4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphthalic anhydride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The potential of 6FDA to induce gene mutation in bacteria was assessed using a GLP- compliant study performed in accordance with OECD Testing Guideline 471.

Plate incorporation

A dose range of 1.5 to 5000 µg/plate. When the plate incorporation method was employed, a visible reduction in the growth of the bacterial background lawns noted for all of the tester strains in both the absence and presence of S9-mix from 1500 µg/plate. No test item precipitate was observed on the plates at any of the doses tested either in the presence or in the absence of S9-mix. There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix.

Pre-incubation

A repeat assay was performed employing the pre-incubation modification and an extended dose range of 0.15 to 5000 µg/plate. The lack of toxicity meant that the maximum recommended dose (5000 µg/plate) was not achieved for any of the Salmonella strains. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or in the absence of S9-mix employing the pre-incubation modification. No test item precipitate was observed on the plates at any of the doses tested either in the presence or in the absence of S9-mix. There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix.

 

The test item 6FDA did not induce an increase in frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of the test, 6FDA was considered to be non-mutagenic.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October to November 2021

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:

yes

Remarks:

See 'any other information on materials and methods'

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch number: CT20201015
Purity: 94.18%
Retest date: 10th September 2022
Storage conditions: room temperature in the dark

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

The metabolic activation system was lyophilized phenobarbital/beta-naphthoflavone induced rat liver S9 and cofactors mix (MutazymeTM) reconstituted with cold, sterile water to provide a 10% phenobarbital/beta-naphthoflavone induced rat liver S9 and cofactors mix. Lot No. 4500 was used in this study.

The S9-mix was prepared by adding 20 mL of cold, sterile water to each vial.

Final concentrations of S9-mix of approximately 10% (v/v), MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium phosphate buffer (100 mM, pH 7.4) and was maintained on ice for the duration of the test.

A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine (for S. typhimurium strains) or tryptophan (for E.coli strain) supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Test concentrations with justification for top dose:

A dose range of 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate was employed.

Test item concentrations were selected based on the results from experiments 1,2 and 3(see details on test system and experimental conditions) in order to ensure the study achieved at least four non-toxic dose levels as required by the test guideline.

Vehicle / solvent:

DMSO was selected as the solvent.

Sterile distilled water was not selected as a potential vehicle as the substance is an anhydride which in the presence of water will ring open to form the associated diacid.

Test item was fully soluble in dimethyl sulphoxide (DMSO) 50 mg/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other:

Details on test system and experimental conditions:

Test for Mutagenicity: Experiment 1 – Plate Incorporation Method

The test item was tested using the following method. Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed for each tester strain, using the direct plate incorporation method.

Without Metabolic Activation - A 0.1 mL aliquot of the appropriate concentration of test item, solvent or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Each concentration of the test item, appropriate positive and solvent controls and each bacterial strain, was assayed using triplicate plates. Untreated controls were also performed in triplicate on the same day as the mutation test.

With Metabolic Activation - The procedure was the same as described previously except that untreated controls were not performed and, following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring - All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning of the background bacterial lawn (toxicity). Sporadic manual counts were performed on thirty-two individual plates due to revertant colonies spreading which prevented an accurate automated count. However, this had no adverse consequences for the integrity of the study because the electronic colony counter is calibrated against manual counts and the results of both scoring methods are equivalent.

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method

As the result of Experiment 1 was considered negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation (S9-mix).

The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as follows:

TA1535 (without S9-mix) and TA98 (with and without S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate.
TA1535 (with S9-mix) and TA100 and TA1537 (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate.
WP2uvrA (with and without S9-mix), 1.5, 5, 15, 50, 150, 500 1500 and 5000 µg/plate.

Eight test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least four non-toxic dose levels as required by the test guideline and were selected based on the cytotoxicity noted in Experiment 1 and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

Without Metabolic Activation - A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the appropriate concentration of test item formulation, solvent or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior
to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Each concentration of the test item, appropriate positive and solvent controls and each bacterial strain, was assayed using triplicate plates. Untreated controls were also performed in triplicate on the same day as the mutation test.

With Metabolic Activation - The procedure was the same as described previously except that untreated controls were not performed and, following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring - All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning of the background bacterial lawn (toxicity). Sporadic manual counts were performed on thirty individual plates due to revertant colonies spreading, bubble interference or to confirm colony numbers on the edge of a plate, all of which prevented an accurate automated count. However, this had no adverse consequences for the integrity of the study because the electronic colony counter is calibrated against manual counts and the results of both scoring methods are equivalent.

Test for Mutagenicity: Experiment 3 – Pre-Incubation Method

The test item exhibited no toxicity to any of the bacterial tester strains employing the preincubation method resulting, in some cases, the maximum dose concentration not being achieved. Therefore, the pre-incubation modification was repeated in the presence and absence of metabolic activation (S9-mix) following testing outlined in experiment 2 and a dose range of 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The plates were incubated and scored/assessed as outlined in experiment 2. Sporadic manual counts were performed on nineteen individual plates due to revertant colonies spreading, which prevented an accurate automated count. However, this had no adverse consequences for the integrity of the study because the electronic colony counter is calibrated against manual counts and the results of both scoring methods are equivalent.

Test for Mutagenicity: Experiment 4 – Plate Incorporation Method

To confirm that the test item only exhibited toxicity employing the plate incorporation modification, a further test was performed. Therefore, the plate incorporation modification was repeated in the presence and absence of metabolic activation (S9-mix) following testing outlined in experiment 1 and a dose range of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The plates were incubated and scored/assessed as outlined in experiment 1. Sporadic manual counts were performed on four individual plates due to artefacts (for example marks on the base plate, salt precipitation in the base agar or dust particles etc.), which prevented an accurate automated count. However, this had no adverse consequences for the integrity of the study because the electronic colony counter is calibrated against manual counts and the results of both scoring methods are equivalent.

Evaluation criteria:

A test article is considered to have provided a mutagenic response if the assay data are valid,
and:

1. Treatments with the test article provide a concentration-related increase in revertant numbers at one or more concentrations in at least one strain with or without metabolic activation system

2. An increase in mean revertant colony numbers per plate is observed which is ≥2-fold (in strains TA98, TA100 and WP2 uvrA) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values

3. Any increase in revertant numbers is reproducible, where applicable.

Statistics:

Statistical significance was not included as part of the result evaluation.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: corporation method from 5000 ug/plate but no cytotoxicity was noted during the pre-incubation methods

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: corporation method from 1500 ug/plate but no cytotoxicity was noted during the pre-incubation methods

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Cytotoxicity was noted during the plate incorporation method from 1500 ug/plate but no cytotoxicity was noted during the pre-incubation methods

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Cytotoxicity was noted during the plate incorporation method from 500 ug/plate but no cytotoxicity was noted during the pre-incubation methods

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Cytotoxicity was noted during the plate incorporation method from 500 ug/plate but no cytotoxicity was noted during the pre-incubation methods

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The test item 4,4’-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphthalic anhydride was considered to be non-mutagenic.

Executive summary:

The potential of 6FDA to induce gene mutation in bacteria was assessed using a GLP- compliant study performed in accordance with OECD Testing Guideline 471.

Experiment 1 (plate incorporation)

The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate as recommended for a soluble and nontoxic substance. There was a visible reduction in the growth of the bacterial background lawns noted for all of the tester strains in both the absence and presence of S9-mix from 500 µg/plate for TA1535 and TA98, 1500 µg/plate for TA100 and TA1537 and at 5000 µg/plate for WP2uvrA. No test item precipitate was observed on the plates at any of the doses tested either in the presence or in the absence of S9-mix. There were no biologically relevant increases in the frequency of revertant colonies recorded
 or any of the bacterial strains, with any dose of the test item, either with or without S9-mix.

Experiment 2 (pre-incubation)

Based on the results noted in Experiment 1, the maximum dose level of the test item in the second experiment was 5000 µg/plate or the toxic limit, depending on bacterial strain type and absence/presence of S9-mix. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or in the absence of S9-mix. The results in terms of toxic response contradicted those observed for the first mutation test and, therefore, a repeat of the pre-incubation test was performed for confirmatory purposes. No test item precipitate was observed on the plates at any of the doses tested either in the presence or in the absence of S9-mix. There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix.

Experiment 3 (pre-incubation)

Based on the disparity in toxicity response noted between Experiments 1 and 2, a repeat assay was performed employing the pre-incubation modification and an extended dose range of 0.15 to 5000 µg/plate. Furthermore, the lack of toxicity meant that the maximum recommended dose (5000 µg/plate) was not achieved for any of the Salmonella strains. Confirming the results from Experiment 2, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or in the absence of S9-mix employing the pre-incubation modification. No test item precipitate was observed on the plates at any of the doses tested either in the presence or in the absence of S9-mix. There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix.

Experiment 4 (plate incorporation)

A fourth experiment was performed due to disparity between methodologies and to confirm the toxicity noted in Experiment 1 (plate incorporation method) and a dose range of 1.5 to 5000 µg/plate. Once again, when the plate incorporation method was employed, a visible reduction in the growth of the bacterial background lawns noted for all of the tester strains in both the absence and presence of S9-mix from 1500 µg/plate. No test item precipitate was observed on the plates at any of the doses tested either in the presence or in the absence of S9-mix. There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix.

 

The test item 6FDA did not induce an increase in frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of the test, 6FDA was considered to be non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The test item 6FDA did not induce an increase in frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of the test, 6FDA was considered to be non-mutagenic.