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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The data from the bacterial mutation assays indicated no genotoxic potential of IPPD (Monsanto Co. 1986, JETOC 1996, Monsanto Co. 1976, 1977a, 1977b). This negative finding is confirmed by the results from a mammalian cell mutation assays (Monsanto Co. 1986) and an in vitro rat hepatocyte DNA repair assay (Monsanto Co. 1986); whereas data from an in vitro chromosome aberration assay (NTP 1988) and in vitro sister chromatid exchange assay (NTP 1988) indicated positive effects. No siginifcant increase of SCE frequency was noted in bone marrow cells of treated mice; however the relevance of this finding is limited.
In conclusion, no mutagenic effects of IPPD were noted in bacterial and mammalian cell mutation assay. Clastogenic effects were noted in an in vitro chromosome aberration assay; however the relevance of this finding is unclear. An increase in SCE frequency was noted in CHO cells, whereas no such effect was observed in vivo in BALB/c mice.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

A combined Micronucleus assay according to OECD Guideline 474 and a Comet assay according to OECD Guideline 489 was conducted to evaluate the potential of N-isopropyl-N'phenyl-p-phenylenediamine to induce micronuclei (MN) in the polychromatic erythrocytes (PCE) of the bone marrow of treated rats and/or to induce DNA strand breaks in the liver, stomach and duodenum of the same animals using the alkaline comet assay (Beevers C 2017).

Male SD rats were administered test compound at 0 (Day 1), 24 (Day 2) and 45 (Day 3) hours at three dose levels (37.5, 75 or 150 mg/kg/day). Clinical chemistry and histopapothogy indicates systemic availability of the compound and defined the MTD:

Clinical Chemistry: There was increased AST and ALT activity in animals given 150 mg/kg/day. Increased ALP activity and an increase in total bilirubin were present in animals given 75 or 150 mg/kg/day. Cholesterol was increased in animals from all groups given N-isopropyl-N’phenyl-p-phenylenediamine.

Histopathology: There were no macroscopic findings considered to be related to administration of N-isopropyl-N’phenyl-p-phenylenediamine.

In the liver, hepatocyte cytoplasmic alteration was present in animals administered 75 or 150 mg/kg/day. Increased mitosis was present in animals from all groups administered the test article, with no clear dose effect. Bile duct hyperplasia was present in 2 animals given 150 mg/kg/day. There was a dose-related decrease in incidence and severity of hepatocyte glycogen and an absence of inflammatory cell foci in animals from all groups administered N-isopropyl-N’phenyl-p-phenylenediamine. In the stomach, vacuolar degeneration, forestomach degeneration, forestomach inflammation and/or erosion/ulcer were present in animals administered 150 mg/kg/day.

There were no dose-related increases in hedgehogs in liver, stomach and duodenum, thus demonstrating that treatment with N-isopropyl-N'phenyl-p-phenylenediamine did not cause excessive DNA damage that could have interfered with Comet analysis.

Animals treated with N-isopropyl-N'phenyl-p-phenylenediamine at all doses exhibited tail intensities that were similar to the concurrent vehicle control group and that were comparable with the laboratory's historical vehicle control data. There were no statistically significant increases in tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control.

It is concluded that under the conditions of this study, N-isopropyl-N'phenyl-p-phenylenediamine, did not induce an increase in micronucleated polychromatic erythrocytes of the bone marrow, nor did it induce DNA strand breaks in the liver, stomach and duodenum when tested up to 150 mg/kg/day (an estimate of the maximum tolerated dose for this study).

In a limited documented publication the in vivo SCE formation in bone marrow cells of treated BALB/c mice was evaluated (Gorecka-Turska 1983). Male mice were applied ip. with IPPD dose of 1, 5, 10, 30, 60 and 120 mg/kg bw. The animals were sacrificed 30 hours after test substance application and the bone marrow cells were prepared for SCE evaluation. No significant increase in SCE frequency was noted in bone marrow cells of any of the treated animals. However, the relevance of the finding is limited, because of the limited documentation.

The in-vivo studies (Micronucleus assay according to OECD Guideline 474, 2016 and a Comet assay according to OECD Guideline 489, 2016) were negative.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Non-human information

In vitro data

The mutagenic potential in bacteria of the test substance IPPD was evaluated in a GLP study (Monsanto Co. 1986). Here, the tester strains Salmonella typhimurium TA 98, TA 100, TA 1535, TA and 1537 were used. A dose range finding test was conducted using tester strain TA100 with and without metabolic activation. Toxicity was indicated at levels of 200 µg/plate and above with and without metabolic activation. This maximum level tested was toxic in the plate incorporation tests. No mutagenic activity was observed for the test material in this assay. The authors concluded that the test sample was not mutagenic towards any of the Salmonella typhimurium test strains used with and without metabolic activation.

In addition, no mutagenic response was noted in another bacterial mutation assay (JETOC 1996). Here, the tester strains Salmonella typhimurium TA100, TA1535, TA98, TA1537, TA1538 and E.coli WP2 uvr A were evaluated in presence or absence of metabolic activation. Moreover, in earlier bacterial mutation assays the test substance also indicated a non-mutagenic potential (Monsanto Co. 1976, 1977a, 1977b).

The negative findings from the bacterial mutation assays are confirmed in a mammalian cell mutation assay. The test substance IPPD was negative in a HGPRT assay, done with CHO cells (Monsanto Co. 1986). An initial cytotoxicity experiment was conducted with IPPD in CHO cells at different S9 concentrations; treatment doses of 10 and 30 µg/ml yielded significant cytotoxicity (>50% cell killing) in the absence and presence of S9 metabolic activation, respectively. The potential mutagenicity of IPPD was then tested at 0, 1, 2, 5, and 10% S9 at 3µg/ml, 10µg/ml, and 30 µg/ml. In this experiment IPPD was significantly cytotoxic to CHO cells at level of 10µg/ml and greater in the absence of exogenous metabolic activation. In the presence of S9 activation significant cytotoxicity was observed for treatment levels of 30µg/ml in the presence of S9. No statistically significant increases in mutant frequency were observed in the IPPD treated cultures. The non-mutagenicity of IPPD was confirmed by a subsequent experiment. In this experiment, IPPD was tested at 2, 5, 10, 15 and 30µg/ml in the absence and presence of 5% S9 activation. IPPD was observed to be significantly cytotoxic at levels of 15 and 30µg/ml. No statistically significant increases in mutant frequency were observed in this experiment.

The authors concluded that the test substance IPPD was not mutagenic in CHO cells under the experimental conditions used.

Moreover, no genotoxicity was indicated in an in vitro rat hepatocyte DNA repair assay (Monsanto Co. 1986).

A chromosomal aberration test with CHO cells was performed to determine the clastogenic potential of IPPD (NTP 1988). The test was performed according to the NTP standard protocol. In a first trial cells were treated with 1.6, 3, 5 and 10 µg/ml test substance without metabolic activation; harvest time 13 hours. In a second trial without metabolic activation cells were treated with 3, 5, 7.5 and 10 µg/ml test substance with a harvest time of 17.5 hours. In a third trial cells were treated with 10, 16, 30 and 50 µg/ml test substance with metabolic activation (S9-mix). Concurrent solvent controls and positive were included. 200 metaphase cells were analyzed per treatment group. An increase in aberrant cells were noted in treated cells with and without metabolic activation compared to the corresponding negative control (% aberrant cells 1st trial –S9: 5.5, 6.0, 10.9 vs. 1.5 control, 2nd trial –S9: 5.5, 19.0, 18.0, 14.0 vs. control 0.0; 3rd trial +S9: 5.5, 8.0, 5.09.0 vs. 2.0 control). However the relevance of these findings is questionable because no cytotoxicity is indicated. In a concurrent sister chromatid exchange assay (SCE assay) (NTP 1988), cytotoxicity was indicated at 3 µg/ml and higher by cell cycle delay (-S9). In the SCE assay CHO cells were treated with IPPD with and without metabolic activation. In a first trial without metabolic activation cells were treated with 0.05, 0.16, 0.5, 1.6 and 5 µg/ml test substance. Cytotoxicity was indicated at 5µg/ml. A weakly positive response was indicated by an increase of SCE frequency above 30 % at 1.6µg/ml compared to the corresponding solvent control. In a second trial without metabolic activation cells were treated with 1, 1.6, 3 and 5 µg/ml. Cytotoxicity was indicated at 3µg/ml by cell cycle delay. Increases of the SCE frequency above 20 % were noted in all treatment groups, indicating a positive response. In the trial with metabolic activation cells were treated with 0.5, 1.6, 5, 16, and 50µg/ml. Cytotoxicity was indicated at 50µg/ml. A weakly positive response was indicated by an increase of SCE frequency of 21% above the concurrent solvent control at 16µg/ml.

In vivo data

A combined Micronucleus assay according to OECD Guideline 474 and a Comet assay according to OECD Guideline 489 was conducted to evaluate the potential of N-isopropyl-N'phenyl-p-phenylenediamine to induce micronuclei (MN) in the polychromatic erythrocytes (PCE) of the bone marrow of treated rats and/or to induce DNA strand breaks in the liver, stomach and duodenum of the same animals using the alkaline comet assay (Beevers C 2017).

Male SD rats were administered test compound at 0 (Day 1), 24 (Day 2) and 45 (Day 3) hours at three dose levels (37.5, 75 or 150 mg/kg/day). Clinical chemistry and histopapothogy indicates systemic availability of the compound and defined the MTD:

Clinical Chemistry: There was increased AST and ALT activity in animals given 150 mg/kg/day. Increased ALP activity and an increase in total bilirubin were present in animals given 75 or 150 mg/kg/day. Cholesterol was increased in animals from all groups given N-isopropyl-N’phenyl-p-phenylenediamine.

Histopathology: There were no macroscopic findings considered to be related to administration of N-isopropyl-N’phenyl-p-phenylenediamine.

In the liver, hepatocyte cytoplasmic alteration was present in animals administered 75 or 150 mg/kg/day. Increased mitosis was present in animals from all groups administered the test article, with no clear dose effect. Bile duct hyperplasia was present in 2 animals given 150 mg/kg/day. There was a dose-related decrease in incidence and severity of hepatocyte glycogen and an absence of inflammatory cell foci in animals from all groups administered N-isopropyl-N’phenyl-p-phenylenediamine. In the stomach, vacuolar degeneration, forestomach degeneration, forestomach inflammation and/or erosion/ulcer were present in animals administered 150 mg/kg/day.

Animals treated with N-isopropyl-N'phenyl-p-phenylenediamine at all doses exhibited group mean %PCE that were similar to the concurrent vehicle control group and which were within to the laboratory’s historical vehicle control data, thus confirming there was no evidence of test article related bone marrow toxicity.

Animals treated with N-isopropyl-N'phenyl-p-phenylenediamine at all doses exhibited MN PCE frequencies that were similar to the concurrent vehicle control group and that fell within the laboratory's historical vehicle control data. There were no statistically significant increases in micronucleus frequency for any of the groups receiving the test article, compared to the concurrent vehicle control.

There were no dose-related increases in hedgehogs in liver, stomach and duodenum, thus demonstrating that treatment with N-isopropyl-N'phenyl-p-phenylenediamine did not cause excessive DNA damage that could have interfered with Comet analysis.

Animals treated with N-isopropyl-N'phenyl-p-phenylenediamine at all doses exhibited tail intensities that were similar to the concurrent vehicle control group and that were comparable with the laboratory's historical vehicle control data. There were no statistically significant increases in tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control.

It is concluded that under the conditions of this study, N-isopropyl-N'phenyl-p-phenylenediamine, did not induce an increase in micronucleated polychromatic erythrocytes of the bone marrow, nor did it induce DNA strand breaks in the liver, stomach and duodenum when tested up to 150 mg/kg/day (an estimate of the maximum tolerated dose for this study).

In a limited documented publication the in vivo SCE formation in bone marrow cells of treated BALB/c mice was evaluated (Gorecka-Turska 1983). Male mice were applied ip. with IPPD dose of 1, 5, 10, 30, 60 and 120 mg/kg bw. The animals were sacrificed 30 hours after test substance application and the bone marrow cells were prepared for SCE evaluation. No significant increase in SCE frequency was noted in bone marrow cells of any of the treated animals. However, the relevance of the finding is limited, because of the limited documentation.


Short description of key information:
The data from the bacterial mutation assays indicated no genotoxic potential of IPPD (Monsanto Co. 1986, JETOC 1996, Monsanto Co. 1976, 1977a, 1977b). This negative finding is confirmed by the results from a mammalian cell mutation assays (Monsanto Co. 1986) and an in vitro rat hepatocyte DNA repair assay (Monsanto Co. 1986); whereas data from an in vitro chromosome aberration assay (NTP 1988) and in vitro sister chromatid exchange assay (NTP 1988) indicated positive effects. No siginifcant increase of SCE frequency was noted in bone marrow cells of treated mice; however the relevance of this finding is limited.
In conclusion, no mutagenic effects of IPPD were noted in bacterial and mammalian cell mutation assay. Clastogenic effects were noted in an in vitro chromosome aberration assay; however the relevance of this finding is unclear. An increase in SCE frequency was noted in CHO cells, whereas no such effect was observed in vivo in BALB/c mice. However, the in vivo study is limited and should be used only for supporting reasons.

The in-vivo studies (Micronucleus assay according to OECD Guideline 474 and a Comet assay according to OECD Guideline 489) were negative.


Endpoint Conclusion: Overall, based on the available data no genotoxic potential of IPPD (N-isopropyl-N'phenyl-p-phenylenediamine) is evident and therefore IPPD is regarded as negative.

Justification for classification or non-classification

No classification is required according to regulation no. 1272/2008 (GHS).