4-chloroformylphthalic anhydride

Genetic toxicity: in vitro

Administrative data

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:

20 May 2015-30 July 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-chloroformylphthalic anhydride

EC Number:

214-874-8

EC Name:

4-chloroformylphthalic anhydride

Cas Number:

1204-28-0

Molecular formula:

C9H3ClO4

IUPAC Name:

1,3-dioxo-1,3-dihydro-2-benzofuran-5-carbonyl chloride

Test material form:

solid

Details on test material:

- Name of test material (as cited in study report): Trimellitic anhydride chloride
For more details see Confidential details on test material

Specific details on test material used for the study:

No correction was made for the purity/composition of the test substance. Trimellitic anhydride chloride was dissolved in dimethyl sulfoxide (DMSO, SeccoSolv, Merck, Darmstadt, Germany). The stock solutions were treated with ultrasonic waves until the test substance had completely dissolved. Except in the dose range finding test where the stock solution was already dissolved after vortexing only. Test substance concentrations were used within 2 hours of preparation.

Method

Target gene:

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

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Dose range finding test (without and with 5% (v/v) S9-mix ; TA100 and WP2uvrA): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate.
The highest concentration of the test substance used in the subsequent mutation experiment was 5000 μg/plate or the level at which the test substance inhibited bacterial growth.

Mutation experiments:
Experiment 1 (with pre incubation):
Without and with 5% (v/v) S9-mix ; TA1535, TA1537, TA98, TA100 and WP2uvrA: 17, 52, 164, 512, 1600 and 5000 μg/plate
Experiment 2 (with pre incubation):
Without and with 10% (v/v) S9-mix ; TA1535, TA1537, TA98, TA100 and WP2uvrA: 154, 275, 492, 878, 1568 and 2800 μg/plate
Experiment 3 (with pre incubation):
Without S9-mix ; TA1535: 17, 52, 164, 512, 1600 and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO, SeccoSolv, Merck, Darmstadt, Germany)
- Justification for choice of solvent/vehicle:
Test compound was soluble in DMSO.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9-mix: 5 µg/plate in saline for TA1535

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

without S9-mix: 2.5 µg/plate in DMSO for TA1537

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9-mix: 10 µg/plate in DMSO for TA98

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9-mix: 650 µg/plate in DMSO for TA100

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9-mix: 10 µg/plate in DMSO for WP2uvrA

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation (in agar) and preincubation

DOSE FINDING RANGE TEST (no pre-incubation was performed during the dose range finding test)
S9-mix was prepared immediately before use and kept on ice. S9-mix contained per 10 ml: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 ml or 5.0 ml Milli-Q water (first or second experiment respectively) (Millipore Corp., Bedford, MA., USA); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution (Merck); 1 ml 0.33 M KCl solution (Merck). The above solution was filter (0.22 μm)-sterilized. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 ml of S9-mix components 1.0 ml S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.

MUTATION EXPERIMENT
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain. In the first experiment, the test substance was tested both in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in a pre-incubation experiment. In a follow-up experiment with additional parameters, the test substance was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains in a pre-incubation experiment. An additional experiment was performed with tester strain TA1535 in the absence of S9-mix.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.

Direct plate experiment: Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (10E9 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in dimethyl sulfoxide and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate.

Pre-incubation experiment: Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were pre-incubated for 30 minutes by 70 rpm at 20°C, either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays), 0.1 ml of a fresh bacterial culture (10E9 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in DMSO. After the pre-incubation period the solutions were added to 3 ml molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate.

After solidification of the top agar, the plates were inverted and incubated in the dark at 37°C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli were counted.

COLONY COUNTING
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.
Evidence of test article precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

ACCEPTABILITY OF THE ASSAY
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Test Facility.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the 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 vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up 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 follow up experiment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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:

not applicable

Positive controls validity:

valid

Test results 3

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:

not applicable

Positive controls validity:

valid

Test results 4

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:

not applicable

Positive controls validity:

valid

Test results 5

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of Trimellitic anhydride chloride on the plates was not observed at the start or at the end of the incubation period. Except in the first experiment where at the end of the incubation period Trimellitic anhydride chloride slightly precipitated at the top concentration of 5000 μg/plate in the presence of S9-mix in strains TA1535, TA1537 and TA98.

RANGE-FINDING/SCREENING STUDIES:
- No precipitation was observed at the start of the incubation period in both tester strain. No mutagenicity was observed up to and including the top dose of 5000 µg/plate. Toxicity: In tester strain TA100, a slight reduction of the bacterial background lawn was observed at the test substance concentration of 1600 μg/plate in the absence and presence of S9-mix. A complete lack of any micro-colony background lawn and no revertant colonies were present at 5000 μg/plate in the absence and presence of S9-mix. In tester strain WP2uvrA, an extreme reduction of the bacterial background lawn and an increase in the size of the microcolonies compared to the solvent control plate was observed at the test substance concentration of 5000 μg/plate in the absence and presence of S9-mix.

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

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Toxicity to all strains exihbited at 5000 µg/plate in the first mutation experiment without S9-mix only. Toxicity to majority of strains exhibited at 2800 µg/plate with or without S9-mix.

Remarks on result:

other: Mutagenic

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that Trimellitic anhydride chloride is mutagenic in the Salmonella typhimurium reverse mutation assay (strain TA100 only) and is not mutagenic in the Escherichia coli reverse mutation assay.

Executive summary:

The mutagenic activity of Trimellitic anhydride chloride was investigated using the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay in accordance with OECD Guideline No. 471 and the EU Method B.13/14 and under GLP compliance.

The test substance was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). At first a dose range finding test was performed in the strains TA100 and WP2uvrA in a direct plate experiment both in the absence and presence of S9-mix (rat liver S9-mix induced Aroclor 1254). After that the test was performed with the inclusion of the pre-incubation step in two independent experiments both in the absence and presence of S9-mix (rat liver S9-mix induced Aroclor 1254). An additional experiment was performed with tester strain TA1535 in the absence of S9-mix.

In the dose range finding test, the test substance was initially tested up to concentrations of 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the strains TA100 and WP2uvrA in the direct plate experiment. The test substance did not precipitate on the plates at this dose level. In tester strain TA100, toxicity was observed at dose levels of 1600 and 5000 μg/plate in the absence and presence of S9-mix. In tester strain WP2uvrA, toxicity was observed at the dose level of 5000 μg/plate in the absence and presence of S9-mix. No biologically relevant increase in the number of revertants was observed upon treatment with the test substance.

Based on the results of the dose range finding test, the test substance was tested in the first mutation experiment at a concentration range of 17 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA with the inclusion of a pre-incubation step. Test substance precipitated on the plates in the presence of S9-mix in the tester strains TA1535, TA1537 and TA98. Toxicity was observed in all tester strains. In tester strain TA1535, test substance induced up to 5.1- and 3.4-fold increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively. In tester strain TA100, test substance induced up to 1.9- and 2.7-fold dose related increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively.

In a follow-up experiment with additional parameters, the test substance was tested at a concentration range of 154 to 2800 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation experiment. Toxicity was observed in all tester strains. In tester strain TA100, test substance induced up to 2.0- and 4.0-fold dose related increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively.

To verify the mutagenic response of tester strain TA1535 in the first experiment, an additional pre-incubation experiment was performed with this tester strain at a concentration range of 17 to 5000 μg/plate in the absence of S9-mix. Toxicity was observed and no biologically relevant increase in the number of revertants was observed.

In tester strain TA100, test substance induced up to 2.0- and 4.0-fold dose related, increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively in both pre-incubation experiments. Since the increases observed in tester strain TA100 were above the historical control data range and the results were reproducible in the repeat experiment, these increases are biologically relevant and test substance is considered to be mutagenic in the absence and presence of S9-mix.

The test substance showed increases in the number of revertant colonies with tester strain TA1535. Although the up to 5.1 fold increase in the absence of S9-mix was above the historical control data range, this increase was not seen in two repeat experiments and only observed at one dose level. Therefore, this increase is considered to be not biologically relevant. The up to 3.4-fold increase in the presence of S9-mix was not above the historical control data range and was observed in only one experiment. Therefore, this increase is considered to be not biologically relevant.

All other bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, increase in the number of revertants in follow-up experiments.

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges 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 Trimellitic anhydride chloride is mutagenic in tester strain TA100 of the Salmonella typhimurium reverse mutation assay. Trimellitic anhydride chloride is not mutagenic in the other Salmonella typhimurium tester strains (TA1535, TA1537 or TA98) or Escherichia coli strain reverse mutation assay using strain WP2uvrA.