Nitrapyrin

Endpoint summary

Additional information

IN VITRO

- Bacterial Reverse Mutation Assay (e.g. Ames test)

Four bacterial reverse mutation assay studies are available to assess the mutagenic potential of the test material.

The key study reported by Mecchi (2007) was conducted under GLP conditions and in accordance with the standardised guideline EPA OPPTS 870.5100, OECD 471, EEC: Directive 2000/32/EC, Council Directive 67/548/EEC, EEC Publication No. L136, and MAFF. It was assigned a reliability score of 1 in accordance with the criteria of Klimisch et al. (1997).

During the study test strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and a tester strain of Escherichia coli (WP2uvrA) were exposed to the test material both in the presence and the absence of metabolic activation, with a maximum dose level of 1000 µg/plate. Vehicle and positive controls were run concurrently. Two separate experiments were conducted, an initial mutagenicity assay, and a confirmatory assay. All concentrations were run in triplicate.

In the initial mutagenicity assay, the mean vehicle control value for tester strain TA98 in the absence of S9 mix was not within the acceptable range for this strain. For this reason, the data generated with TA98 in the absence of S9 mix in this trial were not used to evaluate the test material. The test material was re-tested with TA98 in the absence of S9 mix. All other data generated in the initial mutagenicity assay were acceptable, and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix. An increase in the mean number of revertants per plate was observed with TA100 in the presence of S9 mix; however, this increase did not meet the 2-fold criteria required for a positive evaluation with this tester strain. Based on the results of the initial mutagenicity assay, concentrations were selected for the confirmatory assay.

In the confirmatory mutagenicity assay, all data were acceptable and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix. An increase in the mean number of revertants per plate was observed with TA100 in the presence of S9 mix; however, this increase did not meet the 2-fold criteria required for a positive evaluation with this tester strain. In addition, increases in the mean number of revertants per plate were observed with TA98 in the presence and absence of S9 mix; however, these increases did not meet the 3-fold criteria required for a positive evaluation with this tester strain. Based on the results of the initial and confirmatory mutagenicity assays, concentrations were selected for the repeat mutagenicity assay with TA98 in the absence of S9 mix.

In the repeat mutagenicity assay, all data were acceptable and no positive increases in the mean number of revertants per plate were observed with tester strain TA98 in the absence of S9 mix. An increase in the mean number of revertants per plate was observed with TA98 in the absence of S9 mix; however, this increase did not meet the 3-fold criteria required for a positive evaluation with this tester strain.

Under the conditions of this study, the test material did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of metabolic activation.

The supporting studies reported by Kennelley (1985) and Meikle & Griffith (1976) also followed the Ames Test methodology; however, they investigated four and one strain of bacteria, respectively, and were assigned a reliability score of 2 according to the criteria of Klimisch et al. (1997). Both studies produced negative results, further substantiating the findings of the Mecchi study.

A final supporting study is available that was conducted by Zeiger et al. (1988). The mutagenic activity of the test material was evaluated in a bacterial reverse mutation assay conducted in accordance with generally accepted scientific methodology. The test material was evaluated in an in vitro Salmonella assay using a pre-incubation protocol as part of a study investigating the potential mutagenicity of 300 chemicals. It was not possible to obtain the entire report including the full experimental results, therefore the study was assigned a reliability score of 4 according to the criteria of Klimisch et al. (1997).

The test material was examined for mutagenic activity using the histidine-requiring Salmonella typhimurium strains TA97, TA98, TA100, TA1535, and TA1537. Testing was performed in the absence of exogenous metabolic activation and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters.

Under the conditions of the study, the test material has been determined to be mutagenic.

This study was conducted in 1983 prior to the majority of the key genotoxicity studies. In light of the extensive number of studies that suggest the substance does not exhibit mutagenic effects, alongside the fact that it was not possible to obtain the full results, the positive result is not considered to affect the overall conclusion that the substance does not possess genotoxic properties. 

- In Vitro Mammalian Cell Gene Mutation

The genotoxic potential of the test material was investigated in a study which was conducted under GLP conditions and following a method which was similar to that which is outlined in the standardised guideline OECD 476. It was assigned a reliability score of 2 in accordance with the criteria of Klimisch et al. (1997).

The test material was evaluated in an in vitro gene mutation test utilising cultured mammalian cells in the Chinese hamster ovary cell/hypoxanthine guanine-phosphoribosyl transferase (CHO/HGPRT) forward mutation assay. The test material was tested for genotoxic potential in the absence and presence of an externally supplied metabolic activation (S9) system. Test material was dissolved in dimethyl sulfoxide (DMSO) and added to the culture medium at concentrations of 20, 40, 60, 80, and 100 µg/mL in the absence of S9; and 120, 140, 160, 180, and 200 µg/mL in the presence of S9. The dose levels of test material for the mutation assay were based upon the results of a preliminary toxicity assay.

Cultures treated with 621 µg/mL ethyl methanesulfonate (EMS) and 4.03 µg/mL 20-methylcholanthrene (20-MCA) served as positive controls for non-activation and activation assays, respectively. Negative control cultures in both the activation and non-activation assays were treated with 0.5 % DMSO.

There were no significant treatment related increases in mutation frequencies in cultures treated with test material either in the absence or presence of S9 as compared to the negative controls. The positive control chemicals induced the expected increases in mutation frequencies. Hence, under the experimental conditions used, the test material was considered negative in the CHO/HGPRT gene mutation assay.

- In Vitro Unscheduled DNA Synthesis Assay (UDS)

The genotoxic potential of the test material was evaluated in the rat hepatocyte unscheduled DNA synthesis (UDS) assay under GLP conditions. The methodology employed was equivalent to that outlined in the standardised guidelines OECD 482 and EU Method B.18. It was assigned a reliability score of 2 in accordance with the criteria of Klimisch et al. (1997).

Primary cultures of rat hepatocytes were prepared by slight modification of the method described by Williams et al. (1977). The test material was prepared at concentrations of 1 x 10^-4, 3.16 x 10^-5, 1 x 10^-5, 3.16 x 10^-6, 1 x 10^-6, 3.16 x 10^-7 and 1 x 10^-7 M in Williams Medium E with 10 % foetal calf serum containing 0.1 % (v/v) dimethylsulfoxide (DMSO). Concurrent positive (2-acetylaminofluorene, 2-AAF), solvent and media controls were run.

Two mL of medium supplemented with dexamethasone (1 µM in 0.1 % ethanol, final concentration) containing the test material and ³H-thymidine (10 µCi/mL) was applied to triplicate cultures at each dose level. The cultures were then incubated for 18 hours after which they were washed for three 30 minute intervals with 1 mM non-labelled thymidine in WE at 37 °C. The nuclei of the cells were then swelled for 10 minutes (room temperature) with 1.0 % (w/v) sodium citrate and subsequently fixed with a solution of ethanol:acetic acid (3:1) for 30 minutes, (room temperature). The coverslips were allowed to air dry and were then mounted on glass slides. The slides were then examined by micro-autoradiography, with 15 cells on each of two slides being evaluated per dose level.

The test material was observed to be toxic to the hepatocyte cultures at concentrations of 1 x 10^-4 and 3.16 x 10^-5 M. Cultures exposed to 3.16 x 10^-6 to 1 x 10^-7 M test material matched the appearance of the negative control cultures. Thus, a wide spectrum of concentrations was evaluated ranging from toxic to nontoxic.

The test material failed to elicit UDS at any concentration tested compared to the negative control (media + 0.1 % DMSO). In contrast, 2-AAF elicited a significant, dose-related increase in UDS at all concentrations tested when compared to the media + 0.1 % DMSO control.

Under the conditions of the study, the test material has been determined to give a negative response when evaluated using the UDS assay.

IN VIVO

- In Vivo Micronucleus Assay

The clastogenic potential of the test material was investigated in a study which was conducted under GLP conditions and following a method similar to that which is outlined in the standardised guideline OECD 474. It was assigned a reliability score of 2 in accordance with the criteria of Klimisch et al. (1997).

During the study, the test material was evaluated for its ability to induce micronuclei in mice. A maximum tolerated dose of 800 mg/kg was established and this was administered to three groups of mice (5 male and 5 females per group). Test material was administered in corn oil by oral gavage; corn oil alone was administered to control mice.

The three groups of mice were killed after 24, 48 and 72 hours, and slides prepared from bone marrow.

Some evidence of bone marrow toxicity was obtained, evidenced by a slight decrease in the ratio of polychromatic to normochromatic erythrocytes (PCE/NCE). The test material did not produce an increase in micronuclei in either PCE or NCE. It is therefore concluded that the test material is not clastogenic in the micronucleus test.

- In vivo UDS Asay

The test material was tested in the UDS test with mouse liver cells in vivo. The methodology used was comparable to that outlined in the standardised guideline OECD 486. The test material was administered to 6 male mice per dose at doses of 125 and 250 mg/kg/day by a single oral gavage administration in corn oil. Two additional groups of 6 mice each received a single oral dose of 10 mL/kg corn oil or 10 mg/kg dimethylnitrosamine (DMN) which served as the negative and positive control, respectively.

No mortality or clinical signs were observed in any of the test material or control-treated animals immediately following dosing except at dose level 250 mg/kg/day where piloerection was noted. Treated and control animals were sacrificed at either 2 to 4 hours or 12 to 16 hours after dosing. The hepatocytes were isolated after the treatment time and seeded on Petri dishes containing coverslips. The hepatocytes were cultured in the presence of 3H-thymidine for four hours and then with normal thymidine for 17-20 hours. The incorporation of the radiolabel was assessed by autoradiography and scored for the net nuclear grain counts.

The group mean net nuclear grain (NG) counts for animals treated with the test material did not increase when compared to the negative control. For the 2 to 4 hour time point, the group mean NG counts for the test material-treated animals were -1.7 and 1.6 for the 125 and 250 mg/kg/day treated animals, respectively, with 0% of cells in repair (cells with ≥ 5 NG). The group mean NG count for the negative control group was -0.8 with 2% of cells in repair. For the 12- to 16-hour time point, the group mean NG counts for the test material-treated animals were -1.8 and -2.5 for the 125 and 250 mg/kg/day treated animals respectively, with ≤ 3% of cells in repair.

The group mean NG count for the negative control group was - 2.1 with 1% of cells in repair. The positive control group mean NG counts were 13.3 and 13.1 for the 2 to 4 hour and 12 to 16 hour treatment duration, respectively. The percentage of cells in repair for the positive control group was 79 and 67% for the 2 to 4 hour and 12 to 16 hour, respectively.

The test material did not induce a significant increase in the mean number of net nuclear grain counts (i.e., an increase of at least 5 counts over the negative control group) in hepatocytes isolated either 2 to 4 hours or 12 to 16 hours after dose administration. Thus, the test material was negative in the UDS test with mouse liver cells in vivo.

Justification for selection of genetic toxicity endpoint

Multiple studies have been provided to address the different aspects of genetic toxicity. Therefore no single study could be selected as key.

Short description of key information:

IN VITRO

- Bacterial Reverse Mutation Assay (e.g. Ames test)

Negative (TA98, TA100, TA1535, TA1537, WP2uvrA); EPA OPPTS 870.5100, OECD 471, EEC L136, MAFF; Mecchi (2007)

Negative (TA97, TA98, TA100, TA1535); Ames method (similar to OECD 471); Kennelly (1985)

Negative (TA1530); Ames test (similar to OECD 471); Meikle & Griffith (1976)

- In Vitro Mammalian Cell Gene Mutation

Negative (CHO cells); method similar to OECD 476; Linscombe & Gollapudi (1986)

- In Vitro Unscheduled DNA Synthesis Assay (UDS)

Negative (rat hepatocyte); method similar to OECD 482 and EU Method B.18; Mendrala & Schumann (1982)

IN VIVO

- In Vivo Micronucleus Assay

Negative (mouse erythrocytes); method similar to OECD 474; Kirkland (1985)

- In Vivo UDS

Negative (mouse liver); method similar to OECD 486; Pant (2009)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to mutagenicity.

In accordance with the criteria for classification as defined in Annex VI, Directive 67/548/EEC (DSD), the substance does not require classification with respect to mutagenicity.