N-1,3-dimethylbutyl-N'-phenyl-p-phenylenediamine

Endpoint summary

Description of key information

Several oral repeated dose toxicity studies were conducted to evaluate the toxicity of the test substance 6PPD. Overall, oral administration of 6PPD to male and female rats revealed adverse effects on the liver and changes in hematology. Based on the findings from the repeated dose toxicity studies covering sub-acute to chronic exposure a NOAEL of 20 mg/kg bw/day is taken as a starting point for DNEL calculation.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

chronic

Quality of whole database:

Based on the findings from the repeated dose toxicity studies covering sub-acute to chronic exposure a NOAEL of 20 mg/kg bw/day is taken as a starting point for DNEL calculation.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

The only study available is limited documented (e.g. no individual data availabe for organ weights), with methodological deficiencies (e.g. particle size distribution: only <2% of particle-size range within the recommended range of 1.5 to 3.0 µg and whole body exposure); thus the exposure of all relevant regions of the respiratory tract is questionable.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: oral

In a GLP and guideline study (Guidelines for 28-Day Repeat Dose Toxicity Testing of Chemicals, Japan) 5 males and 5 female Sprague Dawley rats were exposed to 6PPD by gavage. The animals were treated with 0, 4, 20 and 100 mg/kg bw and day for 28 days. The control and 100 mg/kg bw and day group included additional 5 males and 5 females that were observed for further 14 days after termination of exposure. In the English summary and tables (detailed publication in Japanese) data on mortality, body weight, organ weights and microscopic and macroscopic are given. In addition, data of haematology, clinical chemistry and urinalysis are available. No effects on survival or body weight gain were reported. In rats doses with 100 mg/kg bw and day relative liver weights were significantly increased for both sexes at the end of administration accompanied by periportal fatty change. During recovery these effects were distinctly reversible but the increase of liver weight was still significant for male rats. The histopathological liver change was also observed in all females of the 20 mg/kg bw and day group, without an increase of liver weight. A significant increase of total serum protein was observed in females given 20 mg/kg bw and day or more (dose-dependent effect) and in males given 100 mg/kg bw and day at the end of administration. No such significant increase was noted in males and females of the highest dose group at the end of the recovery period. Increased proteinuria was found in the high dose group (both sexes), however, in the absence of histological changes of the kidney. There were no effects on weights or histopathologic findings of any other organs. In the high dose group further changes of clinical chemistry and haematological parameters were reported indicating an existing anaemia. The authors derived a NOEL of 4 mg/kg bw and day for this study (Hatano Research Institute 1999). As discussed in OECD SIDS (2005) a sex-specific sensitivity is obvious from the study data, the NOEL of 4 mg/kg bw and day is only valid for the female rats and the effects observed at the LOEL of 20 mg/kg bw and day were of a rather mild nature (reversible periportal fatty change of the liver without an increase of liver weight, increased total serum protein). In contrast, there were adverse effects on a range of different parameters observed at 100 mg/kg bw and day for both sexes so that the LOAEL for both sexes is rather at the dose of 100 mg/kg bw and day and the NOAEL at 20 mg/kg bw and day.

In a screening reproduction toxicity test groups of 12 male and 12 female Crj: CD rats were administered by gavage to 6 PPD (Tanaka et al., 2001). The animals were dosed with 0, 6, 25 and 100 mg/kg bw and day for 48 days (males) and from 14 days before mating until day 3 of lactation (females). Animals were sacrificed on day 48 (males) and on day 4 of lactation (females). From the English summary and tables (detailed publication in Japanese) data on mortality, body weight, food consumption, organ weights (liver, adrenals, testes, epididymides) and macroscopic and microscopic examination (liver, kidney, adrenals, epididymides, skin) are available. 6PPD had no influence on body weight gain but food consumption was increased in high dose males (intermittently) and in all 6PPD-treated females (during lactation only) as compared to controls. 1/12 dams of the high dose group died on gestation day 23. Clinical signs consisted of salivation (25 mg/kg bw and day males, 100 mg/kg bw and day both sexes). Dose dependent increases in liver weight (up to 37 %) were observed at 25 mg/kg bw/day and above in both sexes and increased adrenal weight (18 %) occurred in males of the highest dose. The incidence of vacuolar liver degeneration was dose dependently increased in males at 25 mg/kg bw/day (2/12) and 100 mg/kg bw/day (9/11). There were no adverse effects of 6PPD on the reproduction organs of both sexes and no adverse effects were seen in terms of estrus cycle, copulation and fertility results or findings for delivery. No abnormal findings related to the test substance were noted for external examination, clinical signs, growth or necropsy of the offspring. The NOAEL for repeated dose toxicity is considered to be 6 mg/kg bw/day for both sexes based on salivation and liver effects at 25 mg/kg bw/day. The increased food consumption in all 6PPD treated females during lactation being judged as non-adverse, as discussed by OECD SIDS (2005).

The toxicity of 6PPD was evaluated in a 13-week study with Sprague-Dawley rats (Monsanto Co. 1987a). Males and females (25 per dose and sex) were fed with dosages of 0, 250 1000 or 2500 ppm 6PPD (males: 0, 15.7, 62.3, and 153.8 mg/kg bw/day; females: 0, 18.5, 75.0 and 172.1 mg/kg bw/day). The animals were examined for clinical signs, weight gain, food intake and mortality. Ophthalmic examinations, clinical chemistry and haematological examinations were done. At necropsy weights of brain, kidney, liver, spleen and testes were recorded and an extensive macroscopic and microscopic examination of all organs was performed. No mortality occurred and no test-substance related clinical signs were observed. Animals of the highest dose groups showed reduced body weight gains. This reduction in body weight gain was accompanied by reduced food consumption. At the 1000 ppm and 2500 ppm dose groups changes in clinical chemistry parameters (increase in total protein albumin, globulin, calcium and/or cholesterol for both sexes and increase in total bilirubin in males) were observed. Decreases of SGOT, SGPT, creatinine, and BUN values were also observed sporadically in either or both sexes, usually at the higher dietary levels. However, the biologically relevance of these decreases is questionable. Changes in haematology were seen in mid and high dose animals (anemia, lymphocytopenia and thrombocytosis). The authors concluded that the observed anemia was not caused by decreased red blood cell production but rather by an increased rate of red blood cell destruction due to the absence of histological lesions in bone marrow. Increased relative and/or absolute liver weights were noted at 1000 ppm and higher (25 % and 45 % increases in rel. liver weights at 1000 ppm and 2500 ppm, respectively). These increases were not accompanied by macroscopic or microscopic lesions. Decreased weights of testes and spleen (females) at the high dose in the absence of microscopic changes were not considered as treatment related. Female rats of the low dose group (250 ppm) had mild anemia at interim period sampling (study week 6 to 7) that was reversible within the end of study. Lymphocytopenia was observed in females from all dose groups at the terminal sampling. The toxicological significance and relationship to treatment of thrombocytois and lymphocytopenia in this study are unknown. From these data a NOAEL of 250 ppm (15.7 mg/kg bw/day males, 18.5 mg/kg bw/day females) was suggested, which is based on anemia and increase of liver weights at the higher dose groups.

In a chronic study (Monsanto Chemical Co. 1993) 6PPD was administered orally for 24 months via dietary admixture to rats at dose levels of 0, 50, 250, and 1500 ppm. Homogeneity, stability and actual dietary concentrations were analytically verified throughout the study and confirmed to be within the range of ± 15% of the nominal concentration. The mean test-substance intake over the duration of the study was as follows:

- 50 ppm: 2.6 mg/kg/day (males) and 3.2 mg/kg/day (females)

- 250 ppm: 13.5 mg/kg/day (males) and 16.5 mg/kg/day (females)

- 1500 ppm: 84.8 mg/kg/day (males) and 109.5 mg/kg/day (females)

At the beginning of the experiment 70 rats/sex/group were dosed with 6PPD. At 12 months of treatment approximately 20 rats/sex/group were sacrificed. After 24 months of treatment all survivors were sacrificed. Ophthalmoscopic examinations were reported after 12 months and at study termination. Body weight and food consumption were recorded weekly throughout the first 13 weeks and monthly thereafter. Hematology (20 animals per time point/sex/group), clinical chemistry (10 animals per time point/sex/group), and urine analysis (10 animals per time point/sex/group) were performed prior to study initiation, during months 3, 6, 12, 18, and at termination. Blood was taken after fasting over night, urine was taken from unfastened, water depreciated animals and the following parameters were reported. Hematology: hemoglobin concentration, hematocrit, erythrocyte counts, platelet counts, mean corpuscular volume, mean corpuscular hemoglogin, mean corpuscular hemoglobin concentration, total and differential leucocyte counts. Clinical chemistry: serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, alkaline phosphatase, blood urea nitrogen, fasting glucose, cholesterol, total protein, albumin, globulin, albumin to globulin ratio, total bilirubin, sodium, potassium, chloride, calcium, inorganic phosphorus, creatinine phosphokinase, gamma glutamyl transpeptidase. Urine analysis: specific gravity, protein, pH, glucose, ketones, bilirubin, occult blood, urobilinogen, 16h volume, microscopic analysis. Post mortem gross examination was reported on all animals. Organ weights were recorded for ≥ 10 animals/sacrifice timepoint/sex/dose. The following organs were weighted: adrenals, brain, kidneys, testes with epididymides, liver, ovaries, and spleen. Histopathologic evaluation of all animals in the control and high dose groups were performed and on selected organs in the interim groups. The following organs were evaluated in the control and high-dose groups: adrenals, aorta (abdominal), bone (including the articular surface, sternum and femur), bone marrow (sternum), brain (medulla/pons, cerebellar cortex and cerebral cortex), esophagus, eyes, heart, intestine, cecum, colon, duodenum, ileum, jejunum, rectum, kidneys, liver, lungs (including trachea) – inflated with fixative, lymph nodes (mesenteric, mediastinal), mammary gland (right inguinal), ovaries, pancreas, peripheral nerve – sciatic, taken with biceps femoris, pituitary, accessory genital organs (prostate, seminal vesicles, epididymides), salivary glands (submandibular), skeletal muscle (right biceps femoris), skin, spinal cord (midthoracic, lumbar, cervical), spleen, stomach, testes, thymus, thyroid (including the parathyroids), urinary bladder – inflated with fixative, uterus, all gross lesions and tumors.

There were no statistically significant differences among survivorship or early deaths in any dose group compared to controls. Clinical observations were of the type commonly seen in lifetime rodent studies and no consistent patterns were evident. There was an increase in the incidence of poor condition and emaciation of the high dose females, but not males. No treatment related ophthalmoscopy observations were reported after 12 months or at termination. Mean body weight was consistently lower in males and females at 1500 ppm (mean difference to control – 9.9% and -18.4%, respectively) and lower in females at 250 ppm (-5.4%). Mean food consumption was increased in males and females at 1500 ppm (mean difference to control +5.5% and +17.3%, respectively) and in females at 250 ppm (+4.1%).

Hematologic observations indicate that 6PPD might induce a slight anemia in animals of the high dose groups. Body and liver weights are already affected at the mid dose groups. No histopathologic observations are reported for any blood forming organ; absolute and relative spleen weights are increased only in the high-dosed males at 12 months, but not at termination. Taken together the hematologic observations are considered slight, high-dose effects with no morphologic correlates.

Consistent statistically significant increased cholesterol levels were observed in males and females in the high dose groups at 6, 12, 18, and 24 months.

There were no consistent observations in in urine analysis for any parameter measured.

Mean absolute and relative kidney weights were increased in the high-dose groups in males and females at 12 months, but not at termination. Chronic nephropathy correlated with and increased incidence of irregularities of the kidney surface observed macroscopically in males and females at the high dose groups. The incidence of chronic nephropathy was similar between the control groups and the dose groups. However, the severity of chronic nephropathy increased among the high-dosed animals of both sexes sacrificed after 12 months and at termination. Mean absolute and relative liver weights were increased in males and females after 12 and 24 months in the high-dose groups and in the mid-dose groups at termination. Microscopic observations indicate increased incidence in pigment in the hepathocytes and reticuloendothelial cells and cytoplasmic vacuolization of the liver in females at the high dose. No effect was observed in males or females in the mid dose or males in the high dose. Follicular cell hyperplasia in the thyroid was slightly but not statistically significant increased among treated males. For further details on this observation see chapter carcinogenicity. In summary, the no-observed-effect-level (NOEL) of this oral chronic toxicity study is 50 ppm in males and females (2.6 mg/kg/day in males, 3.2 mg/kg/day in females), based on reduced body weight in females, increased food consumption in females and increased liver weight at 250 ppm in both sexes (13.5-16.5 mg/kg/day). The observations in the mid dose are not regarded to be clearly adverse. At the high dose (1500 ppm; 84.8-109.5 mg/kg/day) adverse liver histopathology in females and slight effects on hematology are observed in both sexes.

In an older chronic two-year study 50 rats per sex and dose were fed with 0, 100, 300 or 1000 ppm 6PPD in the diet (ca. 0, 8, 23 and 75 mg/kg bw/day) (Monsanto Co.1978). No test substance-related clinical signs were observed. Body weights and body weight gains were reduced for males and females of the highest dose group. For females, this persisted throughout most of the study and was statistically significant (p<0.01) for the first 16 months of the study. Body weights and body weight gains for mid and low dose groups were comparable to control groups. Food consumption was lower during the first two weeks for females of the high dose group and to the first four weeks of the study for males of the highest dose group. No test substance-related changes were noted in clinical chemistry and urinalyses. Comparable to the chronic study of Hatato et al. (1999) changes in haematological parameters were observed which indicate slight anemia in the high dosed animals. In high dose males an increase in liver weights (19 %) was seen and in high dose females a decrease in kidney (11 %) and spleen weights (22 %) was noted. A final examination gave no indication for gross pathological findings, histopathological changes and neoplastic alterations caused by 6PPD. Based on the findings in the high dose group (reduced body weight,changes in haematology and liver weight changes) a NOAEL of 300 ppm (ca. 23 mg/kg bw/day) is suggested for this study. The findings observed in this chronic study are in line with the observations in the chronic study of Hatano et al. (1999).

Overall, oral administration of 6PPD to male and female rats revealed adverse effects on the liver and changes in hematology. Based on the findings from the repeated dose toxicity studies covering sub-acute to chronic exposure a NOAEL of 20 mg/kg bw/day is taken as a starting point for DNEL calculation.

Repeated dose toxicity: inhalation

In a limited subacute toxicity inhalation study (Monsanto Co. 1979) male and female Sprague-Dawley rats were exposed for 6 hours per day and 5 days per weeks for 4 weeks (a total of 20 exposures) with test substance dust. Groups of 10 rats (5 males and 5 females) were exposed to 51, 247 and 498 mg/m³. Test animals were exposed in specially constructed stainless steel and glass inhalation chambers, each having a capacity of 500 litres. Untreated control animals received no dust exposures. Dust was generated by passing streams of clean, dry air through the test material powder. Analyses of chamber air and particle size distribution were conducted. Animals were observed for mortality, toxic signs and body weight gain. Hematologic and clinical chemistry studies and urinalyses were conducted. Clinical signs observed in treated animals were hypoactivity, swollen snouts and scratching after treatment. One male from the mid dose group died during the investigation period. Increased liver weights were noted in treated animals; however no individual data are available. Changes in haematology were noted in treated animals. However, the findings of these studies are questionable, because of the unsuitable particle size distribution used. Generally more than 98 % of the particles generated were above the recommended aerodynamic diameters ranging from 1 to 3 µm, thus the exposure of all relevant regions of the respiratory tract is questionable. However, the symptoms and effects recorded are comparable to effects noted in the oral repeated dose studies like changes in haematology parameters and increased lung weights. Because of the whole body exposure chosen, an oral and/or dermal exposure is possible.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
oral administration of 6PPD to male and female rats revealed adverse effects on the liver and changes in hematology. Based on the findings from the repeated dose toxicity studies, including two sub-acute, one sub-chronic and two chronic studies, an overall NOAEL of 20 mg/kg bw/day is taken as a starting point for DNEL calculation.

Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: hematopoiesis; digestive: liver

Justification for classification or non-classification

No classification is required according to the classification criteria 67/548/EEC and regulation no. 1272/2008 (GHS).