1-[difluoro(trifluoromethoxy)methoxy]-1,2,2-trifluoroethylene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

MOVE3 was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

MOVE 3 was not mutagenic at the TK-locus of mouse lymphoma L5178Y cells in the absence and presence of metabolic activation (S9-mix) in the in vitro mammalian cell gene mutation test conducted according to OECD guideline No. 490.

MOVE 3 was not clastogenic and/or aneugenic to cultured human lymphocytes in the absence and presence of metabolic activation (S9-mix) in the in vitro micronucleus test in cultured human lymphocytes conducted according to OECD guideline No. 487.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17-Dec-2015 to 11-Mar-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Sponsor
- lot/batch No.of test material: 150525V10
- Expiration date of the lot/batch: October 2020
- Purity test date: October 2015
- Name of test material (as cited in study report): MOVE3
- Substance type:Clear colourless liquid
- Physical state: Liquid

FORM AS APPLIED IN THE TEST: Liquid

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 94, 469, 938, 1876, 2814, 3752 and 4690 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 938, 1876, 2814, 3752 and 4690 µg/plate
Experiment 2:
Without and with S9-mix: 938, 1876, 2814, 3752 and 4690 µg/plate
Experiment 3:
Without and with S9-mix: 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:
A solubility test was performed. The test item was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other:

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (pre incubation test)

Inclusion of a pre incubation step of 30 and 60 minutes in the first and second experiment respectively. During the pre incubation phase the temperature has been kept at 20˚C to avoid volatilisation of the test item.

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100, WP2uvrA) or more or a three-fold (TA1535, TA1537, TA98) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

Conclusions:

Interpretation of results (migrated information):
negative

Based on the results of this study it is concluded that MOVE3 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

In this study the mutagenic activity of MOVE 3 was evaluated in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay.

The procedures described in this study were based on the most recent OECD and EC guidelines. The study design has been developed considering the physic-chemical properties of the test item (low boiling liquid).

Since the test item is a low boiling liquid the pre-incubation method has been considered the more appropriate test design for guaranteeing the exposure of the bacteria cells.

 

MOVE3 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254) with the inclusion of a pre-incubation step for 30 and 60 minutes in the first and second mutation experiment, respectively. An additional experiment was performed at the top dose of 5000 μg/plate with all five tester strains with the inclusion of a pre-incubation step for 60 minutes. During the pre-incubation phase of the experiments the temperature has been kept at 20°C in order to avoid the volatilisation of the test item.

 

In the dose range finding test, the test item was tested up to concentrations of 4690 μg/plate in the absence and presence of 5% (v/v) S9-mix in the strains TA100 and WP2uvrA (pre-incubated by 70 rpm at 20°C for 30 minutes). MOVE3 did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose range finding test were reported as part of the first mutation assay.

 

Based on the results of the dose range finding test, the test item was tested in the first mutation assay at a concentration range of 938 to 4690 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98 (pre-incubated by 70 rpm at 20°C for 30 minutes).

 

In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 938 to 4690 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA and with an increased pre-incubation period up to 60 minutes.

 

The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

 

Since in the mutation assays the top dose of 5000 μg/plate was not reached, an additional mutation assay was performed in the absence and presence of 10% (v/v) S9-mix with the inclusion of a pre-incubation step for 60 minutes in the tester strains TA1535, TA1537, TA98, TA100 and TA102. In this third mutation assay, the top concentration of 5000 μg/plate was tested. The bacterial background lawn was not reduced and no biologically relevant decrease in the number of revertants was observed.

 

MOVE3 did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in follow-up experiments.

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that MOVE3 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2016 to October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 July 2016.

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Sponsor
- lot/batch No.of test material: 150525V10
- Expiration date of the lot/batch: October 2020
- Purity test date: October 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15 - 25 °C, protected from light
- Stability under test conditions: stable

FORM AS APPLIED IN THE TEST: The substance is a volatile liquid (BP = 19.5°C). It was tested in vapour form.

Target gene:

thymidine kinase (TK) locus on chromosome 11 in mouse lymphoma (L5178Y) cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The mouse lymphoma L5178Y cells (L5178Y tk +/- 3.7.2C line) used in the gene mutation assay were obtained from Dr. J. Cole, MRC Cell Mutation Unit, University of Sussex, United Kingdom. The chromosome number of these cells is 40 (stable aneuploid karyotype, 2n = 40).
The cells were stored as frozen stock cultures in liquid nitrogen. Subcultures were prepared from these stocks (stock 6 November 2009) for experimental use. Each new stock culture was checked for mycoplasma contamination, which was absent for the used batch (Appendix 3).

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

MOVE3 vapours were tested at the following target concentrations:
76, 60, 40, 20 and 10% (v/v)
76% (v/v) is the highest concentrations theoretically achievable, considering that the the atmosphere in the chamber consists of 19% O2, 5% CO2 and the test substance supplemented with N2.
No cytotoxycity pre-test was perfomed.

The actual concentrations were measured at start and end of exposure.
At the start of the exposure, measured concentrations were:
4h-exposure(with and withour S9-mix): 0.0; 9.4, 19.2, 36.4, 57.9, 73.4%
24h-exposure(without S9-mix): 0.0; 9.5, 19.1, 37.4, 57.4, 74.9%
(all within 10% of the target concentrations)
At the end of the exposure, measured concentrations were:
4h-exposure(with and withour S9-mix): 0.0; 7.3*, 18.1, 37.6, 55.3, 73.4%
24h-exposure(without S9-mix): 0.1**; 8.5*, 18.9, 37.4, 56.5, 71.1%
**Concentration probably caused by analyser zero drift.
* Deviation from target concentration more than 10%, reason unknown.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: N2

Untreated negative controls:

yes

Remarks:

Air (19% O2, 5% CO2, 76% N2)

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Remarks:

The exposure to the positive control was not conducted in incubator chambers, but by addition of 100μl stock solution of the control to a final volume of 10 ml containing the cells, followed by incubation at ca37C and ca5% CO2 in a humidified incubator.

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Since the test substance is a volatile liquid with a boiling point of ca. 20°C, cells in culture flasks were exposed in modular incubator chambers (Billups-Rothenburg, USA) to various concentrations. The atmosphere in the chamber consisted of 19% O2, 5% CO2 and the test substance supplemented with N2.

STUDY DESIGN
To 6,000,000 cells in 5 ml growth medium (with 10% horse serum), growth medium (with 0% horse serum) was added for treatment without the S9-mix to a final volume of 10 ml. For treatment with S9-mix, 1 ml 20% (v/v) S9 mix (§ 4.4) was also added.
The cells were exposed to the test substance or the negative (clean air) control for 4 and 24 h in the absence of S9-mix and for 4 h in the presence of S9-mix, at ca. 37°C and ca. 5% CO2 in a humidified incubator. In the absence of S9-mix, one single culture treated with 100 μl MMS for 4 and 24 h was used as a positive control substance at a final concentration of 0.1 mmol/l. In the presence of S9-mix, one single culture treated with 100 μl MCA was used as a positive control substance at a final concentration of 10 μg/ml.
At the start and end of the treatment, all cell cultures were checked visually and selected cultures were checked for viability by trypan blue exclusion.

GENE MUTATION ANALYSIS
The frequency of TFT-resistant mutants and the cloning efficiency of the cells were determined two days after starting the test. The number of cells was counted and the cloning efficiency of the cells was determined. To determine the frequency of TFT-resistant mutants, the cell suspensions were diluted to a density of 10,000 cells per ml in culture medium (with 20% horse serum) containing 4 μg TFT per ml. Portions (200 μl) of each dilution were transferred to each well of two 96-well microtiter plates, and the plates were incubated at ca. 37°C and ca. 5% CO2 in a humidified incubator. The incubation period was 12 and 11 days for the 4 h exposures and 24 h exposure, respectively.
After this period, the number of wells without growth of cells was counted and the cloning efficiency in the TFT plates (mutant cloning efficiency) was calculated.
The mutant colonies of the negative and positive controls were scored using the criteria of small and large colonies. In case the test substance induced a positive response, colony sizing was also performed at concentrations causing a positive response.

DETERMINATION OF CYTOTOXICITY
The cytotoxicity of the test substance was determined by measuring the relative initial cell yield, the relative suspension growth (RSG) and the relative total growth (RTG).
After the treatment period, the cultures were checked for visible aberrant effects (e.g. lysed cells) and the viability of the cells treated with the higher concentrations of test substance was checked. The medium containing the test substance, solvent control or positive control was removed and the cells were washed twice with culture medium (with 10% horse serum). Finally, the cells were suspended in culture medium (with 10% horse serum) and the number of cells was counted. The cell suspensions were diluted to 300,000 cells per ml and the cultures were incubated for about 44-48h at ca. 37°C and ca. 5% CO2 in a humidified incubator to allow near-optimal phenotypic expression of induced mutations.
After 20-24h and 44-48h the number of cells of all remaining cultures was counted. After 20-24h the cell suspensions were diluted, if required, to 300,000 cells per ml and further incubated at ca. 37°C and ca. 5% CO2 as described above. After 44-48h a portion of the cells was diluted in culture medium (with 20% horse serum) to 10 cells per ml for determining the cloning efficiency. The remaining cells were used for determining the frequency of TFT-resistant mutants. Portions (200 μl) of each dilution at 10 cells per ml were transferred to each well of two 96-well microtiter plates and the plates were incubated at ca. 37°C and ca. 5% CO2 in a humidified incubator. The incubation period was 12 and 11 days for the 4 h exposures and 24 h exposure, respectively.
After this period the number of wells without growth of cells was counted and the cloning efficiency was determined using the zero term of the Poisson distribution.

ANALYSIS OF RESULTS
The cloning efficiency of the cells was calculated from the total number of negative wells on the microtiter plates and the number of cells seeded per well.
To assess the cytotoxic effects of the test substance or the positive controls on the cells, the initial cell yield after the treatment period, the RSG and the RTG to that of the negative controls were calculated.
The CE of the cells was used, together with the CE on the TFT-containing plates, to calculate the MF. The MF was expressed as the number of TFT-resistant mutants per 1,000,000 clonable cells.

Rationale for test conditions:

According to OECD 490 conducting a 24h treatment is suggested in cases where the short-term treatment yields negative results and if there is information suggesting the need for longer treatment [e.g., nucleoside analogs, poorly soluble substances]. As the solubility of the test substance vapor in the culture medium was unknown, in this study, the first (4 h treatment groups) and second test (24 h treatment group) were performed simultaneously in one experiment for efficiency reasons.

Evaluation criteria:

A response was considered to be positive if the induced MF (MF above the spontaneous background mutant frequency) was more than 126 mutants per 1,000,000 clonable cells.

Any apparent increase in MF at concentrations of the test substance causing more than 90% cytotoxicity, was considered to be an artefact and not biologically relevant if at least one of the two following conditions was fulfilled:
- At least one test concentration with an RTG between 10% and 20% was tested with no evidence of mutagenicity.
- No evidence of mutagenicity was observed at all other test concentrations with an RGT > 10%.

The mutagenicity assay is regarded inconclusive if less than 4 analysable concentrations are obtained, if the highest concentration - in case it is set by cytotoxicity - results in insufficient cytotoxicity or if a positive response is obtained at only one concentration.

Providing that all acceptability criteria are fulfilled, a test substance is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the Global Evaluation Factor (GEF = 126 mutants per 1,000,000 clonable cells) and the increase is concentration related. The test substance is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test substance is then considered unable to induce mutations in this test system.

Although most studies will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the mutagenic potential of the test substance. Results may remain equivocal in this case.
Both numerical significance and biological relevance were considered together in the evaluation.

Statistics:

No statistical analysis was performed.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight cytotoxicity (RTG = 75%, 79% and 84% compared to the control) at the three highest concentrations (76, 60, 40% (v/v) )

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight cytotoxicity (RTG = 75%, 86% compared to the control) at the two highest concentrations (76, 60% (v/v) )

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

24h-exposure

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

No increase in MF above 126 mutants per 1,000,000 clonable cells compared to the control was observed at the MOVE3 concentrations of 10%(v/v) (RTG=53%, cytotox= 47%), 20%(v/v) (RTG = 36%, cytotox = 64%); 40%(v/v) (RTG = 7%, cytotox = 93%)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Severe cytotoxicity (RTG = 0%, 0% and 7%) at the three highest concentrations (76%, 60% and 40%(v/v)). Cytotoxicity (RTG = 36% and 53%) at the concentrations of 20% and 10%(v/v) respectively

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The OECD guideline 490 states that at least four test concentrations that meet the acceptability criteria (appropriate cytotoxicity, number of cells, etc) should be evaluated following exposure for 4h in the absence and presence of S9-mix and these conditions were met during this test. As the solubility of the test substance vapor in the culture medium was unknown, a longer treatment (24 h without S9) was conducted in parallel with the pulse treatment for efficiency reasons (to obtain additional data in case the results of the pulse treatment needed to be clarified) without a preliminary evaluation of the cytotoxicity.
In the 24 h exposure group, the three highest concentrations induced more than 90% cytotoxicity, which is beyond the acceptability range and therefore cannot be used for evaluation of mutagenicity due to the severity of the cytotoxicity. As a result, this part of the test does not exactly meet the acceptability criteria of evaluating at least four test concentrations. Since however, the cytotoxicity range from 100% to <10% was considered adequately covered under the study, it is concluded that appropriate exposure of cells to MOVE3 is demonstrated by the observed cytotoxicity and that sufficient data were obtained to justify a proper evaluation of mutagenicity. Exposure with the test substance for 4 h was therefore adequate. MOVE 3 is clearly negative under all the suitable conditions and even in case of 93% cytotoxicity for the 24h exposure.

Remarks on result:

other: at the two highest concentrations of 60% and 76% (v/v), an increase in the mean mutant frequency (MF) by more than 126 mutants per 1,000,000 clonable cells was observed.

Remarks:

The increase in mutant frequency was, however only observed at a concentration causing more than 99% cytotoxicity (RTG value <1%). Therefore, this increase is considered to be not biologically relevant.

At the end of the exposure the target concentration was met in all but one case (taking into account the margin of 10%), which was the lowest concentration of both the 4 h and 24 h exposure. In this case the deviation was -27% and -15% for the 4 h and 24 h exposure, respectively. It should be noted that the second sample is taken to demonstrate that the container/incubator was not leaking. The data clearly demonstrated that the container/incubator was not leaking, since a larger reduction in concentration would have been expected in case of leakage. In general, the lower values obtained from the second sample may indicate that the test material was taken up in the culture medium or metabolized during exposure, but the actual reason in this study remains unknown. As the four higher concentrations in the 4h treatment were all within the 10% margin of the target concentrations, and the guideline requires at least four concentrations tested in duplicate in the 4hour treatment for a valid assay, the test was considered valid.

Conclusions:

In both the absence and presence of S9-mix no indication for a mutagenic potential was observed at any of the concentrations tested.
It is concluded that under the conditions used in this study, the test material MOVE 3 is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells in the presence of metabolic activation (S9-mix).

Executive summary:

The test material MOVE 3 was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells according to OECD Guideline method No.490 and in compliance with GLP.

 

A single test was conducted both in the absence and the presence of a metabolic activation system (S9-mix).

In the test, five duplicate cultures were treated for 4 hours in the presence and absence of S9-mix, and 24 hours in the absence of S9-mix. The highest nominal concentration of MOVE 3 evaluated for mutagenicity was 76% both in the absence (4 and 24 hours) and presence (4 hours) of S9-mix. As the atmosphere in the chamber consisted of 19% O2, 5% CO2 and the test material (supplemented with N2 to achieve lower concentrations), 76% (v/v) was the highest achievable concentration. Additional nominal concentrations were 60%, 40%, 20% and 10% (v/v). Target concentrations were analytically verified and the analytical results demonstrated that the actual exposure concentrations were acceptable.

Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and in the presence of S9-mix, respectively and clean air served as negative control in both tests. Negative and positive controls met all the acceptance criteria therefore the results were considered valid.

 

Following 4 h exposure in the absence and presence of S9-mix, the test material was slightly cytotoxic to the cells resulting in a reduction in the RTG. The mean RTG at the highest three concentrations evaluated (76%, 60% and 40%) was 75%, 79% and 84% in the absence and 75%, 86% and 102%, in the presence of S9-mix.

In the absence of S9-mix after 24h exposure severe cytotoxicity was observed, resulting in a reduction in RTG of more than 90% at the highest three concentrations tested (RTG of 0%, 0% and 7% following exposure to 76%, 60% and 40% MOVE 3). As a consequence, these three concentrations were considered not suitable for assessing the mutant frequency (MF) and only the two lower concentrations could be evaluated.

 

Following 4 h exposure in the absence and presence of S9-mix, no increase in the MF by more than 126 mutants per 1,000,000 clonable cells,i.e.no positive response, compared to the negative control was observed at any of the concentrations tested.

Following 24 h exposure in the absence of S9-mix, no increase in the MF by more than 126 mutants per 1,000,000 clonable cells, i.e. no positive response, compared to the negative control was observed at concentrations of 10%(v/v) and 20%(v/v). At the two highest concentrations of 60% and 76% (v/v), an increase in the MF by more than 126 mutants per 1,000,000 clonable cells was observed. The increase in mutant frequency was, however only observed at a concentration causing more than 99% cytotoxicity (RTG value <1%). Therefore, this increase is considered to be not biologically relevant. At a concentration of 40%(v/v), causing 93% cytotoxicity (RTG value ≥7%) no increase in mutant frequency was observed.

 

The OECD guideline 490 states that at least four test concentrations that meet the acceptability criteria (appropriate cytotoxicity, number of cells, etc) should be evaluated following exposure for 4h in the absence and presence of S9-mix and these conditions were met during this test. As the solubility of the test substance vapor in the culture medium was unknown, a longer treatment (24 h without S9) was conducted in parallel with the pulse treatment for efficiency reasons (to obtain additional data in case the results of the pulse treatment needed to be clarified).

 

Exposure with the test substance for 4 h was therefore adequate.

In the 24 h exposure group, the three highest concentrations induced more than 90% cytotoxicity, which is beyond the acceptability range and therefore cannot be used for evaluation of mutagenicity due to the severity of the cytotoxicity. As a result, this part of the test does not exactly meet the acceptability criteria of evaluating at least four test concentrations.

 

Since however, the cytotoxicity range from 100% to <10% was considered adequately covered in the 4h exposure, and since the appropriate exposure of cells in the 24h exposure to MOVE3 is demonstrated by the observed cytotoxicity, it is considered that sufficient data were obtained to justify a proper evaluation of mutagenicity.

MOVE 3 is clearly negative under all the suitable conditions and even in case of 93% cytotoxicity for the 24h exposure.

It is concluded that under the conditions used in this study, the test substance MOVE 3 is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells in the absence and presence of metabolic activation (S9-mix).

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2016 - December 017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Appearance : liquid, colorless
- Source: Sponsor
- lot/batch No.of test material: 150525V10
- Expiration date of the lot/batch: October 2020
- Purity test date: October 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15 - 25 °C, protected from light
- Stability under test conditions: stable

FORM AS APPLIED IN THE TEST: The substance is a volatile liquid (BP = 19.5°C). It was tested in vapour form.

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:Blood samples were obtained by venapuncture from two young healthy, non-smoking donors (28 and 37 years old) with no known recent exposures to genotoxic chemicals or radiation.
- Suitability of cells: Peripheral human lymphocytes are recommended in the international OECD guideline.
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable :non-smoking donors (28 and 37 years old), A different donor was used for the first and second experiment.
- whole blood
- Number of passages if applicable: Not applicable

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The medium for culturing the human peripheral blood lymphocytes consisted of RPMI 1640 medium (with HEPES and Glutamax-I), supplemented with heat-inactivated (30 min, 56ºC) fetal calf serum (20% v/v), penicillin (100 U/ml medium), streptomycin (100 μg/ml medium) and phytohemaglutinin (2.4 μg/ml).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

The S9-mix consisted of a liver homogenate fraction (S9) and cofactors

Test concentrations with justification for top dose:

The test was carried out with five different concentrations of the test substance and appropriate negative (air without test substance) and positive controls.
In the first experiment: 76, 60, 40, 20 and 10% (v/v, all ± 10%).
In the second experiment: in the pulse treatment groups both with and without S9-mix,76, 60, 40, 20 and 10% (v/v, all ± 10%). and in the continuous treatment group were based on the cytotoxicity observed during the first experiment and were as follows: 30, 20, 10, 5 and 2.5% (v/v, all ± 10%)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

clean air

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Vinblastine Sulphate

Details on test system and experimental conditions:

METHOD OF APPLICATION: ince the test substance is a volatile liquid with a boiling point of ca. 20°C, cells in culture flasks were exposed in modular incubator chambers (Billups-Rothenburg, USA) to various concentrations. The atmosphere in the chamber consisted of 19% O2, 5% CO2 and the test substance supplemented with N2. The highest concentration achievable was therefore 76% (v/v).

DURATION
- Preincubation period: Whole blood was incubated in the presence of phytohaemaglutinin for 48 ± 2 hours at ca. 37ºC in humidified air containing ca. 5% CO2.
- Exposure duration: pulse 4h and continuous 24h



NUMBER OF REPLICATIONS: duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Each culture was harvested and processed separately. The cells were harvested by low speed centrifugation, treated with a hypotonic solution (0.075 M potassium chloride), fixed three times with a freshly prepared mixture of methanol and acetic acid, spread on clean slides and air dried. All procedures were performed at ambient temperature. Three slides were prepared from the negative (clean air and solvent) controls, positive controls and from each culture treated with the test substance.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:

DETERMINATION OF CYTOTOXICITY
- Method:Quantitative evaluation of cytotoxicity was performed using the CBPI.

Evaluation criteria:

A response was considered positive if all of the following criteria were met:
- at least one of the test concentrations exhibited a statistically significant increase compared to the concurrent negative control
- the increase was concentration-related in at least in one experimental condition when evaluated with an appropriate trend test
- any of the results were outside the distribution of the historical solvent control data
A response was considered negative if all of the following criteria were met:
- none of the test concentrations exhibited a statistically significant increase compared to the concurrent negative control
- there was no concentration-related increase when evaluated with an appropriate trend test
- all results were inside the distribution of the historical negative control data

Statistics:

The frequencies of micronuclei were compared with those of the concurrent solvent controls using the Chi-Square test (one sided). The results were considered statistically significant if the P-value was less than 0.05.

Key result

Species / strain:

other: cultured human lymphocytes

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not assessed
- Effects of osmolality: Not applicable
- Evaporation from medium: Not applicable
- Water solubility: Not applicable
- Precipitation: Not applicable

RANGE-FINDING/SCREENING STUDIES:

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: see detailed results in section below.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: see table 1 to 5
- Indication whether binucleate or mononucleate where appropriate: see table 1 to 5

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see table 7 and 8
- Negative (solvent/vehicle) historical control data: see table 6


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI
- Other observations when applicable: N/A

In the first experiment, in both pulse treatment groups, the numbers of binucleated cells containing a micronucleus in the negative control cultures (clean air) were outside of the distribution of the historical solvent control data of the test facility. In addition, in the continuous treatment group, the test substance induced cytotoxicity resulting in an insufficient number of suitable concentrations for microscopic evaluation. Hence, all groups had to be repeated. Following the first experiment, a separate non-GLP pilot study was performed to investigate the reason for the fact that the negative control cultures were outside of the historical range. The pilot experiment showed that the increased background levels observed in the first experiment were probably not linked to the donor used for that experiment. In the pilot study a similar increase was observed in 1 on 4 instances of the groups treated within the exposure chamber. Moreover, it was observed that the increase in the number of binucleated cells containing a micronucleus did not correspond to an actual increase of the % aberrant metaphases. Therefore it was concluded that the increased background levels are more a chance finding than a reproducible finding. Consequently, a second experiment was performed including two cultures per group to serve as untreated incubator controls. In the second experiment, the numbers of binucleated cells containing a micronucleus in the untreated incubator control cultures in the pulse treatment groups were within the historical data range of the test facility. The numbers of binucleated cells containing a micronucleus in the incubator control cultures in the continuous treatment group (1.30%) was slightly outside historical data range, but well within the data range published in literature (range 0.4-1.5%) (Hogstedt, 1984, Fenech, 1993). Due to the changes in the OECD guideline, the historical solvent control data of the test facility for 24 hours treatment are limited and results are therefore also compared to the data presented in literature. In the pulse treatment group with S9-mix, the numbers of binucleated cells containing a micronucleus in the negative (clean air) control cultures (1.35%) were slightly outside the historical data of the test facility but clearly within the data range published in literature. In the pulse and continuous treatment groups without S9-mix, the numbers of binucleated cells containing a micronucleus in the clean air control cultures were within the historical data of the test facility. Treatment with the positive control substances (Cyclophosphamide and Vinblasine sulphate), resulted in statistically significant increases in the number of binucleated cells containing micronuclei, when compared to the numbers found in the concurrent control cultures. The response for Vinblastine sulphate (6.85) was slightly higher when compared to the historical positive control data range (2.20 – 6.35), but is considered compatible with this range. Due to the changes in the OECD guideline, the historical positive control data of the test facility for 24 hours treatment are limited. The results however indicate that the system was responsive to a genotoxic substance. Based on the information described above, the experiment is considered valid.

First experiment

In the first experiment in the pulse treatment groups both with and without S9-mix, the test substance was slightly cytotoxic to the cells in a dose related manner. In the absence of S9-mix, the test substance concentrations (76%, 60% and 40% (v/v%)) showed a cytotoxicity of 25%, 19% and 8%, respectively. In the presence of S9-mix, concentrations of 76%, 40% and 20% showed a cytotoxicity of 26%, 3% and 7%, respectively. In both pulse treatment groups, the concentrations (76%, 40% and 20%) together with negative (clean air) control and positive control cultures were selected for analysis of micronuclei induction ( Tables 1 and 2). In the continuous treatment group of the first experiment, during preparation of the slides (prior to CPBI determination), it was observed that at the concentrations of 76% and 60% of the test substance only cell debris was observed. At a concentration of 40%, only a few mononucleated cells were observed (no bi-nucleated cells were present). At a concentration of 20%, cell density was low (approx. 20%) when compared to the negative control. At a concentration of 10% test substance no aberrant findings were observed. As a consequence, an insufficient number of suitable concentrations was available for microscopic evaluation. Therefore, this treatment group was also repeated in the second experiment simultaneously with the pulse treatment groups.

Second experiment

In the second experiment, in the pulse treatment group with S9-mix, a dose related cytotoxicity was observed. In this treatment group, the test substance concentrations (76%, 60%, 40% and 20%) showed a cytotoxicity of 52, 39, 26 and 0%. The observed cytotoxicity is higher than in the first experiment. The difference may be due to the different donor used for the first and second experiment. Other than that, there is no explanation for this variation in cytotoxicity. At the lowest test substance concentration a cytotoxicity of 9% was observed when compared to the concurrent negative (clean air) control cultures. However, a cytotoxicity less than 10% is considered to be a fluctuation of the cytotoxicity baseline and not biologically relevant. In the pulse treatment group without S9-mix, the test substance concentrations (76%, 60%, 40% and 20%) showed a cytotoxicity of 26, 27, 15 and 0% when compared to the concurrent clean air control cultures. At a concentration of 10% no cytotoxicity was observed. In both pulse treatment groups, the concentrations (76%, 40% and 20%) together with negative (clean air) and untreated incubator control and positive control cultures were selected for analysis of micronuclei induction (Tables 3 and 4).

In the continuous treatment group without S9-mix a dose related cytotoxicity was observed. The highest test substance concentration (30%) was severely cytotoxic to the cells. The lower concentrations (20%, 10% and 5%) showed a cytotoxicity of 42%, 15% and 6%, respectively when compared to the concurrent clean air control cultures. Although the observed cytotoxicity of 42% was below the required maximal cytotoxicity of 50±5% according to the OECD guideline 487, the cell density at this concentration on the slides was low, indicating cell death in the cultures and potential underestimation of cytotoxicity. Therefore, the cytotoxicity of 42% was considered acceptable. In this group, the concentrations of 20%, 10% and 5% together with negative (clean air) and untreated incubator control and positive control cultures were selected for analysis of micronucleus induction ( Table 5).

In all treatment groups, the test substance did not show a statistically significant increase in the number of binucleated cells containing micronuclei, at any of the concentrations analyzed when compared to the numbers found in the concurrent control cultures. In addition, no dose related micronucleus induction was observed, except in the pulse treatment group without S9-mix. This observation was further investigated in the continuous treatment group and this result was not reproduced (also when compared to the first experiment). In the second treatment, in the pulse and continuous treatment groups without S9-mix, at the concentrations of 76% an 5% respectively, the observed number of binucleated cells containing micronuclei (1.50% and 1.40%, respectively) were slightly outside the limited historical data with clean air. However the observed micronuclei frequency was within the data presented in the literature (Hogstedt, 1984, Fenech, 1993). Therefore, these findings were considered not biologically relevant.

Table 1: Pulse treatment with metabolic activation (first experiment)

Treatment /recovery time (h)	Dose level (µg/ml)	Cell stage analysis/500 (MO-BN-MU)			BN (%)	CBPI	CBPI (mean)	RI (%)	% Cytotox. (100-RI)	Selected for MN analysis (+/-)	MNBN/ 1000BN	MNBN/ 2000 BN (%)	Statistics1(p-value)
	NC	241	252	7	50.4	1.532	1.528	100	0	+	19	40 (2.00)	-
		246	246	8	49.2	1.524					21
	76%	319	180	1	36.0	1.364	1.392	74	26	+	11	27 (1.35)	n.s.
		291	208	1	41.6	1.420					16
	60%	312	186	2	37.2	1.380	1.391	74	26	-	-	-	-
4/20		300	199	1	39.8	1.402					-
(+S9)	40%	266	232	2	46.4	1.472	1.513	97	3	+	18	29 (1.45)	n.s.
		228	267	5	53.4	1.554					11
	20%	258	239	3	47.8	1.490	1.492	93	7	+	18	36 1.80	n.s.
		254	245	1	49.0	1.494					18
	10%	271	226	3	45.2	1.464	1.477	90	10	-	-	-	-
		261	233	6	46.6	1.490					-
	CP20	399	101	0	20.2	1.202	1.211	40	60	+	64	120	<0.0001
		390	110	0	22.0	1.220					56	(6.00)	***

Treatm: treatment time; Cytotox: cytotoxicity; MO: mononucleated cells; BN: binucleated cells; MU: multinucleated cells; CBPI: Cytokinesis-Block Proliferation Index; RI: Replication index; MN: micronuclei MNBN: micronucleated binucleated cells; NC: negative control (clean air);CP: Cyclophosphamide; n.s: not significant compared to the concurrent control; - : not selected for micronuclei analysis;1Chi-square test (one sided);*** p≤0.0001

Table 2: Pulse treatment without metabolic activation (first experiment)

Treatment /recovery time (h)	Dose level (µg/ml)	Cell stage analysis/500 (MO-BN-MU)			BN (%)	CBPI	CBPI (mean)	RI (%)	% Cytotox. (100-RI)	Selected for MN analysis (+/-)	MNBN/ 1000BN	MNBN/ 2000 BN (%)	Statistics1(p-value)
	NC	262	231	7	46.2	1.490	1.485	100	0	+	26	45 (2.25)	-
		269	222	9	44.4	1.480					19
	76%	309	191	0	38.2	1.382	1.365	75	25	+	19	40 (2.00)	n.s.
		327	172	1	34.4	1.348					21
	60%	319	181	0	36.2	1.362	1.393	81	19	+	17	34 (1.70)	n.s.
4/20		290	208	2	41.6	1.424					17
(-S9)	40%	283	215	2	43.0	1.438	1.445	92	8	+	12	32 (1.60)	n.s.
		276	222	2	44.4	1.452					20
	20%	237	262	1	52.4	1.528	1.521	107	0	-	-	-	-
		246	251	3	50.2	1.514					-
	10%	260	234	6	46.8	1.492	1.482	99	1	-	-	-	-
		272	220	8	44.0	1.472					-

Treatm: treatment time; Cytotox: cytotoxicity; MO: mononucleated cells; BN: binucleated cells; MU: multinucleated cells; CBPI: Cytokinesis-Block Proliferation Index; RI: Replication index; MN: micronuclei; MNBN: micronucleated binucleated cells; NC: negative control (clean air); ns : not significant compared to the concurrent control; - : not selected for micronucleianalysis

Table 3: Pulse treatment with metabolic activation (second experiment)

Treatment /recovery time (h)	Dose level (µg/ml)	Cell stage analysis/500 (MO-BN-MU)			BN (%)	CBPI	CBPI (mean)	RI (%)	% Cytotox. (100-RI)	Selected for MN analysis (+/-)	MNBN/ 1000BN	MNBN/ 2000 BN (%)	Statistics1(p-value)
	NC	274	225	1	45.00	1.454	1.450	100	0	+	15	27 (1.35)	-
		278	221	1	44.20	1.446					12
	NC*	271	228	1	45.60	1.460	1.437	97	3	+	12	22 (1.10)	n.s.
		295	203	2	40.60	1.414					10
	76%	378	122	0	24.40	1.244	1.215	48	52	+	9	21 (1.05)	n.s.
		407	93	0	18.60	1.186					12
4/20	60%	358	140	2	28.00	1.288	1.273	61	39	-	-	-	-
(+S9)		371	129	0	25.80	1.258					-
	40%	319	181	0	36.20	1.362	1.335	74	26	+	11	23 (1.15)	n.s.
		349	148	3	29.60	1.308					12
	20%	275	225	0	45.00	1.450	1.482	107	0	+	12	22 (1.10)	n.s.
		243	257	0	51.40	1.514					10
	10%	294	206	0	41.20	1.412	1.409	91	9	-	-	-	-
		297	203	0	40.60	1.406					-
	CP20	410	90	0	18.00	1.180	1.165	37	63	+	27	58	<0.0001
		425	75	0	15.00	1.150					31	(2.90)	***

Treatm: treatment time; Cytotox: cytotoxicity; MO: mononucleated cells; BN: binucleated cells; MU: multinucleated cells; CBPI: Cytokinesis-Block Proliferation Index; RI: Replication index; MN: micronuclei; MNBN: micronucleated binucleated cells; NC: negative control (clean air); NC*: negative control (incubator control); CP: Cyclophosphamide; n.s: not significant compared to the concurrent control; - : not selected for micronuclei analysis; 1Chi-square test (one-sided); *** p≤0.0001

Table 4: Pulse treatment without metabolic activation (second experiment)

Treatment /recovery time (h)	Dose level (µg/ml)	Cell stage analysis/500 (MO-BN-MU)			BN (%)	CBPI	CBPI (mean)	RI (%)	% Cytotox. (100-RI)	Selected for MN analysis (+/-)	MNBN/ 1000BN	MNBN/ 2000 BN (%)	Statistics1(p-value)
	NC	261	238	3	47.41	1.486	1.458	100	0	+	10	19 (0.95)	-
		288	209	3	41.80	1.430					9
	NC*	274	221	5	44.20	1.462	1.462	101	0	+	10	19 (0.95)	n.s.
		272	225	3	45.00	1.462					9
	76%	352	147	1	29.40	1.298	1.337	74	26	+	18	30 (1.50)	n.s.
		312	188	0	37.60	1.376					12
4/20	60%	370	130	0	26.00	1.260	1.335	73	27	-	-	-	-
(-S9)		295	205	0	41.00	1.410					-
	40%	283	217	0	43.40	1.434	1.389	85	15	+	10	23 (1.15)	n.s.
		328	172	0	34.40	1.344					13
	20%	262	234	4	46.80	1.484	1.470	103	0	+	9	18 (0.90)	n.s.
		277	218	5	43.60	1.456					9
	10%	271	228	1	45.60	1.460	1.465	102	0	-	-	-	-
		265	235	0	47.00	1.470					-

Treatm: treatment time; Cytotox: cytotoxicity; MO: mononucleated cells; BN: binucleated cells; MU: multinucleated cells; CBPI: Cytokinesis-Block Proliferation Index; RI: Replication index; MN: micronuclei; MNBN: micronucleated binucleated cells; NC: negative control (clean air); NC*: negative control (incubator control); n.s: not significant compared to the concurrent control; - : not selected for micronuclei analysis

Table 5: Continuous treatment without metabolic activation (second experiment)

Treatment /recovery time (h)	Dose level (µg/ml)	Cell stage analysis/500 (MO-BN-MU)			BN (%)	CBPI	CBPI (mean)	RI (%)	% Cytotox. (100-RI)	Selected for MN analysis (+/-)	MNBN/ 1000BN	MNBN/ 2000 BN (%)	Statistics1(p-value)
	NC	212	272	16	54.4	1.608	1.584	100	0	+	10	23 (1.15)	-
		231	258	11	51.6	1.560					13
	NC*	258	235	7	47.0	1.498	1.485	83	17	+	11	26 (1.30)	n.s.
		270	224	6	44.8	1.472					15
	30%	#	#	#	-	-	-	-	-	-	-	-	n.s.
		#	#	#	-	-					-
24	20%	328	172	0	34.4	1.344	1.337	58	42	+	12	23 (1.15)	n.s.
(-S9)		340	155	5	31.0	1.330					11
	10%	253	239	8	47.8	1.510	1.498	85	15	+	10	18 (0.90)	n.s.
		267	223	10	44.6	1.486					8
	5%	252	236	12	47.2	1.520	1.548	94	6	+	15	28 (1.40)	n.s.
		220	272	8	54.4	1.576					13
	2.5%	216	266	18	53.2	1.604	1.577	99	1	-	-	-	-
		235	255	10	51.0	1.550					-
	VB	426	72	2	14.4	1.152	1.177	30	70	+	75	137	<0.0001
	0.0125	403	93	4	18.6	1.202					62	(6.85)	***

Treatm: treatment time; Cytotox: cytotoxicity; MO: mononucleated cells; BN: binucleated cells; MU: multinucleated cells; CBPI: Cytokinesis-Block Proliferation Index; RI: Replication index; MN: micronuclei; MNBN: micronucleated binucleated cells; NC: negative control (clean air); NC*: negative control(incubatorcontrol);VB:Vinblastinesulphate;n.s:notsignificantcomparedtotheconcurrentcontrol;-:notselectedforslidepreparation;1Chi- square test (one-sided); # : no cells available on the slides; not selected for micronuclei analysis ***p≤0.0001

Historical data of the in vitro micronucleus test

Table 6 : Historical positive control data of the indirect acting clastogen Cyclophosphamide (in the presence of metabolic activation)

Treatment time / Recovery time (h)	Number of tests (n)	Dose level (µg/ml)	% of binucleated cells containing micronuclei/2000 binucleated cells (mean ± S.D.)	Range (%)
4 / 20 (+S9)	36	20	3.52 ± 1.02	1.80 – 6.30

Table 7 :Historical positive control data of the aneugenic compound Vinblastine sulphate (in the absence of metabolic activation)

Treatment time / Recovery time (h)	Number of tests (n)	Dose level (µg/ml)	% of binucleated cells containing micronuclei/2000 binucleated cells (mean ± S.D.)	Range (%)
24 /- (-S9)	6	0.0125	4.49 ±1.14	2.20 – 6.35

Tablz 8: Historical negative controls

Treatment time / Recovery time (h)	Number of tests (n)	Vehicle	% of binucleated cells containing micronuclei/2000 binucleated cells (Mean ± S.D.)	Range (%)
4 / 20 (+S9)	38	*	0.70 ± 0.17	0.40 – 1.20
4 / 20 (-S9)	38	*	0.69 ± 0.20	0.20 – 1.15
24 /- (-S9)	11	*	0.88 ± 0.15	0.65 – 1.15
24/- (-S9)	7	2% DMSO	1.11 ± 0.27	0.90-1.65

Conclusions:

It is concluded that the test substance MOVE 3 was not clastogenic and/or aneugenic to cultured human lymphocytes, under the conditions used in this study.

Executive summary:

The test substance, MOVE 3 was examined for its potential to induce micronuclei in cultured binucleated human lymphocytes, in the absence and presence of a liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix). The pulse treatment groups both with and without S9-mix and the continuous treatment group were performed simultaneously in both experiments. Duplicate cultures were used. The treatment/recovery time of the pulse and continuous treatment group was 4/20 and 24/0 hours, respectively. Cytotoxicity was determined from the Cytokinesis-Block Proliferation Index (CBPI).

In the first experiment, in both pulse treatment groups, the numbers of binucleated cells containing a micronucleus in the negative control cultures (clean air) were outside of the historical data range of the test facility. In addition, in the continuous treatment group, the test substance induced cytotoxicity resulting in an insufficient number of suitable concentrations for microscopic evaluation. Hence, all groups had to be repeated.

As the distribution of the historical solvent control data of the test facility for exposure to clean air are very limited, additional untreated incubator controls (without use of exposure chamber) were included. In the second experiment, the numbers of binucleated cells containing a micronucleus in the negative control (clean air) and untreated incubator control cultures were within historical data range of the test facility and / or within the data range published in literature (control data were also compared to literature data as the historical data of the test facility were limited). In both experiments, treatment with the positive controls Cyclophosphamide and Vinblastine sulphate resulted in statistically significant increases in the numbers of binucleated cells containing micronuclei, when compared to the numbers observed in the concurrent negative (clean air) control cultures. Therefore, the test was considered valid.

In the first experiment, the highest nominal concentration of the test substance evaluated was 76% both in the pulse and continuous treatment groups. As the atmosphere in the chamber consisted of 19% O2, 5% CO2 and the test material (supplemented with N2 to achieve lower concentrations), the highest achievable concentration was 76%. In the second experiment, the highest nominal concentration used in pulse (± S9-mix) and continuous treatment groups were 76% and 30 %, respectively. The highest concentration (30%) used in the continuous treatment group was based on the cytotoxicity observations made in the first experiment.

In the first experiment in both pulse treatment groups, the test substance was slightly cytotoxic to the cells in a dose related manner. The concentrations selected for analysis of micronucleus induction in the pulse treatment group with S9-mix (76%, 60% and 40% (v/v%)) showed a cytotoxicity of 26%, 3% and 7% respectively. In the pulse treatment group without S9-mix, concentrations selected (76, 40 and 20%), showed a cytotoxicity of 25%, 19% and 8%, respectively. In the continuous treatment group, it was observed that the test substance induced severe cytotoxicity resulting in an insufficient number of suitable concentrations for microscopic evaluation.

In the second experiment, in both pulse treatment groups, a dose related cytotoxicity was observed. The concentrations selected for analysis of micronucleus induction in the pulset reatment group with S9-mix (76%, 40% and 20%) showed a cytotoxicity of 52, 26 and 0%. The concentrations selected for analysis of micronucleus induction in the pulse treatment group without S9-mix (76%, 40% and 20%(v/v%)) showed a cytotoxicity of 26, 15 and 0% when compared to the concurrent clean air control cultures. In the continuous treatment group without S9-mix, also a dose related cytotoxicity was observed. The highest test substance concentration (30%) was severely cytotoxic to the cells and therefore not suitable for scoring. The concentrations selected for analysis of micronucleus induction in the continuous treatment group without S9-mix (20%, 10% and 5%) showed a cytotoxicity of 42%, 15% and 6% when compared to the concurrent clean air control cultures. Although the observed cytotoxicity of 42% was below the aimed maximal cytotoxicity of 55±5% according to the OECD guideline 487, the cell density at this concentration on the slides was low, indicating cell death in the cultures and potential underestimation of cytotoxicity. Therefore, the cytotoxicity of 42% was considered acceptable.

In both experiments in the all treatment groups, the test substance did not show a statistically significant increase in the number of binucleated cells containing micronuclei, at any of the concentrations tested when compared to the numbers found in the concurrent control (clean air) cultures. In addition, no dose related micronuclei induction was observed in treatment groups, except in the pulse treatment group without S9-mix. This observation was further investigated in the continuous treatment group and this result was not reproduced (also when compared to the first experiment). Therefore, it was considered not biologically relevant.

From the results obtained in this in vitro micronucleus test, it is concluded that the test substance MOVE 3 was not clastogenic and/or aneugenic to cultured human lymphocytes, under the conditions used in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo data are requested to be generated for the tonnage band of registration.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Three in vitro studies were conducted with MOVE 3:

1)     Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay.

The study design has been developed considering the physic-chemical properties of the test item (low boiling liquid). Since the test item is a low boiling liquid the pre-incubation method has been considered the more appropriate test design to guarantee the exposure of the bacteria cells. The test was performed in two independent experiments in the presence and absence of S9-mix with the inclusion of a pre-incubation step for 30 and 60 minutes in the first and second mutation experiment, respectively. An additional experiment was performed at the top dose of 5000 μg/plate with all five tester strains with the inclusion of a pre-incubation step for 60 minutes. During the pre-incubation phase of the experiments the temperature has been kept at 20°C in order to avoid the volatilisation of the test item.

MOVE3 did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in all the experiments. Acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that MOVE3 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

2)     In Vitro Mammalian Cell Gene Mutation Test at the TK-locus of L5178Y Cells

As MOVE 3 is a low boling point liquid, the study was conducted with MOVE 3 in vapour form in order to guarantee the appropriate exposure of the cells.

A single test was conducted both in the absence and the presence of a metabolic activation system (S9-mix).

In the test, five duplicate cultures were treated for 4 hours in the presence and absence of S9-mix, and 24 hours in the absence of S9-mix. The highest nominal concentration of MOVE 3 evaluated for mutagenicity was 76% both in the absence (4 and 24 hours) and presence (4 hours) of S9-mix. As the atmosphere in the chamber consisted of 19% O2, 5% CO2 and the test material (supplemented with N2 to achieve lower concentrations), 76% (v/v) was the highest achievable concentration. Additional nominal concentrations were 60%, 40%, 20% and 10% (v/v). Target concentrations were analytically verified and the analytical results demonstrated that the actual exposure concentrations were acceptable.

Following 4 h exposure in the absence and presence of S9-mix, the test material was slightly cytotoxic to the cells. No increase in the MF by more than 126 mutants per 1,000,000 clonable cells,i.e.no positive response, compared to the negative control was observed at any of the concentrations tested.

The longer treatment (24 h without S9) was conducted in parallel with the pulse treatment for efficiency reasons (to obtain additional data in case the results of the pulse treatment needed to be clarified).

In the absence of S9-mix after 24h exposure severe cytotoxicity was observed, resulting in a reduction in RTG of more than 90% at the highest three concentrations tested (RTG of 0%, 0% and 7% following exposure to 76%, 60% and 40% MOVE 3). As a consequence, these three concentrations were considered not suitable for assessing the mutant frequency (MF) and only the two lower concentrations could be evaluated. no increase in the MF by more than 126 mutants per 1,000,000 clonable cells, i.e. no positive response, compared to the negative control was observed at concentrations of 10%(v/v) and 20%(v/v). At the two highest concentrations of 60% and 76% (v/v), an increase in the MF by more than 126 mutants per 1,000,000 clonable cells was observed. The increase in mutant frequency was, however only observed at a concentration causing more than 99% cytotoxicity (RTG value <1%). Therefore, this increase is considered to be not biologically relevant. At a concentration of 40%(v/v), causing 93% cytotoxicity (RTG value ≥7%) no increase in mutant frequency was observed.

MOVE 3 was clearly negative under all the suitable conditions and even in case of 93% cytotoxicity for the 24h exposure.

3)     In vitro micronucleus test in cultured human lymphocytes.

 As MOVE 3 is a low boling point liquid, the study was conducted with MOVE 3 in vapour form in order to guarantee the appropriate exposure of the cells.

Two experiments were performed as in the first experiment, in both pulse treatment groups, the numbers of binucleated cells containing a micronucleus in the negative control cultures (clean air) were outside of the historical data range of the test facility. In addition, in the continuous treatment group, the test substance induced cytotoxicity resulting in an insufficient number of suitable concentrations for microscopic evaluation. Hence, all groups had to be repeated.

In the first experiment, the highest nominal concentration of the test substance evaluated was 76% both in the pulse and continuous treatment groups. As the atmosphere in the chamber consisted of 19% O2, 5% CO2 and the test material (supplemented with N2 to achieve lower concentrations), the highest achievable concentration was 76%. In the second experiment, the highest nominal concentration used in pulse (± S9-mix) and continuous treatment groups were 76% and 30 %, respectively. The highest concentration (30%) used in the continuous treatment group was based on the cytotoxicity observations made in the first experiment.

As the distribution of the historical solvent control data of the test facility for exposure to clean air are very limited, additional untreated incubator controls (without use of exposure chamber) were included in the second experiment. In the second experiment, the numbers of binucleated cells containing a micronucleus in the negative control (clean air) and untreated incubator control cultures were within historical data range of the test facility and / or within the data range published in literature (control data were also compared to literature data as the historical data of the test facility were limited).

In both experiments in the all treatment groups, the test substance did not show a statistically significant increase in the number of binucleated cells containing micronuclei, at any of the concentrations tested when compared to the numbers found in the concurrent control (clean air) cultures. In addition, no dose related micronuclei induction was observed in treatment groups, except in the pulse treatment group without S9-mix. This observation was further investigated in the continuous treatment group and this result was not reproduced (also when compared to the first experiment). Therefore, it was considered not biologically relevant.

It was concluded that the test substance MOVE 3 was not clastogenic and/or aneugenic to cultured human lymphocytes.

 

Justification for classification or non-classification

Based on the available data, no self-classification is proposed for the substance for the hazard class Germ Cell Mutagenicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the UN GHS.