2-Chloro-5-chloromethylpyridine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

2-Chloro-5-chloromethylpyridine showed a mutagenic effect in a Salmonella/microsome test (strains TA 98, TA 100, TA 1535 and TA 1537) in strain TA 100 without and with metabolic activation (Herbold, 1991). In a HPRT test (V79 cells; +/- S9 mix) a negative result was observed after exposure to 2-Chloro-5-chloromethylpyridine (Herbold, 2003). A chomosome aberration test in vitro (V79 cells) revealed a clastogenic effect in the absence and in the presence of S9 mix after 4h treatment starting at 14 µg/ml (Nern, 2005).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

July to Aug 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

yes

Remarks:

no strains with an AT base pair at the primary reversion site (E. coli WP2 strains or S. typhimurium TA 102) were used to detect cross-linking mutagens

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from Aroclor 1254 induced rat livers

Test concentrations with justification for top dose:

40, 60, 80, 1000, 1400, 1800 µg/plate (first test);
400, 600, 800, 1000, 1400, 1800 µg/plate (repeat test)

Vehicle / solvent:

Solvents used: ethanol (test substance), DMSO (positive controls)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, nitrofurantoin and 4-nitro-1,2-phenylene diamine were only used without S9 mix and 2-aminoanthracene was only used with S9 mix

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice the amount of negative controls, whereas for TA 1537, at least a threefold increase should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Statistics:

not specified

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

weak strain-specific bacteriotoxic effect at 400 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA 98, TA 1535, TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

weak strain-specific bacteriotoxic effect at 400 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

2-Chloro-5-chloromethylpyridine was investigated using the Salmonella/microsome test for point mutagenic effects in doses up to 1800 µg per plate on four Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98.

Doses up to and including 80 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a weak, strain-specific bacteriotoxic effect, so that this range could nevertheless be used for assessment purposes.

Evidence of mutagenic activity of 2-chloro-5-chloromethylpyridine was seen. On Salmonella typhimurium TA 100, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Both with and without S9 mix, there was a positive response and the effect was comparable. The lowest reproducible effective dose was 1000 µg per plate for Salmonella typhimurium TA 100. The Salmonella/ microsome test thus showed 2-chloro-5-chloromethylpyridine to have a weak but definite mutagenic effect.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Conclusions:

positive

Executive summary:

The mutagenic potential of 2-chloro-5-chloromethylpyridine was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471. Doses up to and including 80 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a weak, strain-specific bacteriotoxic effect. Evidence of mutagenic activity of 2-chloro-5-chloromethylpyridine was seen. On Salmonella typhimurium TA 100, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Both with and without S9 mix, there was a positive response and the effect was comparable. The lowest reproducible effective dose was 1000 µg per plate for Salmonella typhimurium TA 100. The Salmonella/ microsome test thus showed 2-chloro-5-chloromethylpyridine to have a weak but definite mutagenic effect.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Dec 2002 to March 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male rat liver S9 mix

Test concentrations with justification for top dose:

1, 2, 3, 4, 5, 6, 9, 12, 15 µg/ml (-S9) and 3, 5, 6, 10, 12, 18, 20, 24, 30, 36, 40, 60, 80 µg/ml (+S9);

Vehicle / solvent:

Vehicle(s)/solvent(s) used: ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethylmethanesulfonate (EMS, 900 µg/ml, -S9); dimethylbenzanthracene (DMBA, 20 µg/ml, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 hours
- Expression time (cells in growth medium): normally 6 days
- Selection time (if incubation with a selection agent): 6 to 8 days

NUMBER OF REPLICATIONS: at least 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

- Mutant frequencies will only be used for assessment, if at least 5 dishes per culture were available and relative survival to treatment, relative population growth and absolute cloning efficiency were 10 % or greater.
- A trial will be considered positive if a concentration-related and in parallel cultures reproducible increase in mutant frequencies is observed. To be relevant, the increase in mutant frequencies should be at least two to three times that of the highest negative or negative control value observed in the respective trial. If this result can be reproduced in a second trial, the test substance is considered to be mutagenic.
- Despite these criteria, a positive result will only be considered relevant, if no significant change in osmolality compared to the negative control can be observed. Otherwise, unphysiological culture conditions may be the reason for the positive result (Scott et al, 1991).
- A test substance will be judged as equivocal if there is no strictly concentration related increase in mutation frequencies but if one or more concentrations induce a reproducible and biologically relevant increase in mutant frequencies in all trials.
- An assay will be considered negative if no reproducible and relevant increases of mutant frequencies were observed.

Statistics:

The statistical analysis relies on the mutant frequencies which are submitted to a weighted analysis of variance as well as to a weighted recursive regression, both with Poisson derived weights (Hsie et al., 1981; Arlett et al., 1989).

According to the acceptance criteria mutant frequencies based on less than 5 plates and/or on a relative survival to treatment and/or a relative population growth and/or an absolute cloning efficiency below 10% are not included in the statistical analysis.

All acceptable groups are included in the weighted analysis of variance followed by pairwise comparisons to the vehicle control on a nominal significance level of a = 0.05 using the Dunnett test (Dunnett, 1955). The regression analysis part is performed on the basis of the actual concentrations thereby omitting the positive, negative and vehicle controls. If there is a significant concentration related increase of the mutant frequency (a = 0.05) in the main analysis the highest concentration will be dropped and the analysis will be repeated. This procedure will be repeated until p > 0.05. In that way eliminated concentrations are flagged correspondingly.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 10 µg/ml and above (- S9 mix) or at 50 µg/ml and above (+ S9 mix) or

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

SELECTION OF TEST CONCENTRATIONS:
A preliminary cytotoxicity test was conducted without and with metabolic activation using concentrations of 2-Chlor-5-chlormethylpyridin ranging from 5 ug/ml to 4000 ug/ml. Concentrations of up to 4000 ug/ml 2-Chlor-5-chlormethylpyridin did not change the pH in the medium of the pre-test. The osmolality in the medium of the pre-test was only slightly altered by 4000 ug/ml 2-Chlor-5-chlormethylpyridin. Precipitation of 2-Chlor-5-chlormethylpyridin in the culture medium started at 4000 ug/ml. Without S9 mix cytotoxicity was evident at 10 ug/ml and above. With S9 mix cytotoxic effects of 2-Chlor-5-chlormethylpyridin were observed at 50 ug/ml and above. Due to these findings, 2-Chlor-5-chlormethylpyridin was tested in the mutation experiments in concentrations ranging from 1 ug/ml to 15 ug/ml without metabolic activation and from 3 ug/ml to 80 ug/ml with metabolic activation.

CLONING EFFICIENCY:
The absolute cloning efficiencies for the vehicle controls in the mutation experiments varied from 51.0 % to 70.3 % without activation and from 51.8 % to 113.7 % with activation, thus demonstrating good cloning conditions for the experiments.

CONCURRENT VEHICLE NEGATIVE AND POSITIVE CONTROL DATA:
The positive controls EMS and DMBA had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant frequencies as compared to the corresponding negative controls and thus demonstrated the sensitivity of the test system and the activity of the used S9 mix.

Without and with S9 mix 2-Chlor-5-chlormethylpyridin induced decreases in survival to treatment and decreases in relative population growth. These results revealed a significant concentration-related cytotoxicity of 2-Chlor- 5-chlormethylpyridin. Precipitation of 2-Chlor-5-chlormethylpyridin in the culture medium was not observed.

Without and with S9 mix there was no biologically relevant increase in mutant frequency above that of the vehicle controls.

Based on these results, 2-Chlor-5-chlormethylpyridin is considered to be non-mutagenic in the V79/HPRT Forward Mutation Assay, both with and without metabolic activation.

Conclusions:

negative

Executive summary:

2-Chlor-5-chlormethylpyridin was evaluated for point mutagenic effects at the hypoxanthine-guanine phosphoribosyl transferase locus (forward mutation assay) in V79 cell cultures according to OECD TG 476. Without and with S9 mix the test substance induced decreases in survival to treatment and decreases in relative population growth. These results revealed a significant concentration-related cytotoxicity of the test substance. Without and with S9 mix there was no biologically relevant increase in mutant frequency above that of the vehicle controls. Based on these results, the test substance was considered to be non-mutagenic in the V79/HPRT Forward Mutation Assay, both with and without metabolic activation.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

May to July 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: PAA Ready Mix (PAA, Paching, Austria), consists of MEM, Earle, with supplements.
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from the liver of Aroclor 1254 induced male Sprague Dawley rats

Test concentrations with justification for top dose:

4 hr treatment and 18 hr harvest:
- S9 mix: 3, 6, 12, 13, 14 µg/mL; + S9 mix: 7, 14, 28, 33, 38 µg/mL

4 hr treatment and 30 hr harvest:
- S9 mix: 12, 13, 14 µg/mL; + S9 mix: 28, 33, 38 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test substance was soluble in ethanol up to 500 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

PRE-TEST FOR CYTOTOXICITY:
The selection of the concentrations used for the main study was based on the results of a pre-test in which cells were exposed with and without S9 mix for 4 hours to variousconcentrations of the test substance. Duplicate cultures were tested for every concentration and control. As indicators of cytotoxic effects, mitotic indices and numbers of surviving cells (survival index) were used. The mitotic index was determined by counting a total of 1000 cells per culture. The results of the solvent controls were set 100% and compared to the 2-Chlor-5-Chlormethylpyridin treated cultures.

Evaluation criteria:

ASSESSMENT CRITERIA:
An increased incidence of gaps of both types without concomitant increase of other aberration types was not considered as indication of a clastogenic effect.A test was considered positive, if there was a relevant and statistically significant increase in the aberration rate. A test was considered negative, if there was no such increase at any time interval. A test was also considered negative, if there were statistical significant values, which were, however, within the range of historical negative controls. A test was considered equivocal, if there was an increase above the range of historical negative controls which was statistically significant but not considered relevant, or if an increase occurred, which was considered relevant, but which was not statistically significant.

ASSAY ACCEPTANCE CRITERIA:
An assay was acceptable, if there was a biologically relevant increase in chromosome aberrations induced by the positive controls and if the numbers of aberrations for the negative controls were in the expected range based on results from our laboratory and from published studies.

Statistics:

The statistical analysis was performed by pair-wise comparison of 2-Chlor-5- Chlormethylpyridin-treated and positive control groups to the respective solvent control group. The mitotic index was statistically analyzed (provided that it was reduced compared to the mean of the respective solvent control) using the one-sided chi2-test. The numbers of metaphases with aberrations (including and excluding gaps) and of metaphases with exchanges were compared (provided that these data superceded the respective solvent control). The statistical analysis followed the recommendations outlined by Richardson et al. (1989). The one-sided chi2-test was used for the statistical evaluation. A difference was considered to be significant if the probability of error was below 5%.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Without S9 mix cytotoxic effects were observed at 12 ug/ml and above. With S9 mix cytotoxic effects were observed at 28 ug/ml and above. Precipitation in the medium was not observed.

Therefore, concentrations of 3, 6 and 14 ug/ml of 2-Chlor-5-Chlormethylpyridin were chosen for reading in the absence of S9 mix. In the presence of S9 mix 14, 28 and 38 ug/ml of 2-Chlor-5-Chlormethylpyridin were employed. All of these cultures harvested 18 hours after the beginning of the treatment were included. In addition, cultures treated in the absence of S9 mix with 14 ug/ml and harvested 30 hours after the beginning of the treatment were used. The same was true for cultures treated in the presence of S9 mix with 38 ug/ml.

Cultures treated with 2-Chlor-5-Chlormethylpyridin in the absence and in the presence of S9 mix showed biologically relevant and statistically significant increased numbers of aberrant metaphases, starting at 14 ug/ml 2-Chlor-5-Chlormethylpyridin.

The positive controls mitomycin C and cyclophosphamide induced clastogenic effects and demonstrated the sensitivity of the test system and the activity of the used S9 mix.

Conclusions:

positive

Executive summary:

The clastogenic potential of 2-Chlor-5-Chlormethylpyridin was evaluated in a chromosome aberration test on Chinese hamster V79 cells according to OECD TG 473. Without S9 mix cytotoxic effects were observed at 12 ug/ml and above. With S9 mix cytotoxic effects were observed at 28 ug/ml and above. Precipitation in the medium was not seen. Cultures treated with 2-Chlor-5-Chlormethylpyridin in the absence and in the presence of S9 mix showed biologically relevant and statistically significant increased numbers of aberrant metaphases, starting at 14 ug/ml. Based on this test, the test substance is considered to be clastogenic for mammalian cells in vitro.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The mutagenic potential of 2-chloro-5-chloromethylpyridine was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471 (Herbold, 1991). Doses up to and including 80 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a weak, strain-specific bacteriotoxic effect. Evidence of mutagenic activity of 2-chloro-5-chloromethylpyridine was seen. On Salmonella typhimurium TA 100, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Both with and without S9 mix, there was a positive response and the effect was comparable. The lowest reproducible effective dose was 1000 µg per plate for Salmonella typhimurium TA 100. The Salmonella/ microsome test thus showed 2-chloro-5-chloromethylpyridine to have a weak but definite mutagenic effect.

2-Chlor-5-chlormethylpyridin was evaluated for point mutagenic effects at the hypoxanthine-guanine phosphoribosyl transferase locus (forward mutation assay) in V79 cell cultures according to OECD TG 476 (Herbold, 2003). Without and with S9 mix the test substance induced decreases in survival to treatment and decreases in relative population growth. These results revealed a significant concentration-related cytotoxicity of the test substance. Without and with S9 mix there was no biologically relevant increase in mutant frequency above that of the vehicle controls. Based on these results, the test substance was considered to be non-mutagenic in the V79/HPRT Forward Mutation Assay, both with and without metabolic activation.

The clastogenic potential of 2-Chlor-5-Chlormethylpyridin was evaluated in a chromosome aberration test on Chinese hamster V79 cells according to OECD TG 473 (Nern, 2005). Without S9 mix cytotoxic effects were observed at 12 ug/ml and above. With S9 mix cytotoxic effects were observed at 28 ug/ml and above. Precipitation in the medium was not seen. Cultures treated with 2-Chlor-5-Chlormethylpyridin in the absence and in the presence of S9 mix showed biologically relevant and statistically significant increased numbers of aberrant metaphases, starting at 14 ug/ml. Based on this test, the test substance is considered to be clastogenic for mammalian cells in vitro.

Justification for classification or non-classification

Based on the study results a classification according to Regulation (EC) No. 1272/2008 (CLP) is not required.