2-methyl-p-phenylenediamine

Carcinogenicity

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study report which meets basic scientific principles.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

yes

Remarks:

(only two dose levels tested; food consumption not monitored for all animals; feed efficiency not determined; high dose animals tested several months after the initiation of the low dose group; the treatment duration was less than 24 months)

GLP compliance:

no

Remarks:

pre-GLP

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Charles River Breeding Laboratories, Inc., Wilmington, Massachusetts, United States.(high dose rats and their controls) and Laboratory Supply Co., Inc., Indianapolis, Indiana, United States (Low dose rats and their controls). Except for the low dose rats and their controls, all the other dosed animals were received in separate shipments from their respective controls.- Age at study initiation: Approx. 6 weeks - Weight at study initiation: Not reported- Housing: Animals were housed in groups of 5 per cage by sex. During quarantine and for the first 13 months of study, low dose rats and their controls were kept in galvanized- or stainless-steel wire-mesh cages suspended above newspapers. High dose rats and their controls were housed in galvanized-steel wire-mesh cages during quarantine and for the first 11 months of study. Newspapers under cages were replaced daily and cages and racks washed weekly. For the remainder of the study, all rats were housed in suspended polycarbonate cages equipped with disposable nonwoven filter sheets. Animals were provided appropriate bedding material. Clean cages, lids, filters, and bedding were provided twice weekly.- Diet: Pelleted Wayne Lab-Blox was provided to all animals during the initial quarantine and final observation periods. During the dosing period, all animals were fed Wayne Lab-Blox meal containing the appropriate concentration of the test material. Control animals had untreated meal available ad libitum. Meal was supplied to low dose and their controls in Alpine aluminum feed cups (Curtin Matheson scientific, Inc., Woburn, Massachusetts) containing stainless steel baffles. High dose rats and their controls were fed from Alpine feed cups for the first 14 months of study and from stainless steel gangstyle hoppers (Scientific Cages, Inc., Bryan, Texas) for the remainder of the study. During the final observation period, animals were fed pellets on the cage floor. Food hoppers were changed twice weekly. Food was replenished daily in Alpine feed cups.- Water: Water was available from 250 mL water bottles equipped with rubber stoppers and stainless steel sipper tubes. Bottles were replaced twice weekly and refilled as needed between changes.- Acclimation period: 2 wk prior to initiation of treatment.- Health Inspection: Upon arrival, a sample of animals was examined for parasites and other signs of disease.ENVIRONMENTAL CONDITIONS- Temperature: 23-34°C- Humidity: Not reported- Air changes: 6 air changes per hour. Incoming air was filtered through Tri-Dek 15/40 denier Dacron filters (Tri-Dim Filter Corp., Hawthorne, New Jersey)- Photoperiod (hrs dark / hrs light): 12 h fluorescent lighting/12 h darkness per day

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION- Rate of preparation of diet: Diets were prepared weekly.- Mixing appropriate amounts with (Type of food): Wayne Lab-Blox (Allied Mills, Inc., Chicago, Illinois). The test substance was mixed with the feed in a blender (Patterson-Kelley standard model stainless steel twin-shell V-blender with 6 kg capacity). After 20 min. of blending, the mixtures were placed in double plastic bags and stored.- Storage temperature of food: At 4°C for no longer than 2 wk.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

78 wk

Frequency of treatment:

The test substance was available continuously in the animal's diets. Food was replenished daily.

Post exposure period:

Low dose control group: Males: 29 wk; Females: 30 wkHigh dose control group: 31 wk (males and females)Low dose group: Males: 28 wk; Females: 29 wkHigh dose group: Males: 30 wk; Females: 31 wk

Remarks:

Doses / Concentrations:Low dose Control group: 0% Basis:nominal in diet

Remarks:

Doses / Concentrations:High dose Control group: 0% Basis:nominal in diet

Remarks:

Doses / Concentrations:Low dose group: 0.06% Basis:nominal in diethigh and low time-weighted average concentrations

Remarks:

Doses / Concentrations:High dose group: 0.2% Basis:nominal in diethigh and low time-weighted average concentrations

No. of animals per sex per dose:

350 animals (50 per sex per dose for all groups except for high dose control group which comprised of 25 animals per sex)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Initially, based on the results of a 4 wk sub-chronic dietary study in Fischer 344 rats at dose levels of 0.02, 0.05, 0.08, and 0.11%, the highest dose selected for the main study was 0.05% and the lower dose was 0.03%. During the course of the main study, because of a lack of observed mean body weight depression or other clinical signs at a level of 0.05%, the group receiving 0.03% was discontinued 2 months after initiation. A new group was initiated at a concentration of 0.2%. The rats initially receiving a concentration of 0.05% were given 0.06% from Week 15.- Rationale for animal assignment: Animals were assigned to groups and distributed among cages so that the average body weight per cage was approx. equal for a given sex and species.High dose rats and their controls were placed on test 11 months after low dose rats and their controls. Each dosed rat group was placed on test during the same week as its respective control group.

Observations and examinations performed and frequency:

MORTALITY: Yes - Time schedule: Twice dailyDETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: Once every month- Examinations: The presence of tissue masses and lesions was determined by monthly observation and palpation of each animal.BODY WEIGHT: Yes - Time schedule for examinations: Animals were weighed immediately prior to initiation of the experiment. Body weights were recorded twice weekly for the first 12 wk of the study and at monthly intervals thereafter.FOOD CONSUMPTION AND COMPOUND INTAKE:- Food consumption: Yes; Food consumption for two cages from each group was monitored for seven consecutive days once a month for the first nine months of the bioassay and for three consecutive days each month thereafter.- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: YesFOOD 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: Not reportedOPHTHALMOSCOPIC EXAMINATION: NoHAEMATOLOGY: NoCLINICAL CHEMISTRY: NoURINALYSIS: NoNEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes- How many animals: All animals- Method of euthanasia: Carbon dioxide inhalation - Tissues examined: Major tissues, organs, and gross lesionsHISTOPATHOLOGY: Yes- Tissues examined microscopically: Skin, subcutaneous tissue, lungs and bronchi, trachea, bone marrow, spleen, lymph nodes, thymus, heart, salivary gland, liver, pancreas, esophagus, stomach, small intestine, large intestine, kidney, urinary bladder, pituitary, adrenal, thyroid, parathyroid, testis, prostate, seminal vesicle, brain, tunica vaginalis, muscle, ear, uterus, mammary gland, and ovary- Preservation of tissue/organ samples: Tissues were preserved in 10% buffered formalin the histopathologic examination consisted of gross and microscopic examination of major tissues, organs, and gross lesions taken from sacrificed animals and, whenever possible, from animals found dead.Tissues were preserved in 10 percent buffered formalin, - Preparation of slides: The tissues samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. An occasional section was subjected to special staining techniques for more definitive diagnosis. .A few tissues were not examined for some animals, particularly for those that died early. Also, some animals were missing, cannibalized, or judged to be in such an advanced state of autolysis as to preclude histopathological interpretation. Thus, the number of animals for which particular organs, tissues, or lesions were examined microscopically varied and does not necessarily represent the number of animals that were placed on experiment in each group.

Statistics:

- Probabilities of survival were estimated by the product-limit procedure of Kaplan and Meier. Statistical analyses for a possible dose-related effect on survival used the method of Cox when testing two groups for equality and used Tarone's (1975) extensions of Cox's methods when testing a dose-related trend. - One-tailed P-values were reported for all tests except the departure from linearity test, which was only reported when its two-tailed P-value was less than 0.05. - The one-tailed Fisher exact test was used to compare the tumor incidence of a control group to that of a group of treated animals at each does level. The Cochran-Armitage test for linear trend in proportions, with continuity correction, was also used, when appropriate. - The standard procedures for analyses of the incidence of tumors (Fisher exact tests, Cochran-Armitage tests, etc.) were followed. - Curves of the proportions surviving without an observed tumor were computed as in Saffiotti et al. (1972). - Cox's methods of comparing these curves were used for two groups; Tarone's extension to testing for linear trend was used for three groups. The approximate 95 percent confidence interval for the relative risk of each dosed group compared to its control was calculated from the exact interval on the odds ratio. - When the lower limit of the confidence interval was greater than one, it could be inferred that a statistically significant result (a P<0.025 one-tailed test when the control incidence was not zero, P<0.05 when the control incidence was zero) had occurred. When the lower limit was less than unity but the upper limit was greater than unity, the lower limit indicated the absence of a significant result while the upper limit indicated that there was a theoretical possibility of the induction of tumors by the test substance which could not be detected under the conditions of this test.- The details of statistical analysis are provided in study report.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

only isolated clinical signs were observed in few animals and no effect on survival

Mortality:

mortality observed, treatment-related

Description (incidence):

only isolated clinical signs were observed in few animals and no effect on survival

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS: Only isolated clinical observations were reported. These included crusted lesions on the dorsolateral surface in one low dose control male and one low dose male and on the side of the head in one low dose female; tissue masses on the ear in two low dose males and on the foreleg in one low dose male; a subcutaneous mass under the base of the tail in one high doss male; and growths on the foreleg in three low dose females and on the ear in another low dose female.MORTALITY: There was no significant association between dosage and mortality for male or female rats. 5 males and 5 females from the high dose group died in Week 78.BODY WEIGHT AND WEIGHT GAIN: Mean body weight depression was observed in high dose female group. However, this trend was not as evident in other groups of dosed rats.FOOD CONSUMPTION AND COMPOUND INTAKE: No dataFOOD EFFICIENCY: No dataGROSS PATHOLOGY: No dataHISTOPATHOLOGY: NON-NEOPLASTIC: No treatment-related non-neoplastic lesions were observed.HISTOPATHOLOGY: NEOPLASTIC: No convincing carcinogenic effects were observed. The tumorigenic effects were as follows:- Interstitial-cell tumors: The incidence of interstitial-cell tumors was significant in high dose males. The effect was not considered treatment related due to high variation in the spontaneous incidence of this tumor.- Pituitary adenoma: The incidence of pituitary adenoma observed in low dose females was significantly lower than the control group. The comparison was not significant in the high dose group.

Relevance of carcinogenic effects / potential:

- The incidence of interstitial-cell tumors was significant in high dose males. The effect was not considered treatment related due to high variation in the spontaneous incidence of this tumor.- The incidence of pituitary adenoma observed in low dose females was significantly lower than the control group. The comparison was not significant in the high dose group.- No convincing carcinogenic effects were observed.

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Conclusions:

2,5-Toluenediamine sulphate was not carcinogenic in Fischer 344 rats. Despite the limitations of the study, the US NTP designates the findings of this study as negative in male and female rats.

Executive summary:

The carcinogenic potential of 2,5-Toluenediamine sulphate was determined following OECD guideline 451 (Carcinogenicity studies)

A total of 350 Fischer 344 rats (approx. 6 wk old) were acclimatized for 2 wk for this study. Animals were housed in groups of 5 per cage by sex. Appropriate bedding was provided. Animals were fed pelleted Wayne Lab-Blox meal during initial quarantine and final observation period. During the dosing, animals were fed with Wayne Lab-Blox containing test material.

The test material was fed at dose levels of 0 (low dose control), 0 (high dose control), 0.06 and 0.2%.

Initially, based on the results of a 4 wk sub-chronic dietary study in Fischer 344 rats at dose levels of 0.02, 0.05, 0.08, and 0.11%, the highest dose selected for the main study was 0.06% and the lower dose was 0.03%. During the course of the main study, because of a lack of observed mean body weight depression or other clinical signs at a level of 0.05%, the group receiving 0.03% was discontinued 2 months after initiation. A new group was initiated at a concentration of 0.2%. The rats initially receiving a concentration of 0.05% were given 0.06% from Week 15.

In this study, 50 animals per sex per dose were used except for the high dose control group which comprised of 25 animals per sex. The following parameters were evaluated in the study: clinical signs, mortality, body weight, food consumption and histopathologic findings (gross and microscopic examination of major tissues, organs, and gross lesions).

No treatment related effects on survival were observed. Isolated clinical signs were observed in a few treated and control animals. Treatment related body weight depression was observed in females treated with 0.2% test material.

No treatment related non-neoplastic lesions were observed. The occurrence of interstitial-cell tumors in high dose males was not considered treatment-related. The pituitary adenoma in females was not significant in comparison to control.

Under the conditions of the test, 2,5-Toluenediamine sulphate was not carcinogenic to Fischer 344 rats. Despite the limitations of the study, the US NTP designates the findings of this study as negative in male and female rats.

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

The carcinogenic potential of 2-methyl-p-phenylenediamine sulphate was determined following OECD guideline 451 (Carcinogenicity studies) on rats and mice

A total of 400 B6C3F1 mice (approx. 6 wk old) were acclimatized for 2 wk for this study. Low dose mice and their controls were housed ten per cage for the first 18 months of study and five per cage thereafter. The number of high dose mice per cage was reduced from ten to five after 13 months. The number of high dose controls per cage was reduced to five after 12 months. Animals were fed pelleted Wayne Lab-Blox meal during initial quarantine and final observation period. During the dosing, animals were fed with Wayne Lab-Blox containing test material.

The test material was fed at dose levels of 0(low dose control), 0(high dose control), 0.06 and 0.1%.

Initially, based on the results of a 4 week sub-chronic dietary study in C57BL/6 mice at dose levels of 0.02, 0.05, 0.08, and 0.11%, the highest dose selected for the main study was 0.06% and the lower dose was 0.03%. During the course of the main study, because of a lack of observed mean body weight depression or other clinical signs, the group receiving 0.03% was discontinued 6 months after initiation and a new group was initiated at a concentration of 0.1%.

In this study, 50 animals per sex per dose were treated to assess the carcinogenic potential. The following parameters were evaluated in the study: clinical signs, mortality, body weight, food consumption and histopathologic findings (gross and microscopic examination of major tissues, organs, and gross lesions).

No treatment related effects on the clinical signs and mortality were observed. The treatment related body weight depression was observed in females treated with 0.1% test material.

The non-neoplastic lesions were not considered treatment related. Hepatocellular carcinoma was observed in males was not significant in comparison to the control group. The combined incidence of pituitary adenoma NOS or pituitary carcinoma NOS in the high dose females was significantly lower than the high dose control while in low dose group, the comparison was not significant.

The incidence of alveolar/bronchiolar neoplasms was significantly higher in treated females in comparison to the control group. However, it should be noted that high dose control mice were housed in a separate room from dosed mice and received in separate shipments from dosed mice. Because of these factors, this increased incidence does not provide sufficient evidence of a compound-related effect.

Under the conditions of the test, the carcinogenic potential of 2,5-Toluenediamine sulphate could not be established. Despite the limitations of the study, the US NTP designates the findings of this study as negative in male and female mice

The carcinogenic potential of 2,5-Toluenediamine sulphate was determined following OECD guideline 451 (Carcinogenicity studies)

A total of 350 Fischer 344 rats (approx. 6 wk old) were acclimatized for 2 wk for this study. Animals were housed in groups of 5 per cage by sex. Appropriate bedding was provided. Animals were fed pelleted Wayne Lab-Blox meal during initial quarantine and final observation period. During the dosing, animals were fed with Wayne Lab-Blox containing test material.

The test material was fed at dose levels of 0 (low dose control), 0 (high dose control), 0.06 and 0.2%.

Initially, based on the results of a 4 wk sub-chronic dietary study in Fischer 344 rats at dose levels of 0.02, 0.05, 0.08, and 0.11%, the highest dose selected for the main study was 0.06% and the lower dose was 0.03%. During the course of the main study, because of a lack of observed mean body weight depression or other clinical signs at a level of 0.05%, the group receiving 0.03% was discontinued 2 months after initiation. A new group was initiated at a concentration of 0.2%. The rats initially receiving a concentration of 0.05% were given 0.06% from Week 15.

In this study, 50 animals per sex per dose were used except for the high dose control group which comprised of 25 animals per sex. The following parameters were evaluated in the study: clinical signs, mortality, body weight, food consumption and histopathologic findings (gross and microscopic examination of major tissues, organs, and gross lesions).

No treatment related effects on survival were observed. Isolated clinical signs were observed in a few treated and control animals. Treatment related body weight depression was observed in females treated with 0.2% test material.

No treatment related non-neoplastic lesions were observed. The occurrence of interstitial-cell tumors in high dose males was not considered treatment-related. The pituitary adenoma in females was not significant in comparison to control.

Under the conditions of the test, 2,5-Toluenediamine sulphate was not carcinogenic to Fischer 344 rats. Despite the limitations of the study, the US NTP designates the findings of this study as negative in male and female rats. No classification was applied according to CLP criteria.

Additional information

Justification for selection of carcinogenicity via oral route endpoint:
reliability 2