Reaction mass of Benzeneethanol, 2-chloro-α-(1-chlorocyclopropyl)-α-(chloromethyl)- and Oxirane, 2-(1-chlorcyclopropyl)-2-[(2-chlorphenyl)methyl]-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic potential of the test material was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471. Doses up to and including 16 µg per plate did not cause any bacteriotoxic effects. At higher doses, the substance had a strong, strain-specific bacteriotoxic effect, so that this range could only be used to a limited extent up to and including 512 ug per plate for assessment purposes.

The positive controls increased mutant counts to well over those of the solvent controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix. None of the five strains used showed a dose-related and biologically relevant increase in mutant counts over those of the solvent controls in the preincubation test. This applied both to the tests with and without S9 mix. Due to these results the test item has to be regarded as non-mutagenic in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 13 May 2011 to 08 September 2011

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline S2A

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline S2B

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Metabolic activation system:

Rat liver derived metabolic activation system

Test concentrations with justification for top dose:

First test
In the absence of S9 mix - 3 hour treatment, 18 hour recovery: 60, 65 and 70 μg/mL.
In the presence of S9 mix (2% v/v) - 3 hour treatment, 18 hour recovery: 70, 80 and 100 μg/mL.
Second test
In the absence of S9 mix - 21 hour continuous treatment: 40, 45 and 52.5 μg/mL.
In the presence of S9 mix (5% v/v) - 3 hour treatment, 18 hour recovery: 140, 170 and
190 μg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

Experimental design: Duplicate human peripheral blood culture, were exposed to the solvent, test substance or positive control substances at appropriate concentrations in the following experiments
1. A cytogenetic experiment was conducted using a sample of pooled blood in the presence and absence of S9-mix. Cells were exposed to the test substance and control for a period of 3-h both in the presence and absence of S9-mix. Solvent, untreated and positive control cultures were included.
2. A second independent cytogenetic experiment was conducted using a sample pooled blood in the presence and absence of S9-mix. Cells were exposed to the test substance and control substances for a period of 3-h in the presence of S9-mix and 20-h in the absence of S9-mix. Solvent, untreated and positive control cultures were included. Treatment of the cultures started approx 48-h after culture initiation. A single sampling time, 20-h after the start of treatment was used. The sampling time of 20-h after the start of treatment used in this study was based on a measured mean cell cycle time for cultured human lymphocytes of 13.5-h. In both experiment, a range of concentration of the test substance was used in order to define suitable concentrations for chromosomal aberration analysis.

Culture establishment: Human blood samples were obtained by venepunture in lithium heparin tubes on the days of culture from healthy, non-smoking donors. Equal volume of blood from three females donors were polled together for each experiment. All donors have a previously established low incidence of chromosomal aberrations in their peripheral blood lymphocytes. At 0-h, cultures (10 mL) were established by the addition of 0.5 mL of whole blood to RPMI-1640 tissue culture medium supplemented with approx 10% foetal bovine serum (FBS), 1.0 IU/mL heparin, 100 IU/mL penicillin, and 100 µg/mL streptomycin. The lymphocytes were stimulated to enter cell division by addition of phytohaemagglutinin (5% v/v) and were maintained at 37⁰C for 48-h with gentle mixing.

Metabolic activation system: The metabolic activation system was prepared as a 1:1 mixture of S9 fraction and cofactor solution. S9 was prepared from male SD rats dosed once daily by oral gavage for 3 d with a combined phenobarbital and β-napthoflavone corn-oil preparation. The treated animals were sacrificed on the day following the third dose. A 25% w/v homogenate was prepared. The co-factor solution was prepared as a single stock solution of Na2HPO4, KCl, glucose-6-phosphate NADP (Na salt) and MgCl2 in sterile double deionized water and adjusted to a final pH of 7.4.

Culture treatment: Prior to treatment, the cultures to be treated for a 20-h period were centrifuged and the culture medium was replaced with fresh supplemented RPMI-1640 culture medium. The cultures to be treated for a 3-h period did not receive a medium change at this point. Approx 48-h after culture establishment, aliquots of the test substance, solvent control or positive controls were administered to duplicate cultures. In addition, 200 µL of a 1:1 mix of S9 and co-factor solution was added to each culture to be treated in the presence of S9-mix. Cultures from both experiments in the presence of S9-mix and cultures from experiment 1 in absence of S9-mix were treated for a period of 3-h at 37⁰C, after which the culture medium was removed following centrifugation and replaced with fresh supplemented medium. The cultures were re-incubated at 37⁰C for a remainder of the 68-h growth period. Cultures from experiment 2 in the absence of S9-mix were treated for a period of 20-h until, the end of the 68-h growth period. The effect of the test substance on the pH and osmolality of the culture medium was investigated using single cultures containing medium only as changes in pH and increases in osmolality have been reported to result in the production on chromosomal aberrations.

Culture harvesting: Approx 2-h prior to harvesting the cultures were treated with colcimid. 68-h after the culture establishment the culture were centrifuges, the supernatant was removed and the cells were re-suspended in approx 10 mL of 0.075 KCl at room temperature for approx 10 min. The cultures were centrifuged, the supernatant was removed and the remaining cells fixed in freshly prepared metnanol/glacial acetic acid fixtative added dropwise and make up to a volume of approx 10 mL. The fixative was removed following centrifugation and replaced with freshly prepared fixative. After at least 2 subsequent changes of foxtative, slides were made by dropping the cell suspension on to clean moist labeled microscope slides. The slides were air dried, stained in filtered Giemsa stain double ionized water for min, rinsed in water, air-dried and mounted with cover slip in DPX.

Evaluation criteria:

The percentage of aberrant metaphases was calculated for each treatment scored both including and excluding cells with only gap-type aberrations. The Fischer Exact Probability test (one-sided) was used to evaluate statistically the percentage or metaphase showing aberration (excluding cell with only gap-type aberrations). Data from each treatment group in the presence and absence or S9-mix was compared with the respective solvent control group values. The data was interpreted as follows:
1. No statistically significant increase in the percentage or aberrant cells at any concentration above concurrent solvent control values –Negative.
2. A statistically significant increase in the percentage of aberrant cells above concurrent solvent control values which falls within the laboratory solvent control range-Negative.
3. An increase in the percentage of aberrant cells at least at one concentration which is substantially greater than the laboratory historical solvent control value-Positive.
4. A statistically significant increase in the percentage of aberrant cells which is above concurrent solvent values and which is above the historical solvent control range upper value but below that described above may require further evaluation.

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

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

Additional information on results:

Determination of mitotic indices and selection of concentrations: The highest concentration selected for chromosomal aberration was 500 µg/mL following a 3-h treatment period and 100 µg/mL following a 20-h treatment period. Concentration related reduction in mitotic activity and cytotoxic effects on the chromosomal morphology were observed in cultures from both experiments, thus demonstrating that the test substance is biologically active in this test system. Significant reductions in mean mitotic activity, compared to the solvent control values, were observed in cultures from both experiments treated with the highest concentration of the test substance selected for chromosomal aberration analysis. Cultures treated in higher concentration of the test substance were considered not to be suitable for chromosomal aberration analysis due to lack of metaphase as a result of toxicity or severe cytotoxic effects on chromosome structure. No significant effect on osmolality was observed.

Results of chromosomal aberration analyses: Statistically significant increase in the percentage of aberrant cells, above the solvent control values were recorded in cultures in experiment 1 and 2 treated with the test substance in the presence and absence of S9-mix.

Conclusions:

Under the test conditions, the test material has shown no evidence of causing an
increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test
system in the presence and absence of S-9.

Executive summary:

The potential of structural chromosome aberrations was determinded in an in vitro cytogenetic test with human lymphocytes according to OECD Guideline 473. 

Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin, and exposed to the test substance both in the absence and presence of S9 mix derived from rat livers. Solvent and positive control cultures were also included. Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal
damage.
In order to determine the toxicity of 2-(2-Chlorbenzyl)-2-(1 chlorcyclopropyl) oxiraneto cultured human lymphocytes, the mitotic index was assessed for all cultures treated with the test substance and the solvent control,
dimethyl sulphoxide (DMSO). Justification for concentration selection was based on
cytotoxicity.
On the basis of these data, the following concentrations were selected for metaphase analysis:
First test
In the absence of S9 mix - 3 hour treatment, 18 hour recovery: 60, 65 and 70 μg/mL.
In the presence of S9 mix (2% v/v) - 3 hour treatment, 18 hour recovery: 70, 80 and 100 μg/mL.
Second test
In the absence of S9 mix - 21 hour continuous treatment: 40, 45 and 52.5 μg/mL.
In the presence of S9 mix (5% v/v) - 3 hour treatment, 18 hour recovery: 140, 170 and 190 μg/mL.
In both the absence and presence of S9 mix, 2-(2-Chlorbenzyl)-2-(1-chlorcyclopropyl)oxirane caused no statistically significant
increases in the proportion of metaphase figures containing chromosomal aberrations, at any concentration, when compared with the solvent control, in either test.
No statistically significant increases in the proportion of polyploid cells were observed during metaphase analysis, in either test.
All positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.
It is concluded that the test item has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March - April 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Solubility and stability of the test substance in the solvent/vehicle: A stability test in the solvent did not reveal significant degradation of the active ingredient

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from male Sprague Dawley rats (for induction one single i.p. injection of Aroclor 1254)

Test concentrations with justification for top dose:

First test: 0, 16, 50, 158, 500, 1581 and 5000 µg/plate (+/- S9 mix). Due to bacteriotoxic effects at doses > 16 µg/plate this range could only be used to limited extent up to and including 512 µg per plate.
Repeat test: 0, 4, 8, 16, 32, 64, 128, 256 and 512 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

cumene hydroperoxide

mitomycin C

other: Nitrofurantoin, 4-Nitro-1,2-phenylene diamine, 2-Aminoanthracene

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 that of negative controls, whereas for TA1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Executive summary:

The mutagenic potential of the test material was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471. Doses up to and including 16 µg per plate did not cause any bacteriotoxic effects. At higher doses, the substance had a strong, strain-specific bacteriotoxic effect, so that this range could only be used to a limited extent up to and including 512 ug per plate for assessment purposes.

The positive controls increased mutant counts to well over those of the solvent controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix. None of the five strains used showed a dose-related and biologically relevant increase in mutant counts over those of the solvent controls in the preincubation test. This applied both to the tests with and without S9 mix. Due to these results the test item has to be regarded as non-mutagenic in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the study result for Genetic toxicity in vitro no classification according to Regulation (EC) No. 1272/2008 (CLP), ANNEX I, is warranted.