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Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay (Ames test): Key study (41102163)

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with Carbohydrazide using the Ames plate incorporation method at up to six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors) The dose range for Experiment 1 (range-finding test) was determined in a preliminary toxicity assay and ranged between 15 and 5000 µg/plate, depending on strain type. The experiment was not repeated on a separate day due to the results of the main experiment.

An additional dose level (15 µg/plate) was included and an expanded dose range was selected in order to achieve four non-toxic dose levels and the toxic limit of the test item.

Results.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item caused a visible reduction in the growth of the bacterial background lawn at 5000 µg/plate to all of the strains in the absence of S9 but only in WP2uvrA and TA1537 in the presence of S9-mix. A substantial reduction in revertant colony frequency was also observed at 5000 µg/plate in TA98 (with S9 mix only). Therefore the test item was tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either

the presence or absence of S9-mix.

There were dose related and statistically significant increases in the frequency of TA100 and TA1535 revertant colonies at sub-toxic test item dose levels, in both the absence and presence of S9-mix. The increases observed for TA1535 were particularly large and in excess of the in-house historical control ranges with increases greater than 12 fold over the concurrent vehicle control. Therefore, the test item was considered to be mutagenic under the conditions of the test. The OECD 471 test guideline and

the study plan permits non-repetition of the experiment when a clear, positive response is obtained in the first experiment, therefore a second confirmatory experiment was not required.

Conclusion.

The test item, Carbohydrazide was considered to be mutagenic under the conditions of this test.

Bacterial reverse mutation assay (Ames test): Supporting study

Test method.

The study was conducted in accordance with Ames test method specified under item 4 of article 34 of Industrial Safety and Health Law.

Bacterial strains used for the test.

Six bacterial strains, Salmonella typhimurium TA100, TA98, TA1535, TA1537, TA1538 and Escherichia coli WP2uvrA were used for this study.

Testing conditions.

With metabolic activation (S9 mix): Six concentrations of test material at 10, 50, 100, 500, 1000 and 5000 µg/plate were used.

Without metabolic activation (without S9 mix): Six concentrations of test material at 10, 50, 100, 500, 1000 and 5000 µg/plate were used.

Test results

No mutagenicity was confirmed for any of the 6 bacterial strains used with any dose of test material between 10 to 5000 μg/plate either with or without S9 mix.

Chromosome Aberration Test in Human Lymphocytes in vitro 

Introduction.

The report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations

Methods.

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration); whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

 

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

 

Group

Final Concentration of Test Item (µg/ml)

4(20)-hour without S9

28.13, 56.25, 112.5, 225, 450, 900

4(20)-hour with S9 (2%)

28.13, 56.25, 112.5, 225, 450, 900

24 hour without S9

14.06, 28.13, 56.25, 112.5, 225, 450

4(20)-hour with S9 (1%)

28.13, 56.25, 112.5, 225, 450, 900

 

Results.

All vehicle (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

 

All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

 

The test item induced a very modest but statistically significant increase in the frequency of cells with aberrations in the 24 hour continuous exposure group. This was proven to be reproducible when the slides from the preliminary toxicity test were scored for the presence of aberrations. It should be noted that the responses only occurred at a single dose level which exhibited optimum levels of toxicity and, therefore, may be due to a cytotoxic mode of action rather than a true genotoxic mechanism. No increases in the frequency of aberrations were observed in any of the 4(20)-hour exposure groups.

 

Conclusion.

 The test item was considered to be weakly clastogenic to human lymphocytes in vitro.

CHO HPRT Forward Mutation Assay

Introduction.

The study was conducted to assess the potential mutagenicity of the test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells.

Methods.

Chinese hamster ovary (CHO) cells were treated with the test item at six dose levels, in duplicate, together with vehicle (solvent) and positive controls. Four treatment conditions were used for the test, i.e. In Experiment 1, a 4 -hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

 

 The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows:-

 

Exposure Group

Final concentration of test item (µg/ml)

4-hour without S9

28.13, 56.25, 112.5, 225, 450, 900,

4-hour with S9 (2%)

28.13, 56.25, 112.5, 225, 450, 900,

24 hour without S9

28.13, 56.25, 112.5, 225, 450, 900,

4-hour with S9 (1%)

28.13, 56.25, 112.5, 225, 450, 900,

 

Results.

The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus.

 

The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.

 

The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

 

Conclusion.

 The test item was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of the test.


Justification for selection of genetic toxicity endpoint
All studies, apart from the supporting Ames study, were conducted to GLP and are reliability 1. All studies followed standard guidelines.

Short description of key information:
OECD Guideline 471 Bacterial Reverse Mutation Test "Ames Test" (Key study)
The test material was considered to be mutagenic under the conditions of this test.

A second Ames study found the substance to be non-mutagenic.

OECD Guideline 473 Chromosome Aberration Test in Human Lymphocytes in vitro:
The test item was considered to be weakly clastogenic to human lymphocytes in vitro.

OECD 476 In Vitro Mammalian Cell Gene Mutation Tests (CHO HPRT Forward Mutation Assay):
The test item was considered to be non-mutagenic to CHO cells at the HPRT locus.

Endpoint Conclusion:Adverse effect observed (positive)

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Single Cell Gel Electrophoresis (Comet) Assay in the Rat: in vivo: Key study (PS81LR)

Methods.

A range-finding test was performed to find suitable dose levels of the test item. The Comet assay main test was conducted at the maximum tolerated dose (MTD) 720 mg/kg with 360 mg/kg and 180 mg/kg as the lower dose levels. Animals were killed 4 hours after the second dose administration, the glandular stomach and liver tissues sampled and processed, the slides were then prepared prior to scoring for the presence of Comets.

Further groups of rats were given a double oral dose of water (seven rats) or N-Nitroso-N-methylurea (5 rats), to serve as vehicle and positive controls respectively.

Bone marrow was taken from the MTD and the vehicle control animals and processed for bone marrow smears to assess the PCE to NCE ratio.

Results

The presence of clinical signs at the MTD dose level indicated that systemic absorption had occurred and the results of the plasma analysis demonstrated the presence of Carbohydrazide in the blood plasma. The assessment of the bone marrow slides from the vehicle and MTD dose groups also demonstrated some toxicity in the MTD dose group indicating that absorption had occurred.

The test item was tested up to the maximum tolerated dose level of 720 mg/kg using male animals only. Three premature deaths occurred in the main test at the 720 mg/kg dose level after the second dose was administered and before the 28 hour time point. There was no evidence of an increase in the percentage tail intensity or median percentage intensity in animals dosed with the test item dose groups when compared to the concurrent vehicle control group. The positive control material produced a marked increase in the percentage tail intensity and median percentage tail intensity in both the liver and glandular stomach, indicating that the test method was working as expected.

Conclusion

The test item did not induce any increases in the percentage tail intensity or median percentage tail intensity in the liver or glandular stomach and therefore the test item was considered to be unable to induce DNA strand breakage to these tissues in vivo, under the conditions of the test.

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

Additional information

Justification for classification or non-classification

Based on the results of in-vitro testing the substance needs to be considered for classification as a germ cell mutagen, due to the following positive results:

OECD Guideline 471 Bacterial Reverse Mutation Test "Ames Test": The test material was considered to be mutagenic under the conditions of this test.

OECD Guideline 473 Chromosome Aberration Test in Human Lymphocytes in vitro: The test item was considered to be weakly clastogenic to human lymphocytes in vitro.

However, a secondary Ames study was negative and the substance was non-mutagenic to CHO cells at the HPRT locus (in a CHO HPRT Forward Mutation Assay). Therefore, the in-vitro study results are not conclusive for classification based on the following information.

- one positive and one negative Ames study

- the weak positive result to human lymphocytes in vitro was only observed in the continuous exposure group, at a single dose level which exhibited optimum levels of toxicity and therefore may be due to a cytotoxic mode of action rather than a true genotoxic mechanism.

Classification should be based on a total weight of evidence available. When classification is based on single tests (i.e. the positive results above) they should provide clear and unambigously positive results. It is considered that the results of the in-vitro studies do not provide unambigously positive results.

In addition, these in-vitro results are not supporting any in-vivo data and there is no known chemical structure activity relationship to known germ cell mutagens.

Based the in vitro information available, a further in-vivo testing was proposed to decide on a definitive classification. In the Single Cell Gel Electrophoresis (Comet Assay) conducted in the Rat, the test item was tested up to the maximum tolerated dose level of 720 mg/kg and did not induce any increases in the percentage tail intensity or median percentage tail intensity in the liver or glandular stomach. Therefore, the test item was considered to be unable to induce DNA strand breakage to these tissues in vivo.

Based on the available results, is considered that the substance does not meet the criteria for classification as a germ cell mutagen.