Trifluoroiodomethane

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:

31 Oct 2018 to 19 Nov 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ISO/IEC 17025:2005

Version / remarks:

2005

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Trifluoroiodomethane

EC Number:

219-014-5

EC Name:

Trifluoroiodomethane

Cas Number:

2314-97-8

Molecular formula:

CF3I

IUPAC Name:

trifluoroiodomethane

impurity 1

Reference substance name:

Carbon dioxide

EC Number:

204-696-9

EC Name:

Carbon dioxide

Cas Number:

124-38-9

Molecular formula:

CO2

impurity 2

Reference substance name:

Trifluoromethane

EC Number:

200-872-4

EC Name:

Trifluoromethane

Cas Number:

75-46-7

Molecular formula:

CHF3

IUPAC Name:

Trifluoromethane

Test material form:

gas

Method

Target gene:

his (Salmonella), trp (E. coli)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: The S9 (Lot No. 3999, Exp. Date: 29 Aug 2020) was purchased commercially from MolTox (Boone, NC).
- method of preparation of S9 mix: Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice.
- concentration S9 in the final culture medium: 10% (v/v)
- quality controls of S9: Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and 2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.

Test concentrations with justification for top dose:

1.4, 2.8, 6.9, 14, 21, 28 and 37 mmol/L

Vehicle / solvent:

Air

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

air

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

FREQUENCY AND ROUTE OF ADMINISTRATION
The test system was exposed to the test substance via the desiccator methodology, a modification of the plate incorporation methodology originally described by Ames et al. (1975) and updated by Maron and Ames (1983). The desiccator methodology has been shown to be an effective method for detecting the genotoxic activity of volatile and gaseous test substances (Wagner et al., 1992).

INITIAL TOXICITY-MUTATION ASSAY TO SELECT DOSE LEVELS
The initial toxicity-mutation assay was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the untreated control, positive controls and seven dose levels of the test substance, in duplicate, in the presence and absence of Aroclor-induced rat liver S9. Dose levels for the confirmatory mutagenicity assay were based upon post-treatment toxicity.

CONFIRMATORY MUTAGENICITY ASSAY
The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test substance. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the untreated control, positive controls and seven dose levels of the test substance, in triplicate, in the presence and absence of Aroclor-induced rat liver S9.

UNTREATED AND POSITIVE CONTROL TREATMENT
One half milliliter (0.5 mL) of S9 mix or Sham mix, 100 μL of tester strain and 50.0 μL of positive control were added to 2.0 mL of molten selective top agar at 45±2°C. When plating the untreated controls (2 plates), the addition of positive control was omitted. The mixture was vortex mixed and overlaid onto the surface of a minimal bottom agar plate. After the overlay was solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2°C. One set of air control plates was plated as indicated for the untreated control plates. The overlaid plates was inverted and placed uncovered in the appropriate number of 9-liter desiccators.

TEST SUBSTANCE TREATMENT
One half milliliter (0.5 mL) of S9 mix or Sham mix and 100 μL of tester strain were added to 2.0 mL of molten selective top agar at 45±2°C. The mixture was vortex mixed and overlaid onto the surface of a minimal bottom agar plate. The overlaid plates was inverted and placed uncovered in the appropriate number of 9-liter desiccators. An appropriate quantity of the test substance was introduced into each desiccator by withdrawing an appropriate amount of air and replacing it with test substance. The desiccators were incubated for 24±1 hours at 37±2°C. Following the 24-hour incubation, the plates were removed from the desiccators and incubated with the lids replaced at 37±2°C for a total of 48 to 72 hours.

SCORING
The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the untreated control plate using the codes shown in the following table. As appropriate, colonies were enumerated either by hand or by machine.

Evaluation criteria:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:

- Strains TA1535 and TA1537: Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean untreated control value and above the corresponding acceptable untreated control range.

- Strains TA98, TA100 and WP2 uvrA: Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean untreated control value and above the corresponding acceptable untreated control range.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

STERILITY RESULTS
No contaminant colonies were observed on the sterility plates for the S9 and Sham mixes.

INITIAL TOXICITY-MUTATION ASSAY
No precipitate was observed. Toxicity was observed as a reduction in revertant counts, in Salmonella strains TA100 and TA1535 in the absence and presence of S9-mix at 37 mmol/L and in TA100 in the absence of S9-mix also at 28 mmol/L and higher. Positive mutagenic responses (2.6- through 20.3- fold, maximum increases) were observed with tester strains TA98, TA1537 and WP2 uvrA in the presence and absence of S9 activation and TA1535 in the absence of S9 activation.

CONFIRMATORY MUTAGENICITY ASSAY
Based upon the results of the initial toxicity-mutation assay, the dose levels selected for the confirmatory mutagenicity assay were 1.4, 2.8, 6.9, 14, 21, 28 and 37 mmol/L. No precipitate was observed. Toxicity was observed as a reduction in revertant counts, in Salmonella strains TA100 and TA1535 in the absence and presence of S9-mix at 37 mmol/L and in TA100 in the absence of S9-mix also at 28 mmol/L and higher. Positive mutagenic responses (3.3- through 21.1- fold, maximum increases) were observed with tester strains TA98, TA1537 and WP2 uvrA in the presence and absence of S9 activation.

Applicant's summary and conclusion

Conclusions:

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, the test substance did cause a positive mutagenic response with TA98, TA1537 and WP2 uvrA in the presence or absence of Aroclor-induced rat liver S9.

Executive summary:

The gaseous test substance was tested to evaluate its mutagenic potential according to OECD TG 471 and in compliance with GLP by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

In the initial toxicity-mutation assay, the dose levels tested were 1.4, 2.8, 6.9, 14, 21, 28 and 37 mmol/L. No precipitate was observed. Toxicity, as a reduction in revertant count, was observed at 28 and/or at 37 mmol/L with tester strains TA100 and TA1535 in the presence and absence of S9 activation. Positive mutagenic responses (2.6- through 20.3- fold, maximum increases) were observed with tester strains TA98, TA1537 and WP2 uvrA in the presence and absence of S9 activation and TA1535 in the absence of S9 activation. Based upon these results, the maximum dose tested in the confirmatory mutagenicity assay was 37 mmol/L. In the confirmatory mutagenicity assay, the dose levels tested were 1.4, 2.8, 6.9, 14, 21, 28 and 37 mmol/L. No precipitate was observed. Toxicity, as a reduction in revertant count, was observed at 28 and/or at 37mmol/L with tester strains TA100 and TA1535 in the presence and absence of S9 activation. Positive mutagenic responses (3.3- through 21.1- fold, maximum increases) were observed with tester strains TA98, TA1537 and WP2 uvrA in the presence and absence of S9 activation.

These results indicate that the test substance was positive for the ability to induce reverse mutations at selected loci of some strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.