Ethane, 1,1,2-trichloro-1-fluoro-2-(trifluoromethoxy)-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 July 2012 - 3 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (1R)-1,2,2-trichloro-2-fluoroethyl trifluoromethyl ether and (1S)-1,2,2-trichloro-2-fluoroethyl trifluoromethyl ether

EC Number:

938-115-1

Cas Number:

94720-90-8

Molecular formula:

C3HCl3F4O

IUPAC Name:

Reaction mass of (1R)-1,2,2-trichloro-2-fluoroethyl trifluoromethyl ether and (1S)-1,2,2-trichloro-2-fluoroethyl trifluoromethyl ether

Test material form:

liquid

Details on test material:

- Description: colourless liquid
- Batch 02072012

Method

Target gene:

Reverse mutation assays employ bacterial strains which are already mutant at a locus whose phenotypic effects are easily detected.
The Salmonella tester strains have mutations causing dependence on a particular amino acid (histidine) for growth. The ability of test items to cause reverse mutations (reversions) to histidine-independence can easily be measured.
The E. coli tester strains of the WP2 series are similarly mutant at the tryptophan locus.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 5

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue fraction from rat liver induced by Phenobarbital and 5,6-Benzoflavone

Test concentrations with justification for top dose:

A preliminary experiment ("toxicity test") was conducted at the maximum recommended dose levels to define the dose leeves for the main first experiment. This assay was conducted at the following dose levels:
All the strains, with and without S9: 5000 - 1580 - 500 - 158 and 50 µg/plate

On the basis of the cytotoxicity obtained in the preliminary toxicity test, the first main assay ("Main Assay I"), using the plate incorporation method, was conducted at the following dose levels:
TA1535, without S9: 500 - 250 - 125 - 62.5 and 31.3 µg/plate
TA1535, with S9: 2000 - 1000 - 500 - 250 - 125 and 62.5 µg/plate
TA1537, with and without S9: 1000 - 500 - 250 - 125 - 62.5 and 31..3 µg/plate
TA98, without S9: 2000 - 1000 - 500 - 250 - 125 and 62.5 µg/plate
TA98, with S9: 2000 - 1000 - 500 - 250 and 125 µg/plate
TA100, without S9: 1000 - 500 - 250 - 125 and 62.5 µg/plate
TA100 with S9: 2000 - 1000 - 500 - 250 and 125 µg/plate
WP2 uvrA, with and without S9: 2000 - 1000 - 500 - 250 and 125 µg/plate

As no relevant increase in revertant numbers was observed at any concentration tested, a second main assay using the pre-incubation method (Main Assay II) was conducted with a dose-range slightly modified to take into account the toxicity results of Main Assay I:
TA1535 and TA1537 without S9: 500 - 250 - 125 - 62.5, 31.3 and 15.6 µg/plate
TA1535 and TA1537, with S9: 1000 - 500 - 250 - 125 - 62.5 and 31.3 µg/plate
TA98, with and without S9: 1000 - 500 - 250 - 125 - 62.5 and 31.3 µg/plate
TA100, without S9: 250 - 125 - 62.5, 31.3 - 15.6 and 7.81 µg/plate
TA100 with S9: 1000 - 500 - 250 - 125 - 62.5, 31.3 and 15.6 µg/plate
WP2 uvrA, with and without S9: 1000 - 500 - 250 - 125 - 62.5 and 31.3 µg/plate

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: DMSO is compatible with the survival of the bacteria and the S9 metabolic activity. The test item was found to be soluble at 100 mg/mL. This result permitted a maximum concentration of 5000 µg/plate to be used in the toxicity test.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

yes

Remarks:

untreated plate

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535 without S9

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Remarks:

untreated plate

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

TA1535 with S9

Controls 3

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Remarks:

untreated plate

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 without S9

Controls 4

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Remarks:

untreated plate

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

TA1537 with S9

Controls 5

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Remarks:

untreated plate

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 without S9

Controls 6

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Remarks:

untreated plate

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

TA98 with S9

Controls 7

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

yes

Remarks:

untreated plate

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100 without S9

Controls 8

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Remarks:

untreated plate

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

TA100 with S9

Controls 9

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

yes

Remarks:

untreated plate

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA without S9

Controls 10

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Remarks:

untreated plate

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

WP2 uvrA with S9

Details on test system and experimental conditions:

PRELIMINARY TOXICITY PLATE:
- A preliminary toxicity test was undertaken in order to select the concentrations to be used in the main assays.
- The "untreated" plates received no treatment while the plates at dose level 0.00 are solvent control plates.
- A wide range of dose levels of the test item, set at half-log intervals, were used: 50 - 158 - 500 - 1580 and 5000 µg/plate.
- Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method
- A single plate was used at each test point and positive controls were not included.
- Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

MAIN EXPERIMENTS
- The main experiments were conducted on the basis of the preliminary toxicity test.
- Three replicates plates were performed at each test point.
- The "untreated" plates received no treatment while the plates at dose level 0.00 are solvent control plates. Posiitve controls were added for each strain, and both in absence and in presence of metabolic activation.
- Plate incorporation method (first main experiment): The mixture (overlay agar + test or control item + S9 mix or phosphate buffer + bacterial suspension) was directly poured onto the surface of a minimal medium agar plate and allowed to solidify prior to incubation.
- Preincubation method (second main experiment): The mixture (test or control item + S9 mix or phosphate buffer + bacterial suspension) was directly placed for 30 minutes at 37°C. Then overlay agar was added and the mixture was poured onto the surface of a minimal medium agar plate and allowed to solidify
- The prepared plates were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, plates were held at 4°C for 24 hours or immediately scored by counting the number of revertant colonies on each plate.
- Cytotoxicity: It was indicated by thinning of the background lawn and/or reduction in revertant numbers or microcolony formation. it was assessed all tester strains, both in the absence and presence of S9 metabolism, in the preliminary experiment and in the main experiments.
- Genotoxicity: It was indicated by the increase in the revertants number compared to the negative control. The number of colonies were counted for both the main experiments in each individual plate. The mean and standard error of the mean for each test point, together with a statistical analysis were calculated.

Rationale for test conditions:

The concentration ranges were defined following a cytotoxicity assay. As no relevant increase in revertant numbers was observed at any concentration tested in the first assay, a pre-incubation step was included for all treatments in a second assay.

Evaluation criteria:

- For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.
- The mean plate counts for untreated and positive control plates must be fall within the Testing Facilities historical control data

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

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

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

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:

SOLUBILITY:
- Vehicle (DMSO): The test item was found to be soluble in DMSO at 100 mg/mL.
- Precipitation in the test: No precipitation of the test item was observed at the end of the incubation period at any concentration.

RANGE-FINDING STUDIES (PRELIMINARY EXPERIMENT):
The test item was assayed in the toxicity test (preliminary experiment at a maximum dose level of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate.
In the absence of metabolic activation, toxicity (indicated by the decrease of the revertants number and/or the thining of the background lawn) was observed at the three highest dose levels for the the strains TA 1535, TA 1537 and TA 100. For the strains WP2 uvrA and TA 98, toxicity was only noted at the two highest dose levels.
In the presence of metabolic activation, toxicity was observed at the three highest dose levels for the the strain TA 1537 and at the two highest dose levels for the other strains..

MAIN EXPERIMENT:
No relevant increase in the number of revertant colonies was observed in the plate incorporation or pre-incubation assay, at any dose level, with any tester strain, in the absence or presence of S9 metabolism.
Results show that mean plate counts for untreated and positive control plates fell within the Testing Facilities acceptance criteria based on historical control data
The study was therefore accepted as valid.

Any other information on results incl. tables

Preliminary experiment - Without metabolic activation

Dose level (µg/plate)	TA-1535 (revertants/palte)	TA-1537 (revertants/plate	TA-98 (revertants/plate)	TA-100 (revertants /plate)	WP2 uvrA (revertants /plate)
Untreated	17	12	24	174	34
Vehicle	14	15	26	180	33
50	13	10	30	171	36
158	22	12	28	149	37
500	6 *	11 *	15	124 *	32
1580	8 *	3 *	19 *	118 *	22 *
5000	9 *	7 *	20 *	122 *	25 *

*: thinning of the background lawn

Preliminary experiment - With metabolic activation

Dose level (µg/plate)	TA-1535 (revertants/palte)	TA-1537 (revertants/plate	TA-98 (revertants/plate)	TA-100 (revertants /plate)	WP2 uvrA (revertants /plate)
Untreated	19	23	28	184	37
Vehicle	16	28	31	187	39
50	19	22	27	183	32
158	10	19	35	175	41
500	12	12 *	28	169	31
1580	13 *	13 *	30 *	139 *	29 *
5000	19 *	14 *	24 *	121 *	26 *

*: thinning of the background

First main experiment - Plate incorporation method - With and Without metabolic activation - Mean result for each point

Dose level     (µg/plate)	TA 1535 (mean revertants/plate)		TA 1537 (mean revertants/plate)		TA 98 (mean revertants/plate)		TA 100 (mean revertants/plate)		TA 100 (mean revertants/plate)
	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Untreated	16	20	21	24	34	41	117	134	31	36
Vehicle	15	18	22	26	29	39	129	134	32	37
31.3	15	NT	20	26	NT	NT	NT	NT	NT	NT
62.5	15	20	22	24	36	NT	121	NT	NT	NT
125	18	17	21	25	27	42	120	117	29	35
250	17	15	18	24	29	34	104 *	118	28	36
500	17 *	14	16 *	21	27	41	87 *	118	33	37
1000	NT	16 *	10 *	20 *	24 *	34 *	68 *	97 *	25 *	37 *
2000	NT	17 *	NT	NT	20 *	33 *	NT	66 *	17 *	35 *
Positive control	529	116	220	110	145	532	588	1142	205	271

*: thinning of the background lawn / NT: not tested

Second main experiment - Preincubation method - With and Without metabolic activation - Mean result for each point

Dose level     (µg/plate)	TA 1535 (mean revertants/plate)		TA 1537 (mean revertants/plate)		TA 98 (mean revertants/plate)		TA 100 (mean revertants/plate)		TA 100 (mean revertants/plate)
	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Untreated	18	20	22	24	29	44	154	139	28	34
Vehicle	16	19	18	26	28	38	119	123	29	30
7.81	NT	NT	NT	NT	NT	NT	112	NT	NT	NT
15.6	16	NT	20	NT	NT	NT	109	125	NT	NT
31.3	16	17	17	23	27	37	112	115	25	33
62.5	16	16	18	22	27	36	111 *	108	32	32
125	16 *	18 *	15 *	23	28	36	97 *	93 *	27	33
250	13 *	15 *	11 *	16 *	25 *	38	101 *	99 *	28 *	32 *
500	M	15 *	4 *	16 *	32 *	27 *	NT	84 *	27 *	31 *
1000	NT	17 *	NT	19 *	23 *	30 *	NT	88 *	30 *	24 *
Positive control	516	100	189	99	151	543	609	941	181	237

*: thinning of the background lawn / NT: not tested / M: microcolony formation

Applicant's summary and conclusion

Conclusions:

The test item did not induce two-fold increases in the number of revertant colonies, at any dose level, in any tester strain, in the absence or presence of S9 metabolism. On the basis of the stated criteria it must be concluded that the test item TRICHLOROETHYLIC ADDUCT is not mutagenic to S. typhimurium or E. coli, under the reported experimental conditions.

Executive summary:

The test item TRICHLOROETHYLIC ADDUCT was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. This study was conducted according to the OECD guideline 471.

The test item was used as a solution in dimethylsulfoxide (DMSO).

The test item was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations: 1580, 500, 158 and 50.0 µg/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration. Toxicity was observed with all tester strains at the two or three highest dose levels, both in the absence and presence of S9 metabolism.

On the basis of toxicity test results, a first main assay was conducted using the plate incorporation method over the dose range 31.3 - 2000 µg/plate.

Toxicity was observed with all tester strains at higher dose levels both in the absence and presence of S9 metabolism. No precipitation of the test item was observed at the end of the incubation period at any concentration. No relevant increase in revertant numbers was observed at any concentration tested.

A second main assay was conducted using the preincubation method over the dose range 15.6 - 1000 µg/plate.

Toxicity was observed with all tester strains at higher dose levels, both in the absence and presence of S9 metabolism. No precipitation of the test item was observed at the end of the incubation period at any concentration. No relevant increase in revertant numbers was observed at any concentration tested.

In conclusion, the test item did not induce increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.