3,7-dinitroso-1,3,5,7-tetraazabicyclo[3.3.1]nonane

Carcinogenicity

Description of key information

REACH_not carcinogenic up to 9 mg/animal/d | rat (male/female) | 1y study | Hadidian et al.

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:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

1-year chronic toxicity / carcinogenicity study

GLP compliance:

not specified

Species:

rat

Strain:

other: Fischer

Details on species / strain selection:

Fischer rats are reasonably small; the average weight of the males was no more than 436 g, that of the females 326 g. Another advantage is that they are sensitive to toxic materials but do not respond excessively to carcinogenic stresses, thus avoiding the problem of false positives.

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals were Fischer strain weanling rats, obtained from the Charles River Breeding Laboratories, Wilmington, Mass. After 1 week in a holding facility, they were placed on
experiment. Groups of 2 or 3 rats of the same sex were housed in galvanized cages with screen bottoms, in quarters with controlled temperature and humidity. They had free access to Purina Laboratory Chow and water.

Route of administration:

oral: gavage

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Part 1 (toxicity): 8 w
Part 2 (MTD): 8 w
Part 3 (Main Study): 52 w

Frequency of treatment:

5 times per week

Post exposure period:

6 m

Dose / conc.:

300 other: mg/animal/d

Remarks:

PART 1 (toxicity test)

Dose / conc.:

100 other: mg/animal/d

Remarks:

PART 1 (toxicity test)

Dose / conc.:

30 other: mg/animal/d

Remarks:

PART 1 (toxicity test)

Dose / conc.:

10 other: mg/animal/d

Remarks:

PART 1 (toxicity test)

Dose / conc.:

9 other: mg/animal/d

Remarks:

MAIN STUDY
ca. 22 mg/kg/d males and 31 mg/kg/d females based on final body weight

Dose / conc.:

3 other: mg/animal/d

Remarks:

MAIN STUDY (1/3 MTD)
ca. 7 mg/kg/d males and 10 mg/kg/d females based on final body weight

Dose / conc.:

0.03 other: mg/animal/d

Remarks:

MAIN STUDY
ca. 0.07 mg/kg/d males and 0.1 mg/kg/d females based on final body weight

No. of animals per sex per dose:

MAIN STUDY
- 3 in the dose group of 9 mg/animal/d
- 15 in the 1/3 MTD dose of 3 mg/animal/d
- 12 in the dose group of 0.03 mg/animal/d

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Positive control:

2 mg per day of the versatile carcinogen N-2-fluorenylacetamide (12 males/females)

Observations and examinations performed and frequency:

Animals were examined 5 times per week during the gavaging process, and any abnormality was carefully recorded.
Body weight was measured every other week.

Sacrifice and pathology:

Where the condition of an animal was such that survival seemed unlikely, the rat was killed and dissected. All other animals were killed at the end of the planned period.
A sample of 3 males and 3 females was killed after 1 year, the end of compound administration. The main group was necropsied 6 months later. The terminal body weight and the weights of the liver, kidneys, spleen, adrenal glands, and the pituitary were obtained. These tissues and in addition lungs, gastrointestinal tract (esophagus, stomach, and intestines), bladder, gonads, thyroids, mammary glands, and tissues with lesions were fixed in 10% formalin. Grossly abnormal tissues were examined microscopically. In addition, histologic sections of a random sample of 25% of grossly normal tissues were read. If any additional abnormalities turned up in this survey, the remaining tissues of the appropriate groups were also studied. Sections were prepared and routinely stained with hematoxylin and eosin.

Clinical signs:

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

PART 1 (toxicity test):
300 mg/animal/d 3/3; 100 mg/animal/d 3/3; 30 mg/animal/d 3/3; 10 mg/animal/d 0/3

MAIN STUDY:
Average survival period for the complete study was 550 days. Mean survival period [d] in detail for all dose groups was:
- 9 mg/animal/d: males 556, females 553
- 3 mg/animal/d: males 524, females 509
- 0.03 mg/animal/d: males 556, females 557
One female on the 0.03 mg level, dead after 492 days, showed a squamous cell carcinoma of the tongue obstructing the oral cavity.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- 9 mg/animal/d: Initial mean bw males 55g, females 53g; final mean bw males 405g, females 289g
- 3 mg/animal/d: Initial mean bw males 60g, females 53g; final mean bw males 422g, females 309g
- 0.03 mg/animal/d: Initial mean bw males 65g, females 52g; final mean bw males 412g, females 300g

Food efficiency:

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Mean liver weights [g]:
- 9 mg/animal/d: males 14 (12-17), females 12 (9-15)
- 3 mg/animal/d: males 17 (14-20), females 12 (7-14)
- 0.03 mg/animal/d: males 17 (14-20), females 11 (9-13)

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- 9 mg/animal/d (3 animals per sex): no findings
- 3 mg/animal/d (15 animals per sex): Males no findings; Females 5 polyp uterus, (spontaneous occurring tumors), 1 hepatotoxic
- 0.03 mg/animal/d (12 animals per sex): Males no findings; Females: 2 polyp uterus, (spontaneous occurring tumors)

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- 9 mg/animal/d (3 animals per sex): Males: 1 interstitial cell tumor testes, 1 cyst adenoma lung; Females no findings
- 3 mg/animal/d (15 animals per sex): Males: 6 interstitial cell tumor testes (typical aged male Fischer rats); Females: 1 mammary fibrodadenoma, 1 mammary adenomasarcoma, 1 sarcoma
- 0.03 mg/animal/d (12 animals per sex): Males: 2 interstitial cell tumor testes, 1 cystadenoma lung; Females: 1 squamous cell carcinoma tongue

Other effects:

not specified

Relevance of carcinogenic effects / potential:

No significant increase of carcinogenic effects compared to controls.

Key result

Dose descriptor:

NOAEL

Effect level:

9 other: mg/animal/d

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

histopathology: non-neoplastic

Remarks on result:

other: no significant increase of carcinogenic effects

Key result

Critical effects observed:

no

Negative and Solvent Controls

Relatively few lesions were seen in younger animals. None of the relatively large number of male and female Fischer strain rats in these control groups bore hepatomas or other liver lesions in a period of over 600 days. Mammary adenocarcinomas were comparatively infrequent, affecting only 6 of a total of 160 rats autopsied between 531 and 600 days. Fibroadenoma likewise was found mostly in old females. There were a number of other tumors in a variety of sites,

chiefly in the endocrine glands or in the respiratory system, and some lymphomas.

Both vehicle and untreated control male rats exhibited a progressively increasing proportion of interstitial cell tumors of the testis. This lesion was diagnosed in almost all the males autopsied at 600 days. Within the designated experimental period of approximately 600 days, the Fischer strain rats had a fairly low spontaneous tumor rate, except for the interstitial cell tumor, which is typical for aged male Fischer rats.

Mean Bodyweight (survival period 531 - 599 d):

Males initial bw 62 +/- 4.9; final bw 419 +/- 33.4

Females inital bw 54 +/- 4.2; final bw 323 +/- 25.5

Liver weights (survival period 531 - 599 d):

Males 16.0 +/- 1.3

Females 12.3 +/- 0.97

Noncancerous Lesions

Among the noncancerous lesions in female rats of the Fischer strain, there was a fair incidence of uterine polyps, hyperplastic mammary glands, and endometrial hyperplasia. Small numbers of males had testicular atrophy and some showed interstitial tissue hyperplasia. In this extensive study involving almost 6,000 rats, a relatively low incidence (about 4%) of pulmonary disease was seen over the 18-month experimental period, testifying to the value of the newer cesarianderived,

barrier-bred rats used in the tests and also to the careful management of the animal facilities.

Positive Control (N-2 -Fluorenylacetamide)

In the positive control series encompassing 6 groups of 12 male and 12 female Fischer rats, quite reproducible tumor patterns were obtained.

With the 2 mg level of N-2 -fluorenylacetamide, the survival time of the males ranged from 209-365 days (average of the 6 groups, minimum 238 and maximum 343 days). 70 of 71 males exhibited hepatoma, and 54 of these lesions metastasized, generally to the lung and in a few cases to the adrenals. Relatively few other primary tumor sites were affected. Six rats bore tumors of the external ear duct, 1 had a mammary adenocarcinoma, and 10 developed a variety of neoplasms at several sites. Of interest are tumors of the lips which are not generally seen with this compound. They may have been specifically caused by a combination of carcinogenicity from the compound and repeated friction trauma from the intubations.

Although females were somewhat less responsive to this agent, the survival time was not much longer than with males, from 229-384 days or an average minimum of 240 and maximum of 353 days. The liver was less involved; 46 of 71 females had hepatoma and only 4 of these metastasized. On the other hand, 21 of the rats had ear duct tumors, 28 had mammary adenocarcinoma, and 3 additional females had fibroadenomas. A wide variety of lesions at various sites were observed in 20 additional rats, including 4 tumors of the skin, not usually seen with this compound, and 2 of the tongue or lip.

Conclusions:

The test material showed no significant increase of carcinogenic effects compared to controls up to a daily dose of 9 mg/animal (ca. 22 mg/kg bw/d for males and 31 mg/kg bw/d for females based on final body weight).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

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

DNPT has been tested in the rats after oral and intraperitioneal application. No carcinogenicity has been observed.

However, studies suffer from short duration. In the key study the maximally tolerated chronic dosage was determinedin dose range finding studies. Fischer rats were treated 5 times a week for 52 weeks, for a total of 260 individual doses, unless mortality occured.

No increases in the incidence of tumors in groups of 12 males and 12 females, 15 males and 15 females as well as 3 males and 3 females receiving 0.03 mg, 3 mg and 9 mg/animal were observed. A squamous cell carcinoma of the tongue was seen in one female receiving the lowest dose.

Based on the existing studies DNPT does not exert carcinogenic effects.