02). Interstitial cell hyperplasia and neoplasia represent a morphologic continuum, and at the end of 2 years, nearly all male F344/N rats are expected to have interstitial cell tumors. Male rats dying early would be expected to have a greater incidence of hyperplasia and a lower incidence of tumors than those surviving for 2 years.
Relevance of carcinogenic effects / potential:
Under the conditions of these 2year gavage studies, there was no evidence of carcinogenic activity of C.I. Acid Orange 3 for male F344/N rats administered 375 mg/kg; because of a marked reduction in survival and no indication of carcinogenicity, the 750 mg/kg group was considered to be inadequate for assessment of carcinogenic activity.
There was clear evidence of carcinogenic activity of C. I. Acid Orange 3 for female F344/N rats as shown by the occurrence of transitional cell carcinomas of the kidney in the 750 mg/kg group; this group had reduced survival and chemically related non-neoplastic lesions of the kidney.
Dose descriptor:
NOAEL
Effect level:
> 375 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Effect type: toxicity (migrated information)

In the 2-year studies, the administration of 750 mg/kg C.I. Acid Orange 3 reduced the survival of male and female rats.

The primary cause of death in both species was the spectrum of nonneoplastic lesions in the kidney. These included nephropathy, hyperplasia of the pelvic epithelium, papillary necrosis, inflammation, and pigmentation.

Nephropathy is an agerelated disease process characterized by varied degrees of degeneration, regeneration, and atrophy of the tubular epithelium; hyaline tubular casts; glomerulosclerosis: and interstitial fibrosis. Nephropathy was present in nearly all male rats of each group, but the severity of this lesion was judged to be greater in dosed animals. The incidence and severity of nephropathy were also increased in dosed female rats. Hyperplasia of the transitional epithelium overlying the renal papilla frequently accompanies severe nephropathy, and the increased incidences in dosed rats may reflect the enhanced nephropathy.

Suppurative inflammation consisting of focal aggregates ofneutrophils within the lumens of tubules in the papilla, medulla, and cortex also occurred at increased incidences in dosed rats. Necrosis of the renal papilla occurred primarily in eight high dose females that died between weeks 11 and 17 of the study. A single high dose female died with papillary necrosis during week

97. Two dosed males also had papillary necrosis.

No evidence of renal vascular change was observed in animals dosed with C.I. Acid Orange 3, but such changes could have been masked by the extensive pathologic renal effects produced by the dye.

An increased incidence of orange-brown pigment located within the epithelium of cortical tubules and interstitial macrophages was observed in high dose male rats and, to a much lesser extent, in female rats. The amount of accumulated pigment was minimal and may represent hemosiderin and/or C.I. Acid Orange 3 or a metabolite

A spectrum of nonneoplastic lesions characteristic of uremia and renal secondary hyperparathyroidism occurred in male and female rats.

These lesions in dosed rats reflect the increased severity of nephropathy associated with the administration of C.I. Acid Orange 3. Mineralization of the glandular stomach and aorta and erosion and ulcers of the glandular stomach are frequently associated with uremia. Fibrous dysplasia (osteodystrophy and osteitis fibrosa cystica) reflects profound disturbances in divalent ion metabolism. The pathophysiology of this metabolic bone disease is complex. The severe renal disease results in phosphate retention and abnormal vitamin D metabolism wherein formation of the active 1,25-dihydroxy metabolite of vitamin D is diminished. These factors reduce plasma calcium and cause increased secretion of parathyroid hormone and eventually parathyroid hyperplasia. The parathyroid hormone mobilizes calcium from the bone and increases urinary phosphate excretion to return the plasma concentrations of calcium and phosphate to normal. As the kidney loses the ability to compensate and respond to parathyroid hormone, bone becomes more resistant to the effects of parathyroid hormone and the absorption of calcium from the intestine is reduced by the impaired synthesis of 1,25-dihydroxycholecalciferol. This leads to reduced calcification of bone and excessive production of fibrous connective tissue in bone. Mineralization of soft tissues occurs because the high levels of plasma phosphate and the calcium mobilized from bone upset the normal plasma calcium/phosphate ratio.

Parathyroid hyperplasia was diagnosed in eight high dose male rats and one high dose female rat. Although there was no strong correlation between parathyroid hyperplasia and fibrous dysplasia of the femur and mineralization of the glandular stomach and aorta in rats, these latter lesions are considered to be due to hyperparathyroidism secondary to renal disease.

Despite the reduced survival of high dose male rats, the low dose male rat group is considered to be adequate for a long-term study of carcinogenicity because survival in the 375 mg/kg dose group (30/50) was similar to that in vehicle controls (36/50) Deaths in the 750 mg/kg group were chemically related and were probably caused by adverse effects on the kidney. The 375 mg/kg dose, at which notable kidney toxicity was produced, is considered to be the largest dose that could be administered under the conditions ofthese studies.

The administration of C.I. Acid Orange 3 at the high dose produced an increase in the incidence of transitional cell carcinomas of the renal pelvis in female rats (see Table 12). The carcinomas originated from the transitional epithelium of the renal pelvis and exhibited cellular atypia and local invasion of the. submucosa. Renal transitional cell carcinomas have not been previously observed in approximately 1, 700 historical corn oil vehicle control F344/N female rats. In addition, there was a dose-related increase in the incidence of e{lithelial hyperplasia of the renal pelvis. Although epithelial hyperplasia of the renal pelvis was increased in dosed male rats, no kidney neoplasms were found. Perhaps this is a reflection of the reduced survival in high dose males. The first pelvic transitional cell carcinoma was detected in a female rat that died during week 87. Only nine males in the high dose group were alive during week 87, and all were dead by week 97.

Increased incidences oftesticular interstitial cell hyperplasia and concomitant decreases in the incidences of interstitial cell tumors were seen in dosed male rats. These trends are consistent with the reduced survival pattern of the dosed male rats.

Conclusions:
Non-neoplastic lesions of the kidney were observed in both dose groups of both sexes of rats.
Executive summary:

Non-neoplastic lesions of the kidney were observed in both dose groups of both sexes of rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
375 mg/kg bw/day
Study duration:
chronic
Species:
rat

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The results of the test on mice and rats showed non-neoplastic lesions of the kidney in both dose groups of both sexes of rats and mice.

An evaluation between the members of ETAD association, confirmed that there is no evidence to classify Acid Orange 3 as carcinogenic.