N-isopropyl-N'-phenyl-p-phenylenediamine

Endpoint summary

Description of key information

The repeated dose toxicity of IPPD was evaluated in a subacute and in subchronic feeding studies (Monsanto Co. 1989, Monsanto Co. 1990, Duslo 2009).  Administration of IPPD in the diet for four weeks at dose levels of 1000 ppm and above resulted in lower body weights and body weight gain, haematological effects, changes in clinical chemistry and increased liver and spleen weights. The author suggested a NOEL for females of 500 ppm; whereas no NOEL for males was suggested, because of the slight elevation in total protein observed in the males at 500 ppm (43.8 mg/kg bw/d).
Administration of IPPD in the diet at 180, 360 and 720 ppm to rats for three months resulted in lower body weight gains in high dose males, increased in absolute and relative liver weights in mid- and high dose males and all treated females, increases in spleen and kidney weights in high-dose females, mild anemia in mid- and high- dose animals and changes in clinical chemistry values. No treatment-related pathology in any organ or tissue was observed by microscopic evaluation. Based on the observed liver weight changes a LOAEL of 180 ppm is suggested for females. For males a NOAEL of 180 ppm (13.5 mg/kg bw/d) is suggested. The toxicity of IPPD was evaluated in a 90 day gavage study in Wistar rats (Duslo 2009). Changes in liver weights were noted in animals of the low, mid and high dose group. In addition macroscopic and histopathologic changes in the livers of treated animals of the low, mid and high dose groups were observed. However the adversity of these findings is questionable, because of the reversibility observed. In addition, the study documentation is limited and a re-evaluation of histopathologic data is needed for concluding assessment. 
In conclusion, the 90 day feeding study in rats (Monsanto Co. 1990) is the most relevant study. Based on the findings of this study, the suggested LOAEL of 180 ppm (ca. 13.5 mg/kg bw/day) is used for DNEL calculation. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Dose descriptor:

LOAEL

13.5 mg/kg bw/day

Additional information

The repeated dose toxicity of IPPD was evaluated in a subacute feeding study in Sprague-Dawley rats (Monsanto Co. 1989). Five male and five female rats per treatment group were feed with IPPD in the diet for 28 days. The animals were feed with 0, 500, 1000, 1750 and 2500 ppm (males: 0, 43.8, 92.6, 149.5, 215.1; females: 0, 48.1, 92.8, 160.3, 231.5 mg/kg/d) Control animals (5 per sex and group) received standard laboratory diet. Physical observations, body weight and food consumption measurements were performed on all animals pretest and at selected intervals during the treatment period. Hematology and clinical chemistry determinations were performed on all animals at study termination. After at least four weeks of treatment, all survivors were sacrificed, selected organs were weighed and organ/body weight ratios calculated. Complete gross postmortem examinations were conducted on all animals. No histopathology was conducted.

No mortality occurred during in the study. In addition, no test substance-related clinical signs were observed during the study in any of the treated animals. Dose related decreases in body weight and body weight gain were seen in both sexes at 1000 ppm and above with the exception of the 1750 ppm females. Differences from control in mean body weights were statistically significant in males of the 1750 ppm and 2500 ppm treatment groups (p<0.01). No significant differences were noted in females. The food consumption of males from the 1750 and 2500 ppm groups was reduced in the first week compared to the control males; mean food consumption values in the treated females were unremarkable. Treatment-related findings were observed in several hematology parameters in the males and females at dose levels of 1000 ppm and greater. The affected parameters included reduced hemoglobin concentration and hematocrit in males and females at 1000, 1750 and 2500 ppm and elevated platelet counts and total leukocyte counts in males at 1000, 1750 and 2500 ppm. For the most part these changes were statistically significant. The increases noted in the total leukocyte counts of the males at dose levels of 1000 ppm and greater were associated with an increase in the mean absolute segmented neutrophil counts. In addition to these findings erythrocyte morphology revealed slight anisocytosis and/or poikilocytosis treated males and females at 1000 ppm and above. Clinical chemistry showed elevations in total protein, albumin and globulin in both sexes at 1000 ppm and above. Males also had an increase in total protein at 500 ppm (ca. +7%, p<0.05). Glucose was slightly reduced in males at 1750 and 2500 ppm. Calcium was increased in males and females at 1000 ppm and above. No gross abnormalities related to treatment were noted at necropsy. Males and females showed an increase in liver weights and liver body weight ratios at 1000 ppm and above; males also had increased spleen weights at these dose levels.

The authors concluded that the no effect level in the females was found to be 500 ppm; whereas no NOEL for males was suggested, because of the slight elevation (statistically significant) in total protein observed in the males at 500 ppm (43.8 mg/kg bw/d).

A three-months feeding study with IPPD was conducted to assess the potential subchronic toxicity of IPPD (Monsanto Co. 1990). Sprague Dawley rats (10 per dose and sex) were administered orally, via dietary admixture, at dose levels of 0, 180, 360 and 720 ppm (males: 0, 13.5, 26.5, 54.0; females: 0, 15.6, 30.0, 59.0 mg/kg/d) for a period of three months. Control animals (10 per sex) received standard laboratory diet. Physical observations, body weight and food consumption measurements were performed on all animals pre-test and at selected intervals during the treatment period. Ophthalmic examinations were conducted prior to initiation and terminal sacrifice. Hematology and clinical chemistry evaluations were performed on all animals at months 1.5 and three. After three months of treatment, all animals were sacrificed, selected organs were weighed and organ/body and organ/brain weight ratios calculated. Complete gross post-mortem examinations were conducted on all animals.

Histopathological evaluation of selected tissues was performed for all control and high-dose animals (selected tissues: brain, pituitary, thyroid, thymus, heart, bone marrow, salivary glands, liver, spleen, kidneys, adrenals pancreas, testes (with epididymides), ovaries, uterus, aorta,esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, lymph nodes, peripheral nerve -sciatic taken with biceps femoris, sternum (with bone marrow), mammary gland female, muscle - biceps femoris, eyes, femur, extraorbital lachrymal glands, accessory genital organs -seminal, skin). The lungs, spleen, liver and kidneys of all animals were examined microscopically. In addition ovaries and uterus of all control and treated females were microscopically evaluated.

One high dose and one mid dose female were found dead on Test Day 93 following collection of terminal blood samples. These deaths were attributed to the stress of bleeding and not the administration of the test substance. All of the remaining and treated animals survived to the duration of the study. With the exception of soft stool observed in 1 to 5 high-dose males, no treatment-related clinical signs of toxicity were observed during the study. The mean body weight and body weight gain of the treated males at 720 ppm were slightly lower than control throughout the study. The mean body weights of the treated females were considered comparable to or exceeded control throughout the study. Mean food consumption values in the treated males and females were unremarkable. Treatment-related findings were observed in several hematology parameters in the males and/or females at dose levels of 360 and 720 ppm, implicating a mild anemia. The affected parameters included significantly reduced hemoglobin concentration and hematocrit values in males and females at 360 and 720 ppm at 6 weeks; significantly reduced (p<0.01) hemoglobin concentration in females at 720 ppm at 13 weeks. The haemoglobin concentration was also decreased in high dose males at study termination (not significant). A significantly increased platelet count was noted in males at 360 and 720 ppm at 6 weeks; and significantly reduced mean erythrocyte counts in females at 360 and 720 ppm at 6 weeks and at 720 ppm at 13 weeks. These changes were statistically significant and were considered treatment-related.

Several clinical chemistry parameters were altered during the course of the study. Although these differences from control did not exhibit a strong dose response in all cases, were often only noted at the interim period, and were not indicative of significant toxicity. Because of the consistency with which the effects occurred, the authors suggested that they were treatment-related. The effects included significant increases in total protein in males and females of the mid and high dose groups at week 6 and in males of the mid and high dose groups at study termination. A significant increase in albumin was noted in all treated males and in mid and high dose females at week 6; a significant increase in mid and high dose males at study termination and in mid and high dose females at week 6. Calcium levels were significant increased in all treated animals at week 6 and in low and high-dose males at termination. The chloride values were significantly decreased in mid and high dose males at study termination and in low and high dose females at week 6 and in all treated females at termination. Also noted but not considered biologically relevant were decreases in SGOT, SGPT and alkaline phosphatase.

Ophthalmoscopic examinations conducted at study termination were unremarkable. No treatment-related effects were evident.

No gross lesions were found at necropsy that was related to treatment. Mean liver weights, liver to body weight and liver to brain weight ratios were significant increased in mid and high dose males (17%, 25%, 35% at 180, 360, 720 ppm, respectively) and in all treated females (27%, 22%, 48% at 180, 360, 720 ppm, respectively). In addition, absolute and relative spleen and kidney weights were elevated in high dose females. There were no microscopic findings associated with these organ weight changes and there was no evidence of any other treatment-related histopathology.

The authors concluded, that based on the increased mean liver weights, liver/body weight and liver/brain weight ratios in all female treated groups a LOAEL of 180 ppm (15.6 mg/kg bw/d) is suggested for females. For males a NOAEL of 180 ppm (13.5 mg/kg bw/d) is suggested.

The toxicity of IPPD was evaluated in a 90 day gavage study in Wistar rats (Duslo 2009). However, the study was made under recent guidelines; the documentation is limited (e.g. gross pathology and histopathology) and thus will only be used for supporting reasons. Male and female rats (10 per dose and sex) were administered once daily by gavage with IPPD dosages of 0, 10, 30 and 90 mg/kg bw and day. A satellite control group and high dose group (90 mg/kg bw and day) were also included in the study. The satellite animals were scheduled scarified after a 28 day recovery period. No treatment related mortality occurred during the study; no treatment-related changes in behavioural assessment, functional observations and ophthamologic examinations were noted in treated animals compared to control. Clinical signs like green colour of fur around anus were transiently noted in high dose males. Sporadic salivation and nervous behaviour were noted in high dose female. No significant changes in body weight, body weight gain and food consumption were observed in treated animals. A significant increase of water consumption was noted in treated males and females of the high dose group. Several changes in haematology were noted in treated males and females, some of the changes were statistically significant. However, all findings were within the historical control data range reported or even slightly below. Several changes were noted in clinical biochemistry. A statistically significant increase (p<0.05) in total cholesterol was noted in low, mid and high dose males and females. No historical control data are given. The relevance of the slight increase noted at 10 mg/kg bw and day is questionable. Some changes of other biochemical parameters were statistically significant, but all of these values were within the laboratory historical control data. Some changes in urinalysis were noted in treated males and females. Protein and leucocytes were detected in treated males and females. In females these changes were reversible; whereas in males these changes are consistent. However, the effects in urinalysis were not dose dependent. Statistically significant increases in absolute and relative liver weights were noted in low, mid and high dose males and in mid and high dose females. The liver weights of the treated males and females from the satellite were slightly increased compared to the concurrent control, but no statistically significant was indicated, indicating reversibility of the effect. Absolute and relative kidney weights were also increased in treated males and females. A statistically significant increase was noted only in high dose males. Macroscopic findings were made in livers of treated males and females of all dose groups (“marked structures”, colour change, remark: no additional data given, uncommon nomenclature). In mid and high dose males an enlargement of liver was seen. No such changes were observed in treated animals of the satellite group, indicated reversibly of the effect. Histopathological changes were noted in the livers of all treated animals (vacuolar dystrophy irregular, vacuolar dystrophy centrolobular, remark: no additional data given, uncommon nomenclature). These changes were reversible indicated by lower incidence in satellite group males. However, the adversity of these findings is questionable because of the reversibility observed. In mid and high dose males dystrophy of adrenal glands were observed and in high dose males dystrophy in cortex of kidney. Several other changes were noted which occurred sporadic in control and treated animals.

In summary, haematological and biochemical changes noted in treated males and females were within the historical control data. The changes noted in urinalysis showed no dose dependency. Changes in liver and kidney weights were noted in treated animals. Significant changes in liver weights were noted in low, mid and high dose males and females; whereas significant changes in kidney weights were noted only in high dose males. Macroscopic and histopathologic changes were observed in the livers of low, mid and high dose males and females. The adversity of these findings is questionable, because of the reversibility noted in satellite group animals; in addition, because of the limited documentation a re-evaluation of the data is needed for a concluding assessment.

Justification for classification or non-classification

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