Registration Dossier

Administrative data

Description of key information

1,4-Benzenediamine, N,N'-mixed Ph and tolyl derivs. induces changes in organ weights, blood serum and haematologic parameters upon repeated oral exposure of rats. Some evidence of macrocytic anemia was demonstrated. Evaluation of the test results leads to a NOAEL of 16.00 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Dose descriptor:
NOAEL
16 mg/kg bw/day

Additional information

Oral route

Three repeated dose oral toxicity studies in Fisher 344 rats are available for assessment: a 3-week oral gavage study (Iatropoulos, 1994b), a 4-week dietary study (Iatropoulos, 1994a) and a 52-week chronic dietary study (Iatropoulos, 1996). All of these repeated dose toxicity studies were conducted according to GLP and international standards and they are leading to similar conclusions. Out of these studies, the 52-week chronic feeding toxicity study with a 38-week interim sacrifice and a 12-week recovery period (Iatropoulos, 1996) was chosen as the key study. It provides data to comprehensively assess the long-term effects of 1,4-Benzenediamine, N,N'-mixed Ph and tolyl derivs. The dietary levels of 53; 310 and 1900 ppm were chosen based upon the results of the 4-week dietary study, and corresponded with mean (male and female) ingested concentrations of 2.65; 16.00 and 100.20 mg/kg bw/day on an average basis.

Effects observed during the 52-week study included haematological changes, changes in the blood serum and elevated organ weights. A proliferative activity towards urinary bladder epithelium was also suggested. The clinical chemistry assessments showed elevated levels of cholesterol in the serum of high dose males and females, but these changes did not persist throughout the observation period. Increased bilirubin levels were found in high dose males, whereas high dose females showed decreased bilirubin levels. However, both deviations reversed during the 12-week recovery period. Elevated organ weights for e.g. liver, kidney and spleen were found in the mid and high dose animals. The histopathologic examination at week 52 showed that the spleen and liver exhibited signs of extramedullary erythropoiesis in the high dose male and female groups. Hematologic changes included elevated mean corpuscular volumes and decreased mean corpuscular hemoglobin in high dose males and females. These findings suggest a macrocytic anemia in the high dose animals. Based on these observations, an NOAEL of 16.00 mg/kg bw/day was established for 1,4-benzenediamine, N,N’-mixed Ph and tolyl derivs.

The results of the supporting 3-week oral gavage (Iatropoulos, 1994b) and the 4-week dietary studies (Iatropoulos, 1994a) are in good agreement with the observations described above. The 3-week oral gavage study revealed the test chemical to have target activity towards the liver in that an increase in organ weight plus evidence of proliferative changes in the liver were observed. Oral gavage daily dosing of test chemical at 3000 mg/kg bw/day to rats of both sexes led to 100% mortality within 10 days. The 1000 mg/kg bw dose was lethal to all males by day 7, but only to 6 of 10 females through 21 days. Lower doses (100 and 300 mg/kg day) were not lethal but did induce increased liver weights and signs of proliferative changes within the liver and possibly the urinary bladder. Only the lowest dose was without an effect on body weights.

The 4-week toxicity study was conducted by adding 120; 470 and 1900 ppm of test substance to the animals’ diet, which corresponds to actual ingested doses of 7.5, 30 and 120 mg/kg bw/day. Macrocytic anemia was the primary change in rats related to chemical exposure. These changes were reversible within 2 weeks following dietary exposure cessation, as were liver weight and serum cholesterol elevations. These changes were minor and were associated with no apparent prolonged adverse effects. The PCNA data for liver and bladder indicated increased cellular division at the higher doses, but with a lack of dose-responsiveness. Based upon these findings, the NOAEL for 1,4-benzenediamine, N,N’-mixed Ph and tolyl derivs. in rats is 470 ppm in the diet, corresponding to 30mg/kg bw/day. As this NOAEL is above the NOAEL derived from the 52-week study, the latter is retained in the calculations of the DNELs.

Dermal route

No dermal or inhalation repeated dose toxicity studies are available. According to section R.7.5.5.2 of ECHA’s Guidance on information requirements and chemical safety assessment, new repeated dose toxicity testing is only required, as a last resort, if route-to-route extrapolation starting from the available repeated dose data is not allowed.

The available set of test results for 1,4-Benzenediamine, N,N'-mixed Ph and tolyl derivs. - including a 52-week dietary exposure study and toxicokinetic examinations of both oral and dermal absorption of main constituent(s) of the test substance – does allow oral-to-dermal extrapolation of the chronic oral repeated dose study results towards the relevant DNEL long-term for dermal route-systemic, and as a consequence, no additional dermal repeated dose toxicity study was initialized

Inhalation route

The multi-constituent substance 1,4-Benzenediamine, N,N'-mixed Ph and tolyl derivs. has a very low vapour pressure (see vapour pressure section) and virtually no particles < 100 um are detected upon granulometric sieving experiment (see granulometry section). Moreover, the supported uses and exposure scenarios show negligible exposure to humans via the inhalatory route. As a consequence, exposure of humans via inhalation is not likely and testing of the repeated dose toxicity via inhalation is not appropriate.


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

Justification for classification or non-classification

Three repeated dose oral toxicity studies with rats are available for assessment: a 3-week oral gavage study, a 4-week dietary study and a 52-week chronic dietary study. However, the 3-week study was performed as a range-finding study and the observations made were not of sufficient detail to allow a STOT-RE analysis. As a consequence, only the 4- and 52-week studies were used to assess the STOT-RE properties of 1,4-Benzenediamine, N,N'-mixed Ph and tolyl derivs..

Both the 4- and the 52-week studies showed evidence of changes in organ (liver) weights and macrocytic anemia. In both cases, all changes had minor impact on animal health status, and none were life threatening. The change in liver weight was reversible and was not associated with histopathological changes or liver dysfunction; so the repeated exposure to the substance does not cause serious damage to the organism. The effect of macrocytic anemia was reversible and had a minor impact on animal health. Because of its minimal toxicological importance this effect does not warrant a STOT-RE classification.

As a consequence, it can be concluded that no classification according to CLP Regulation (EC) No. 1272/2008 regarding STOT-RE properties is warranted. Likewise, comparison of the severity of the experimentally observed effects with the criteria described in section 3.2.4 of Annex VI of Directive 67/548/EEC (Dangerous Substances Directive) shows that R48 classification should not be applied.