Reaction mass of 2-(3-ethylphenyl)oxirane and 2,2’-(1,3-phenylene)dioxirane and 2,2'-(1,4-phenylene)dioxirane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 08 June 2009 and 10 September 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 19 August 2008 Date of Signature: 04 March 2009

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-(3-ethylphenyl)oxirane and 2,2’-(1,3-phenylene)dioxirane and 2,2'-(1,4-phenylene)dioxirane

EC Number:

935-853-6

IUPAC Name:

Reaction mass of 2-(3-ethylphenyl)oxirane and 2,2’-(1,3-phenylene)dioxirane and 2,2'-(1,4-phenylene)dioxirane

Constituent 2

Reference substance name:

Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane

IUPAC Name:

Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane

Details on test material:

- Name of test material (as cited in study report): Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane
- Substance type: Multiconstituent
- Physical state: clear extremely pale yellow liquid
- Analytical purity: 80 - 95%
- Lot/batch No.: 200901317-17
- Stability under test conditions : stable
- Storage condition of test material : approximately 4°C in the dark

Method

Target gene:

Not applicable.

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone and beta-naphthoflavone induced rat live, S9

Test concentrations with justification for top dose:

Preliminary Toxicity test:
0, 0.16, 0.31, 0.625, 1.25, 2.5, 5, 10, 15, 20, 30.

Experiment 1:
Without S9-mix: 0*, 1.25*, 2.5*, 5*, 10*, 15, 20, MMC 0.4*
With S9-mix: 0*, 5, 10*, 15*, 20*, 30*, 40, CP 5*
Experiment 2:
Without S9-mix: 0*, 1.25*, 2.5*, 5*, 10*, 15, 20, MMC 0.4*
With S9-mix: 0*, 5, 10*, 15*, 20*, 30*, 40, CP 5*

* Dose levels selected for metaphase analysis

Vehicle / solvent:

The test material was accurately weighed, dissolved in Dimethyl Sulphoxide (DMSO) and serial dilutions prepared. The test material was considered to be a mixture and therefore the maximum dose level was 5000 µg/ml, the maximum recommended dose level.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl Sulphoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9

Migrated to IUCLID6: (CP)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl Sulphoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9

Migrated to IUCLID6: (MMC)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in medium


DURATION
- Preincubation period:
48 hrs

- Exposure duration:
Experiment 1 - 4 hrs with and without S9.
Experiment 2 - 4 hrs with and without S9.

- Expression time (cells in growth medium):
20 hrs for 4 hrs exposure.

- Selection time (if incubation with a selection agent):
Not applicable.

- Fixation time (start of exposure up to fixation or harvest of cells):
24 hrs.


SELECTION AGENT (mutation assays):
No selection agent.

SPINDLE INHIBITOR (cytogenetic assays):
Demecolcine

STAIN (for cytogenetic assays):
When the slides were dry they were stained in 5% Gurrs Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.


NUMBER OF REPLICATIONS:
Duplicate cultures


NUMBER OF CELLS EVALUATED:
100/culture


DETERMINATION OF CYTOTOXICITY
- Method:
mitotic index - A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

-Scoring of Chromosome Damage:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there was approximately 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

OTHER EXAMINATIONS:
- Determination of polyploidy:
Frequency of polyploid cells


OTHER:
None.

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Results and discussion

Test results

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Refer to information on results and attached tables.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test material was dosed into media.
- Effects of osmolality: The osmalality did not increase by more than 50 mOsm.
- Evaporation from medium: Not applicable.
- Water solubility: Not applicable, the test material was accurately weighed, dissolved in Dimethyl Sulphoxide (DMSO)
- Precipitation:
Premlinary toxictiy test: Precipitate observations were recorded at the beginning and end of the exposure periods. No precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure in any of the three exposure groups.

Experiment 1: No precipitate of the test material was observed at the beginning or end of exposure.

- Other confounding effects: Not stated

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was initially 19.53 to 5000 µg/ml. The maximum dose was the maximum recommended dose level. However due to excessive toxicity observed in all dose groups of the preliminary with no surviving dose levels the test was repeated. The revised dose levels were 0.625 to 40 µg/ml for the 4(20)-hour exposure groups and 0.16 to 20 µg/ml for the 24-hour continuous exposure group. No precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure in any of the three exposure groups. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 20 µg/ml in the 4(20)-hour exposure in the absence of metabolic activation (S9) and up to 40 µg/ml in the 4(20)-hour exposure in the presence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was 15 µg/ml. The mitotic index data are presented in Table 1. The test material induced clear evidence of toxicity in all three exposure groups.

The selection of the maximum dose level was based on toxicity and was 20 and 40 µg/ml for the 4(20)-hour with and without S9 exposure groups respectively.

COMPARISON WITH HISTORICAL CONTROL DATA: All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
EXPERIMENT 1:
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present at 20 µg/ml in the 4(20)-hour without S9 exposure group and at 40 µg/ml in the 4(20)-hour with S9 exposure group. No precipitate of the test material was observed at the beginning or end of exposure.

The mitotic index data are given in Table 2 (attachment 1). They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that near optimum mitotic inhibition of 55% was achieved at 10 µg/ml in the absence of S9. In the presence of S9, 31% and 76% mitotic inhibition was achieved at 20 and 30 µg/ml respectively. The higher dose levels in both exposure groups had metaphases suitable for scoring but the toxicity was such that it was beyond the acceptable limits set out in the guidelines.
The maximum dose level selected for metaphase analysis was based on toxicity and was 10 µg/ml for the without S9 exposure group and 30 µg/ml for the with S9 exposure group.

The chromosome aberration data are given in Table 3 and Table 4 (Attachment 1). All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test material induced statistically significant increases in the frequency of cells with aberrations in the both the absence and presence of metabolic activation. There was a clear dose related positive response in both exposure groups. Whilst the toxicity observed at 30 µg/ml (76%) in the presence of S9 was excessive, and the data obtained would be considered to be of little biological relevance, a clear positive response was observed at the next dose level down with only 31% toxicity.

The polyploid cell frequency data are given in Table 5 (attachment 1). The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

The study metaphase analysis was terminated after Experiment 1 because a statistically significant increase in structural aberrations was observed in the 4(20)-hour exposures for Experiment 1. The response was considered to be clear evidence of clastogenic activity and in such cases a second experiment is not necessary and not required by the test guidelines.

EXPERIMENT 2:
The exposure groups for this experiment were performed. However, due to the clear un‑equivocal positive response achieved in the first experiment the slides were not scored and no data is reported.

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Please see the following which are in attachment 1.

For the tables and figures of resluts mentioned above, please refer to the attached background material section for the following tables:

Table 1: Mitotic Index - Preliminary Toxicity Test

Table 2: Mitotic Index - Experiment 1

Table 3: Results of Chromosome Aberration Test - Experiment 1 Without Metabolic Activation (S9)

Table 4: Results of Chromosome Aberration Test - Experiment 1 With Metabolic Activation (S9)

Table 5:  Mean Frequency of Polyploid Cells (%)

Appendix 1: Chromosome Structural Aberrations: Classification, Evaluation Criteria and Historical Control Data

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

The test material induced a statistically significant increase in the frequency of cells with chromosome aberrations in both the absence and presence of a liver enzyme metabolising system.

Executive summary:

Introduction. This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems in so far as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scottet al, 1990). The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008. The study design also meets the requirements of the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.

Methods. Duplicate cultures of human lymphocytes, treated with the test material, 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, 4 hours 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 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours 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. However, due to the clear positive response seen in Experiment 1 metaphase analysis was not performed for the slides of Experiment 2 and therefore no data from this experiment is reported.

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

All the positive control materials 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 material induced clear dose-related statistically significant increases in the frequency of cells with aberrations in the 4(20)-hour exposure groups in the absence and presence of metabolic activation in Experiment 1.

Conclusion. The test material was considered to be clastogenic to human lymphocytes in vitro.