(E)-1,2-difluoroethylene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 July 2019 to 24 September 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC Commission Regulation No. 2017/735. Method B.49: Mutagenicity – In Vitro Mammalian Cell Micronucleus Test. OJ L 112/109

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

(E)-1,2-difluoroethylene

EC Number:

216-629-0

EC Name:

(E)-1,2-difluoroethylene

Cas Number:

1630-78-0

Molecular formula:

C2H2F2

IUPAC Name:

(E)-1,2-difluoroethene

Test material form:

gas

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identity: TKN1
- Batch number: N1190214
- Expiry: Not available, confirmed by Sponsor to be suitable for use
- Appearance: Colourless gas
- Storage conditions: Room temperature
- Purity: 100%
- Date received: 03 April 2019

Method

Species / strain

Species / strain / cell type:

lymphocytes:

Remarks:

Human

Details on mammalian cell type (if applicable):

Human blood was collected from two healthy, non-smoking, adult (between 18-35 years of age) donors, pooled (in equal volumes from each donor) and diluted with HML media.

As lymphocytes do not normally undergo cell division, they were stimulated to do so by the addition of phytohaemagglutinin (PHA), a naturally occurring mitogen.
Cultures were established from the pooled sample and dispensed as 10 mL aliquots (in sterile universal containers) so that each contained blood (0.8 mL), HML media (9 mL) and PHA solution (0.2 mL). All cultures were then incubated at 34 to 39°C, and the cells re-suspended (twice daily) by gentle inversion.

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

S9 Fraction
S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was stored at -90 to -70°C.

Preparation of S9 Mix
S9 mix contained: S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All the cofactors were filter sterilized before use.

Test concentrations with justification for top dose:

Preliminary toxicity test: 0.3125, 0.625, 1.25, 2.5, 5, 25, 50 and 70% v/v
Main tests: -S9 mix (3 hours) 25, 50 and 70% v/v
Main tests: +S9 mix (3 hours) 25, 50 and 70% v/v
Main tests: -S9 mix (20 hours) 0.05, 0.5, 5, 25, 50 and 70% v/v

Top dose justification: Maximum practicable concentration

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile Air

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Sterile air

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Main Test Procedure

3-Hour Treatment in the Absence and Presence of S9 Mix and 20-Hour Treatment in
the Absence of S9 Mix:
The procedure for the main tests was the same as that for the preliminary tests, with the
following exceptions: positive control cultures were included for all tests; duplicate cultures
were prepared for each treatment level and positive control cultures; quadruplicate cultures
were prepared for vehicle controls; two slides were prepared from each culture.

Microscopic Examination:
The prepared slides were examined by fluorescence microscopy. The incidences of
mononucleate, binucleate and polynucleate cells were assessed per culture.
From these results, at least three concentrations were selected for micronucleus analysis. The
highest concentration was intended to be that which caused a depression in the cytokinesisblock proliferative index (CBPI) equivalent to 55 ±5% cytostasis (approximately) when
compared with the concurrent vehicle control or, where no cytostasis was observed, the maximum concentration as recommended in the test guidelines or the limit of solubility. Prior to micronucleus analysis, all slides were randomly coded. Interphase cells were examined by fluorescence microscopy and the incidence of micronucleated cells per 1000 binucleate cells per culture were scored where possible.

Positive control micronucleus counts are reported for the following treatments only:
Cyclophosphamide: 10 μg/mL (3h, +S9 mix)
Mitomycin C: 0.3 μg/mL (3h, -S9 mix); 0.1 μg/mL (20h, -S9 mix)
Colchicine: 0.07 μg/mL (3h, -S9 mix); 0.02 μg/mL (20h, -S9 mix)

Cells were included in the analysis provided the cytoplasm remained essentially intact and
any micronuclei present were separate in the cytoplasm or only just touching the main
nucleus (not connected to the nucleus by a nucleoplasmic bridge). Micronuclei should lie in
the same focal plane as the cell, and should possess a generally rounded shape with a clearly
defined outline. The main nuclei of the binucleate cells scored for micronuclei should be of
approximately equal size. The diameter of the micronucleus should be between 1/16 and 1/3
that of the main nucleus. The color of the micronuclei should be the same or lighter than the
main nucleus. There should be no micronucleus-like debris in the surrounding area.

Acceptance Criteria:
The following criteria were applied for assessment of assay acceptability as per the
requirements of the OECD 487 (2016) test guideline:
-The concurrent vehicle control must be considered acceptable for addition to the
laboratories historical vehicle control database (lie within or close to the 95%
confidence limits).
-Concurrent positive controls must induce responses that are compatible with the laboratories historical positive control database and produce statistically significant increases compared with the concurrent vehicle control.

The criteria for selection of the top dose concentration are consistent with those
outlined in the study plan.

Evaluation criteria:

Analysis of Cytostasis Data:
A CBPI of 1 (all cells of mononucleate) is equivalent to 100% cytostasis. To calculate the CBPI at least 500 cells were assessed per culture.

Criteria for Assessing Genotoxic Potential:

The test item was considered to be clearly positive if, in any of the experimental conditions examined:
• At least one of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent vehicle control.
• The increase in the frequency of micronucleated cells is dose-related when evaluated with an appropriate trend test.
• Any of the results are outside the distribution of the historical vehicle control data (above the upper 95% confidence limit).
If all of these criteria are met, the test item was considered able to induce chromosome breaks and/or gain or loss in the test system.

A negative response will be claimed if, in all of the experimental conditions examined:
• None of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent vehicle control.
• There is no concentration-related increase when evaluated with an appropriate trend test.
• All results are inside the distribution of the historical vehicle control data (below the 95% confidence limit).
If all of these criteria are met, the test item was considered unable to induce chromosome breaks and/or gain or loss in the test system.

Statistics:

An arcsine square-root transformation was used to transform the data. TKN1 treated groups were then compared to control using Williams’ tests (Williams 1971, 1972). Positive controls were compared to control using two-tailed t-tests. Trend tests have also been carried out using linear contrasts by group number. These were repeated, removing the top dose group, until there were only 3 groups.
Statistical significance was declared at the 5% level for all tests.
Data were analyzed using SAS (SAS Institute 2002) and Quasar (Quasar 1.5 2017).

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test:
In all exposure conditions the highest concentration tested was 70% v/v and no precipitate was observed at the end of treatment at 70 % v/v.
After 3-hour treatment in the absence and presence of S9-mix, no significant reduction in the CBPI compared with vehicle control values, was obtained with TKN1 at concentrations up to 70% v/v.

After 20-hour treatment in the absence of S9-mix, a reduction in the CBPI compared with vehicle control values, equivalent to 22.2% cytostasis, was obtained with TKN1 at 70% v/v.
These data were used to select concentrations for the main test.


Main Test:

3-Hour Treatment in the Absence of S9 Mix:
Cytotoxicity:

The highest concentration tested was 70% v/v. No significant reduction in the CBPI compared with vehicle control values, was obtained with TKN1 at concentrations up to 70% v/v. Concentrations of TKN1 selected for micronucleus analysis were 25, 50 and 70% v/v.

Micronucleus Analysis:

TKN1 caused no statistically significant increases in the number of binucleate cells containing micronuclei and there was no evidence of a linear dose-concentration relationship across all concentrations. There was a statistically significant dose-concentration relationship associated to 50% v/v, however this was not considered to be biologically relevant as it was only observed when the maximum concentration was excluded.

The positive control compounds (mitomycin C and colchicine) caused statistically significant increases in the number of binucleate cells containing micronuclei, compatible with the laboratory’s historical positive control data, demonstrating the sensitivity of the test system.

3-Hour Treatment in the Presence of S9 Mix:
Cytotoxicity:

The highest concentration tested was 70% v/v. No significant reduction in the CBPI compared with vehicle control values, was obtained with TKN1 at concentrations up to 70% v/v. Concentrations of TKN1 selected for micronucleus analysis were 25, 50 and 70% v/v.

Micronucleus Analysis:

TKN1 caused no statistically significant increases in the number of binucleate cells containing micronuclei and there was no evidence of a linear dose-concentration relationship.The mean micronucleus frequencies for the vehicle and test item treated cultures were within or close to the laboratory historical 95% confidence limits.

The positive control compound (cyclophosphamide) caused a statistically significant increase in the number of binucleate cells containing micronuclei, compatible with the laboratory’s historical positive control data, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.

20-Hour Treatment in the Absence of S9 Mix:
Cytotoxicity:

The highest concentration tested was 70% v/v. No significant reduction in the CBPI compared with vehicle control values, was obtained with TKN1 at concentrations up to 70% v/v. Concentrations of TKN1 selected for micronucleus analysis were 25, 50 and 70% v/v.

Micronucleus Analysis:

TKN1 caused no statistically significant increases in the number of binucleate cells containing micronuclei and there was no evidence of a linear dose-concentration relationship. The mean micronucleus frequencies for the vehicle and test item treated cultures were within or close to the laboratory historical 95% confidence limits.

The positive control compounds (mitomycin C and colchicine) caused statistically significant increases in the number of binucleate cells containing micronuclei, compatible with the laboratory’s historical positive control data, demonstrating the sensitivity of the test system.

Any other information on results incl. tables

Osmolality and pH Measurements

The osmolality and pH of TKN1 in medium were measured by analysing samples of HML media, dosed at 70% v/v with TKN1, or Sterile Air. For medium dosed with TKN1 at 70% v/v; no fluctuations in osmolality of the medium of more than 50 mOsmol/kg and no fluctuations in pH of more than 1.0 unit were observed compared with the vehicle control. The maximum final concentration tested in the preliminary toxicity test was 70% v/v as this is the maximum practical achievable concentration within this test system.

 

 

 

 

Applicant's summary and conclusion

Conclusions:

It was concluded that TKN1 did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described.

Executive summary:

Introduction

This study was designed to assess the potential of TKN1 to cause an increase in the induction of micronuclei in cultured human peripheral blood lymphocytes in vitro.

Results

Following 3-hour treatment in the absence of S9 mix, no significant reduction in the CBPI
compared with vehicle control values, was obtained with TKN1 at concentrations up to
70% v/v. Concentrations of TKN1 selected for micronucleus analysis were 25, 50 and 70% v/v. TKN1 caused no statistically significant increases in the number of binucleate cells containing micronuclei and there was no evidence of a linear dose-concentration relationship
across all concentrations.

Following 3-hour treatment in the presence of S9 mix, no significant reduction in the CBPI
compared with vehicle control values, was obtained with TKN1 at concentrations up to 70% v/v. Concentrations of TKN1 selected for micronucleus analysis were 25, 50 and 70% v/v. TKN1 caused no statistically significant increases in the number of binucleate cells containing micronuclei and there was no evidence of a linear dose-concentration relationship.

Following a 20-hour exposure in the absence of S9 mix, no significant reduction in the CBPI
compared with vehicle control values, was obtained with TKN1 at concentrations up to 70% v/v. Concentrations of TKN1 selected for micronucleus analysis were 25, 50 and 70% v/v. TKN1 caused no statistically significant increases in the number of binucleate cells containing micronuclei and there was no evidence of a linear dose-concentration relationship.

Conclusion

It was concluded that TKN1 did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described.