4-chloroformylphthalic anhydride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471 and EU Method B.13/14, GLP, Key study, klimisch 1): positive

- Ames Test (OECD 471, GLP, Suportive Study, klimisch 2): positive

- MLC/MLA (OECD 490 and EU Method B.17, GLP, Key , klimisch 1): positive

- HL/MNT in vitro (OECD 487, GLP, Key, klimisch 1): positive

Link to relevant study records

Reference

Reference 1

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21, 1997

Deviations:

no

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

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.

Target gene:

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

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 / 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.

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

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

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

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

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

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.

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

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

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

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 September 2015-02 March 2016

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

Adopted 28 July, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

No correction was made for the purity/composition of the test item.
A solubility test was performed. The test item was dissolved in dimethyl sulfoxide (Merck). The final concentration of the solvent in the exposure medium was 1% (v/v).

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA) (2001)
- Rationale: Recommended test system in international guidelines (e.g. OECD, EC) and literature

MEDIA USED:
- Type of media:
*Horse serum (Life Technologies) was inactivated by incubation at 56°C for at least 30 minutes.
*Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwiindrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).
*Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
*Exposure medium
- For 3 hour exposure: Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
- For 24 hour exposure: Cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
*Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 μg/ml trifluorothymidine (TFT) (Sigma).
*Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3 hours treatment: 17, 52, 164, 512 and 1600 µg/mL
Without S9-mix, 24 hours treatment: 17, 52, 164, 512 and 1600 µg/mL

Mutagenicity test (3-hour exposure only):
Without S9-mix: 5, 10, 25, 50, 75, 100, 115, 130, 150, 175, 200 and 225 μg/ml exposure medium.
With S9-mix: 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 65, 80 and 100 μg/ml exposure medium.

Determination of mutation frequencies at the TK-locus:
Without S9-mix: 25, 50, 75, 130, 150, 175, 200 and 225 μg/ml exposure medium.
With S9-mix: 10, 20, 30, 40, 50, 65, 80 and 100 μg/ml exposure medium.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (Merck Darmstadt, Germany)
- Justification for choice of solvent/vehicle: A solubility test was performed. The test item was dissolved in dimethyl sulfoxide (Merck).

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9-mix: 15 µg/mL in dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9-mix: 7.5 μg/mL in Hanks’ balanced salt solution (HBSS) (Invitrogen Corporation, Breda, The Netherlands) without calcium and magnesium

Details on test system and experimental conditions:

CLEANSING
Prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10 medium containing 10E-4 M hypoxanthine (Sigma), 2 x 10E-7 M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10E-5 M thymidine (Merck) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period cells were returned to R10 medium for at least 1 day before starting the experiment.

DOSE RANGE FINDING TEST
In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 8 x 10E6 cells (10E6 cells/ml for 3 hours treatment) or 5 x 10E6 cells (1.25 x 10E5 cells/ml for 24 hours treatment) with a number of test item concentrations increasing by approximately half log steps. The cell cultures for the 3 hours treatment were placed in sterile 30 ml centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 spm. The cell cultures for the 24 hours treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. The test item was tested in the absence and presence of S9-mix.
Cell cultures were exposed to Trimellitic anhydride chloride in exposure medium for 3 hours in the presence of S9-mix and for 3 and 24 hours in the absence of S9-mix. After exposure, the cells were separated from treatment solutions by 2 centrifugation steps (216 g, 5 min) each followed by removal of the supernatant. The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the final centrifugation step the cells were resuspended in R10 medium. The cells in the final suspension were counted with the coulter particle counter.
For determination of the cytotoxicity, the surviving cells of the 3 hours treatment were subcultured twice. After 24 hours of subculturing, the cells were counted (day 1) and subcultured again for another
24 hours, after which the cells were counted (day 2). The surviving cells of the 24 hours treatment were subcultured once. After 24 hours of subculturing, the cells were counted. If less than 1.25 x 10E5 cells/ml were counted no subculture was performed.
The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity tests. The results of the dose range finding study with the 24 hours treatment period has not been reported, since Trimellitic anhydride chloride already showed clear mutagenic responses after the 3 hours treatment period.

MUTAGENICITY TEST
Eight doses of Trimellitic anhydride chloride were tested in the mutation assay. The test item was tested in the presence and in the absence of S9-mix with a 3- treatment period.
The highest doses that were tested gave a cell survival of approximately 10-20% and the survival in the lowest doses was approximately the same as the cell survival in the solvent control. Also some intermediate doses were tested.

TREATMENT OF THE CELLS
Per culture 8 x 10E6 cells (10E6 cells/ml) were used. The cell cultures were placed in sterile 30 ml centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 spm. Solvent and positive controls were included and the solvent control was tested in duplicate.
Cell cultures were exposed for 3 hours to Trimellitic anhydride chloride in exposure medium in the absence and presence of S9-mix.
After exposure, the cells were separated from treatment solutions by 2 centrifugation steps (216 g, 5 min) each followed by removal of the supernatant. The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the final centrifugation step the cells were resuspended in R10 medium. The cells in the final suspension were counted with the coulter particle counter.
The second mutagenicity assay (prolonged exposure period) was cancelled based on the results of the first mutagenicity assay. Results will not be reported.

EXPRESSION PERIOD
For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 10E6 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test item the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).

DETERMINATION OF THE MUTATION FREQUENCY
For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. 1 cell was added per well (2 x 96-well microtiter plates/concentration) in non selective medium.
For determination of the mutation frequency (MF) a total number of 9.6 x 10E5 cells/concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 105 cells/concentration were plated in ten 96-well microtiter plates, each well containing
1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 days. After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.

ENVIRONMENTAL CONDITIONS
All incubations were carried out in a humid atmosphere (80 - 100%, actual range 41 – 91%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.7 – 37.5°C).

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10E6 survivors and ≤ 170 per 10E6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32.
d) The positive control should demonstrate an absolute increase in the total mutation frequency above the spontaneous background MF (an induced MF (IMF) of at least 300 x 10E-6). Furthermore, the positive control should have an increase in the small colony MF of at least 150 x 10E-6 above that seen in the concurrent solvent/control (a small colony IMF of at least 150 x 10E-6).

DATA EVALUATION
Any increase of the mutation frequency (MF) should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: No differences at the start and after the 3-hour treatment were observed in the pH measurements.
- Effects of osmolality: the osmolarity of the test item had no impact on the study results.
- Precipitation: No precipitation was observed up to the highest concentration.

RANGE-FINDING/SCREENING STUDIES:
- Dose range finding:
The relative suspension growth was 6 and 5% at the test item concentration of 512 μg/ml compared to the relative suspension growth of the solvent control in the absence and presence of S9-mix, respectively. At the test item concentration of 1600 μg/ml, no cell survival was observed in the absence of S9-mix and due to heavy precipitate after the 3 hours treatment no cell count was possible in the presence of S9-mix. The pH showed a drop to 4.1 at the dose level of 1600 μg/ml.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range with metabolic activation. Without metabolic activation, the spontaneous mutation frequencies in the solvent-treated control cultures were slightly above the maximum value of the historical control data (121 and 128 for an upper limit of 118). However, all acceptance criteria were met.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test item was tested up to appropriate cytotoxicity at the highest dose levels tested, Relative total growth (RTG) was reduced to 6 and 17% in the absence and presence of S9-mix, respectively.

Remarks on result:

other: Mutagenic

Conclusions:

It is concluded that Trimellitic anhydride chloride is mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Executive summary:

The mutagenic activity of Trimellitic anhydride chloride was investigated an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells in accordance with OECD Guideline No. 490 and the EU Method B.17 and under GLP compliance.

This report describes the effects of Trimellitic anhydride chloride on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the absence and in the presence of S9-mix with a 3 hours treatment period (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

In the solubility test, the pH and osmolarity of the item concentration of 512 μg/ml (the highest not precipitating dose level) were 6.7 and 0.463 Osm/kg respectively (compared to 7.2 and 0.463 Osm/kg in the solvent control). Since the culture treated with DMSO and the dose level of 512 μg/ml gave the same osmolarity value, the osmolarity of the test item had no impact on the study results.

In the dose range test the pH of all concentrations was determined at the start of the incubation and after 3-hour treatment. No differences at the start and after the 3-hour treatment were observed in the pH measurements. The test item showed a pH drop below 7.0 at dose levels of 512 μg/ml and above.

In the mutation experiment, the pH of the highest dose levels was determined at the start of the incubation and after 3-hour treatment. The following dose levels were determined: 50 to 255 μg/ml in the absence of S9-mix and 50 to 100 μg/ml in the presence of S9-mix (measurements were performed in exposure medium without metabolic activation). No significant differences at the start and after the 3-hour treatment were observed in the pH measurements. The test item showed no pH drop below 7.0 at all dose levels tested after 3-hour treatment.

In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test item range of 17 to 1600 μg/ml in the absence and presence of S9-mix with a 3-hour treatment period The relative suspension growth was 6 and 5% at the test item concentration of 512 μg/ml compared to the relative suspension growth of the solvent control in the absence of presence of S9-mix, respectively. At the test item concentration of 1600 μg/ml, no cell survival was observed in the absence of S9-mix and due to heavy precipitate after the 3 hours treatment no cell count was possible in the presence of S9-mix.

Based on the results of the dose range finding test, the following dose ranges were tested in single cultures in the mutation experiment, 25, 50, 75, 130, 150, 175, 200 and 225 μg/ml in absence of S9-mix and 10, 20, 30, 40, 50, 65, 80 and 100 μg/ml in the presence of S9-mix. The incubation time was 3 hours. Relative total growth (RTG) was reduced to 6 and 17% in the absence and presence of S9-mix, respectively.

The spontaneous mutation frequencies in the solvent-treated control cultures were within the acceptability criteria of this assay.

Mutation frequencies in cultures treated with positive control chemicals were increased 6.4-fold for MMS in the absence of S9-mix, and 16-fold for CP in the presence of S9-mix. In addition the observed mutation frequencies of the positive control items were within the acceptability criteria of this assay. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, Trimellitic anhydride chloride induced an up to 13-fold increase in the mutation frequency. The increase was above the historical solvent control data range and also above the GEF + MF(controls) (251 per 10E6 survivors).

In the presence of S9-mix, Trimellitic anhydride chloride induced an up to 5.4-fold increase in the mutation frequency. The increase was above the historical solvent control data range and also above the GEF + MF(controls) (215 per 10E6 survivors).

It is concluded that Trimellitic anhydride chloride is mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 February 2016 -13 July 2016

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.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

Adopted 26 September 2014

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

No correction was made for the purity/composition of the test item.
A solubility test was performed. In this solubility test the test item formed a suspension at concentrations of 160 mg/ml and above and formed a solution at a concentration of 51.2 mg/ml. Precipitate in the culture medium was observed at concentrations of 1600 μg/ml and above.

Target gene:

Not applicable

Species / strain / cell type:

other: Peripheral human lymphocytes

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Preliminary Toxicity Test: blood cultures were treated with 52, 164, 512, 1600 and 2000 μg Trimellitic anhydride chloride/ml culture medium ; for 3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix.

Experiment 1:
Without and with S9-mix : 10, 50, 75, 100, 125, 150 and 175 μg/ml culture medium (3 hours exposure time, 27 hours harvest time)

Experiment 1A:
Without S9-mix : 10, 100, 150, 175, 200 and 225 μg/ml culture medium (3 hours exposure time, 27 hours harvest time)

Experiment 2:
Without S9-mix : 10, 50, 75, 100, 125 and 150 μg Trimellitic anhydride chloride/ml culture medium (24 hours exposure time, 24 hours harvest time)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (SeccoSolv, Merck, Darmstadt, Germany)
- Justification for choice of solvent/vehicle: A solubility test was performed. The test item was dissolved in dimethyl sulfoxide.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Remarks:

Solvent for positive controls: Hanks’ Balanced Salt Solution (HBSS) (Life Technologies, Bleiswijk, The Netherlands), without calcium and magnesium

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation (S9-mix)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Remarks:

Solvent for positive controls: Hanks’ Balanced Salt Solution (HBSS) (Life Technologies, Bleiswijk, The Netherlands), without calcium and magnesium

Positive control substance:

other: Colchicine

Remarks:

Without metabolic activation (S9-mix)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Remarks:

Solvent for positive controls: Hanks’ Balanced Salt Solution (HBSS) (Life Technologies, Bleiswijk, The Netherlands), without calcium and magnesium

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

TEST SYSTEM: Cultured peripheral human lymphocytes were used as test system. Blood was collected from healthy adult, non-smoking volunteers (aged 18 to 35 years). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2015) are presented below:
- Dose range finding study: age 33, AGT = 12.7 h
- First cytogenetic assay: age 28, AGT = 12.9 h
- Cytogenetic assay 1A: age 21, AGT = 13.0 h
- Second cytogenetic assay: age 35, AGT = 12.9 h

CELL CULTURE: Cells (whole blood cultures) were grown RPMI 1640 medium (Life Technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Life Technologies), L-glutamine (2 mM) (Life Technologies), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) (Life Technologies) and 30 U/ml heparin (Sigma, Zwijndrecht, The Netherlands). All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 55 - 91%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.9 - 37.2°C). The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

DURATION
- Exposure duration: 3 h (± S9) and 24 h continuous exposure (-S9) in preliminary toxicity test; 3 h (± S9) in Experiment 1, 3 h (-S9) in Experiment 1A, 24 h continuous exposure (-S9) in Experiment 2
- At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 h in the presence of Cytochalasin B (5 µg/mL).

SPINDLE INHIBITOR (cytogenetic assays): Prior to the mitosis (after exposure of the test substance) the chemical cytochalasin B (5 μg/mL) was added to the cultures.
STAIN (for cytogenetic assays): after fixation, slides were stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8.

NUMBER OF REPLICATIONS:
- 1 culture per dose for test item, vehicle and positive controls

NUMBER OF CELLS EVALUATED:
- Cytotoxicity: A minimum of 500 cells (with a maximum deviation of 5%) per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells).
- Scoring of Micronuclei: At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were examined by light microscopy for micronuclei. In addition, at least 1000 (with a maximum deviation of 5%) mononucleated cells per culture were scored for micronuclei separately.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity of test item in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
Cytotoxicity = 100-100{(CBPIT – 1)/(CBPIC –1)}
CBPI = [(No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)] / [Total number of cells]
T = test substance treatment culture
C = vehicle control culture

OTHER:
The following criteria for scoring of binucleated cells were used (1 - 2, 6):
- Main nuclei that were separate and of approximately equal size.
- Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
- Main nuclei that were linked by nucleoplasmic bridges.
The following cells were not scored:
- Trinucleated, quadranucleated, or multinucleated cells.
- Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996 (1):
- The diameter of micronuclei should be less than one-third of the main nucleus.
- Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
- Micronuclei should have similar staining as the main nucleus.

ACCEPTABILITY OF THE ASSAY:
An in vitro micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item colchicine induces a statistically significant increase in the number of mononucleated cells with micronuclei and the positive control items MMC-C and CP induces a statistically significant increase in the number of binucleated cells with micronuclei. The positive control data will be analysed by the Chi-square test (one-sided, p < 0.05).

Evaluation criteria:

DATA EVALUATION AND STATISTICAL PROCEDURES:
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with an Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with an Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range. In case the Chi-square test shows that there are statistically significant differences between one or more of the test item groups and the vehicle control group a Cochran Armitage trend test (p < 0.05) will be performed to test whether there is a significant trend in the induction. List of deviations.

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) and ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) were used for statistical analysis of the data.

Key result

Species / strain:

other: Peripheral human lymphocytes

Remarks:

3-hour exposure

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

other: Peripheral human lymphocyte

Remarks:

3-hour exposure

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

other: Peripheral human lymphocyte

Remarks:

24-hour exposure

Metabolic activation:

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
- Effects of pH and osmolality: The test item showed a pH drop (0.5 unit) in cytogenetic assay 1A and 2 at the start of the incubation period at the highest dose level of 225 and 150 μg/ml, respectively. Fluctuations in osmolarity were observed in the first cytogenetic assay at t = 0 at concentrations of 125 and 150 μg/ml, however since these fluctuations were not dose related and not observed after 3 hours, these are not considered biologically relevant. In cytogenetic assay 1A and 2 osmolarity was not influenced by the test item.

RANGE-FINDING/SCREENING STUDIES:
- At a concentration of 2000 μg/ml Trimellitic anhydride chloride precipitated in the culture medium.
- Toxicity had not allowed to prepared slides at 1600 and 2000 µg/mL.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: The number of mono- and binucleated cells with micronuclei found in the solvent control was within the 95% control limits of the distribution of the historical negative control database.

NUMBER OF CELLS WITH MICRONUCLEI
*First cytogenetic assay:
In the first cytogenetic assay, in the absence of S9-mix, at the 3 hours exposure time, Trimellitic anhydride chloride induced a statistically significant increase in the number of binucleated cells with micronuclei at the highest dose level. The number of micronucleated cells was outside the 95% control limits of the distribution of the historical negative control database. In addition, a statistical significant dose related trend was observed in the absence of S9-mix (binucleated cells). These results indicate that Trimellitic anhydride chloride is positive in the in vitro micronucleus study and might be considered a clastogenic compound. The number of mononucleated cells with micronuclei was within the 95% control limits of the distribution of the historical negative control database.
In the presence of S9-mix, Trimellitic anhydride chloride did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei.

*Second cytogenetic assay:
In the second cytogenetic assay with a 24 hours continuous exposure time, Trimellitic anhydride chloride did not induce a statistically significant increase in the number of mononucleated and binucleated cells with micronuclei.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database [see "Any other information on results incl. tables"].
- Negative (solvent/vehicle) historical control data: The number of mono- and binucleated cells with micronuclei found in the solvent control was within the 95% control limits of the distribution of the historical negative control database [see "Any other information on results incl. tables"].

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity of positive results is detailled in the summary table 1 in section "Any other information on results incl. tables".

Remarks on result:

other: clastogenic

Table 1. Number of mononucleated or binucleated cells with micronuclei of human lymphocyte cultures treated with Trimellitic anhydride chloride in the first cytogenetic assay (3 hours exposure time (without S9 -mix), 27 hours harvest time)

Concentration (µg/mL)	Cytostasis (%)	Number of mononucleated cells with micronuclei^1)			Number of binucleated cells with micronuclei^1)			Mean CBPI
		1000	1000	2000	1000	1000	2000
		A	B	A + B	A	B	A + B
0	0	0	0	0	3	4	7	1.88
10	5	1	0	1	2	3	5	1.84
100	35	1	0	1	6	3	9	1.57
150	59	1	0	1	16	15	31***	1.36
0.25 MMC-C	21	0	2	2	24	28	52***	1.70
0.1 Colch	95	32	28	60***	1^2)	2^2)	3	1.04

^*) Significantly different from control group (Chi-square test). * P < 0.05. ** P < 0.01 or *** P < 0.001.

¹⁾ 1000 bi- and mononucleated cells were scored for the presence of micronuclei. Duplicate cultures are indicated by A and B.

²⁾ 284 and 221 binucleated cells were scored for the presence of micronuclei, respectively.

Only the Table with positive results was reported there. The two other conditions (3 hours exposure time with S9 -mix and 24 hours esposure time without S9 -mix) were both negative to clastogenic effects.

Table 2. Historical control data for in vitro micronucleus studies of sovent control

	Mononucleated			Binucleated
	+ S9-mix	- S9-mix		+ S9-mix	- S9-mix
	3 hour exposure	3 hour exposure	24 hour exposure	3 hour exposure	3 hour exposure	24 hour exposure
Mean number of micronucleated cells (per 1000 cells)	0.89	1.07	0.95	3.57	3.77	4.00
SD	0.92	1.10	1.27	2.55	2.48	2.62
n	102	104	99	102	104	99
Upper control limit (95% control limits)	3.04	3.87	3.84	9.19	10.23	10.81
Lower control limit (95% control limits)	-1.25	-1.74	-1.95	-2.05	-2.68	-2.81

SD = Standard deviavtion

n = Number of observations

Distribution historical negative control data from experiments performed between January 2012 and June 2016.

Table 3. Historical control data for in vitro micronucleus studies of positive control substances

	Mononucleated		Binucleated
	- S9-mix		+ S9-mix	- S9-mix
	3 hour exposure	24 hour exposure	3 hour exposure	3 hour exposure	24 hour exposure
Mean number of micronucleated cells (per 1000 cells)	21.11	22.57	26.02	28.78	23.18
SD	28.25	27.75	12.96	25.69	15.59
n	210	204	108	210	204
Upper control limit (95% control limits)	78.68	86.40	48.42	70.48	63.33
Lower control limit (95% control limits)	-41.26	3.61	3.61	-12.92	-16.97

SD = Standard deviation

n = Number of observations

Distribution historical positive control data from experiments performed between January 2012 and June 2016.

Conclusions:

Under the test conditions, trimellitic anhydride mono-chloride was considered to be clastogenic to human lymphocytes in vitro.

Executive summary:

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to test material in the presence and absence of a metabolic activation system (S9 mix). In Experiment 1, test material was tested at the concentrations of 10, 50, 75, 100, 125, 150 and 175 μg/ml culture medium (with and without S9-mix) for a 3 hours exposure time. In the absence of S9-mix no appropriate dose levels could be selected for scoring of micronuclei since at the highest concentration of 175 μg/ml not enough cytotoxicity was observed (46%). The experiment was repeated in cytogenetic assay 1A in which 10, 100, 150, 175, 200 and 225 μg/ml culture medium for 3 hours exposure time without S9 -mix. In Experiment 2, test material was tested at the concentrations of 10, 100 and 125 μg Trimellitic anhydride chloride/ml culture medium (without S9-mix) for 24 hours exposure time. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 h in the presence of Cytochalasin B. After harvesting, the cells were then treated with a hypotonic solution, fixed, stained and examined for micronuclei. Preliminary toxicity test was performed before the main test.

 

All vehicle (DMSO) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

 

In the first cytogenetic assay, in the absence of S9-mix, at the 3 hours exposure time, Trimellitic anhydride chloride induced a statistically significant increase in the number of binucleated cells with micronuclei at the highest dose level. The number of micronucleated cells was outside the 95% control limits of the distribution of the historical negative control database. In addition, a statistical significant dose related trend was observed in the absence of S9-mix (binucleated cells). These results indicate that Trimellitic anhydride chloride is positive in the in vitro micronucleus study and might be considered a clastogenic compound. The number of mononucleated cells with micronuclei was within the 95% control limits of the distribution of the historical negative control database.

In the presence of S9-mix, Trimellitic anhydride chloride did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei.

In the second cytogenetic assay with a 24 hours continuous exposure time, Trimellitic anhydride chloride did not induce a statistically significant increase in the number of mononucleated and binucleated cells with micronuclei.

 

It is concluded that, under the test conditions, this test is valid and that Trimellitic anhydride chloride induces the formation of micronuclei in human lymphocytes in the absence of S9 metabolic activation at the short term incubation period only. In contrast, in the presence of S9-mix no such genetoxic effect was found. Since Trimellitic anhydride chloride induces the micronuclei frequency, it may be considered as an clastogenic compound.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From January 2011 to February 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

The study was performed according to a standard method and in compliance with Japanese GLP. There are some lack of translation from the original Japanese report.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

4-chloroformylphthalic anhydride

Target gene:

Histidine gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rat liver induced with a mixture of phenobarbital and 5,6-benzoflavone.

Test concentrations with justification for top dose:

Dose-finding-study: 0.00305, 0.0122, 0.0488, 0.195, 0.781, 3.13, 12.5 and 50 mg/mL in the presence (S9 mix) and in the absence of metabolic activation.
Final examination : 0.195, 0.391, 0.781, 1.56, 3.13, 6.25, 12.5, 25, and 50 mg/mL in the presence (S9 mix) and in the absence of metabolic activation.
Identification test: 1.56, 3.13, 6.25.12.5, 25, and 50 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the substance decomposes violently into water and 50mg/mL or more of test item dissolve in DMSO.

Untreated negative controls:

yes

Remarks:

Dimethyl sulfoxide (DMSO)

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) :
45µL of strain suspension was directly inoculated into 20mL of nutrient broth (Nutrient broth No.2, Oxoid) and culture was carried out at 37°C and 50 rpm for 10 hours.
The turbidity of the bacterial culture is measured after termination of the culture and the number of bacilli after culture is 1.0x10^9 by the conversion from OD value. It was checked that there were nine or more pieces/mL.
For the direct plate incorporation test without metabolic activation, 500µL of 100 mM-phosphate buffer (pH 7.4) and 100 µL of bacterial culture were added to 100 µL of test solution. It was shook for 20 minutes at 37°C. Then 2 mL of a solution which mixed soft-agar liquid with 0.5mM biotin-0.5mM histidine for Salmonella typhimurium (0.5mM tryptophan in the case of E. coli) at a ratio of 1:10 was added. The plate was incubated for 48 hours at 37°C.
For tests with metabolic activation, 500 µL of S9 mix and 0.1 µL of bacterial culture were added to 100 µL of test solution. It was shook for 20 minutes at 37°C. Then 2 mL of a solution which mixed soft-agar liquid with 0.5mM biotin-0.5mM histidine for Salmonella typhimurium (0.5mM tryptophan in the case of E. coli) at a ratio of 1:10 was added. The plate was incubated for 48 hours at 37°C.

DURATION
- Incubation period: 48 hours at 37°C

DETERMINATION OF CYTOTOXICITY
Background lawn was observed with the stereoscopic microscope and the existence of growth inhibition was checked.
The revertant colony count was performed using the colony analyzer (CA-l1DS, system science incorporated company).

It checked about the existence of precipitation of a test article by viewing after culture

Evaluation criteria:

Average value (below decimal point rounding off) was calculated from the reversion colony count of each plate, and positivity of the test is where all the following standards were met.
1): The number of revertant colonies in a test item group is more than twice of a negative control group.
2): Dose-dependency is accepted.
3): Reproducibility is accepted in the dose finding study and the final examination.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: not clear in the report

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: not clear in the report

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: not clear in the report

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: not clear in the report

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

In the dose finding study, the final examination and the identification test, all test conditions were fulfilled.

The increase of revertant colonies was found more than twice the negative control for TA100 (with direct method and metabolic activation method), in TA1535, WP2uvrA, and TA98.

Precipitation of the test article was observed in any dose.

Conclusions:

Interpretation of results : positive

It was considered that trimellitic anhydride chloride induced gene mutation in the conditions of the test.

Executive summary:

This study was performed to investigate the potential of trimellitic anhydride chloride to induce reverse mutation in S. typhimurium and E. coli. The study was performed according to a standard method and in compliance with Japanese GLP.

 

A preliminary toxicity test was performed to define the dose-levels of the test item to be used for the mutagenicity study. The test item was then tested according to the direct plate incorporation, with and without a metabolic activation system, the S9 mix, prepared from rat liver induced with a mixture of phenobarbital and 5,6-benzoflavone.repared. Four strains of bacteria Salmonella typhimurium TA100, TA1535, TA98 and TA1537 and one strain ofE. coli WP2 uvr A were used. Each strain was exposed to nine dose-levels of the test item. After 48 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. DMSO was used as the negative control while four positive controls were used: 2-aminoanthracene; 9-aminoacridine; 2-nitrofluorene and N-ethyl-N'-nitro-N-nitrosoguanidine

The increase of revertant colonies was found more than twice the negative control for TA100 (with direct method and metabolic activation method), in TA1535, WP2uvrA, and TA98. Precipitation of the test article was observed in any dose. In the dose finding study, the final examination and the identification test, all test conditions were fulfilled.

 

Thus, it is considered that trimellitic anhydride chloride induced gene mutation in the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Following positive effects observed in the in vitro studies and the testing proposal accepted by ECHA, in vivo micronucleus test and in vivo comet assay were conducted (2022):

- In vivo micronucleus test (OECD 474): negative

- In vivo comet assay (OECD 489): negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: 27 June to 18 August 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Remarks:

/ in vivo Micronucleus test combined with in vivo Comet assay

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016

Deviations:

no

Principles of method if other than guideline:

This micronucleus test was combined with the comet assay (according to the guideline OECD 489).

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo, Blackthorn, UK.
- Age at study initiation: 6 to 8 weeks old for the range-finder experiment and 7 to 8 weeks old for the main experiment
- Weight at study initiation: 182-197 g (males) or 162-173 g (females) for the range-finder experiment and 215-256 g (males) for the main experiment
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: in wire topped, solid bottomed cages, with three animals of the same sex per cage.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Animals were acclimatised for at least 5 days and a health inspection was performed before the start of dosing to ensure their suitability for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 40-70%
- Air changes (per hr): 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours dark and 12 hours light

IN-LIFE DATES:
Study Initiation Date: 20 June 2022
Experimental Start Date: 27 June 2022
In-life Start Date: 27 June 2022
In-life End Date: 14 July 2022
Experimental Completion Date: 18 August 2022

Route of administration:

oral: gavage

Vehicle:

- Vehicle: corn oil
- Justification for choice of solvent/vehicle: common organic solvent used for oral dosing in rodents and has been used in previous in vivo toxicity studies with compounds that were close analogues of the test article.
- Concentration of test material in vehicle: 35, 70 and 140 mg/mL
- Dose volume: 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article was weighed out into a pre-labelled container and vehicle added to achieve the final volume. The mixture was stirred vigorously and heated to approximately 75°C (77°C, 78°C or 88°C for Main Experiment formulations) until visibly homogenous, then allowed to cool to room temperature whilst stirring. Formulations were then aliquoted as required.

STABILITY OF DOSING FORMULATIONS
Prior to the Main Experiment, Trimellitic anhydride chloride (TMAC) formulations in the vehicle at 10, 100 and 200 mg/mL were confirmed as stable and homogenous for 24 hours when stored at 15 to 25⁰C, and for 4 days when stored refrigerated (2 to 8°C) and protected from light.
All formulations used for animal dosing in the Main Experiment were protected from light and stored 2-8°C when not required for dosing. On each day of dosing of the Main Experiment, an aliquot of each formulation was held at room temperature in the animal facility and used on the day of removal from refrigerated conditions.
All formulations were used within 3 days of preparation.

Duration of treatment / exposure:

once daily administration, for 3 days (at 0, 24 and 45h)

Frequency of treatment:

daily

Post exposure period:

Not applicable

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control group

Dose / conc.:

350 mg/kg bw/day (nominal)

Remarks:

measured dose (actual dose): 266 mg/kg bw/day

Dose / conc.:

700 mg/kg bw/day (nominal)

Remarks:

measured dose (actual dose) = 700 mg/kg bw/day

Dose / conc.:

1 400 mg/kg bw/day (nominal)

Remarks:

measured dose (actual dose) = 1400 mg/kg bw/day

No. of animals per sex per dose:

6 males/group

Control animals:

yes, concurrent vehicle

Positive control(s):

ethyl methanesulfonate
- Route of administration: oral (gavage)
- Doses / concentrations: 150 mg/kg bw/day
- Prepared in water at the concentration of 15 mg/mL

Tissues and cell types examined:

Bone marrow cells were isolated from one femur

Details of tissue and slide preparation:

SAMPLING:
The isolated femur was cleaned of adherent tissue and the ends removed from the shanks. Using a syringe and needle, bone marrow was flushed from the marrow cavity with 2 mL fetal bovine serum into appropriately labelled centrifuge tubes. The samples were filtered through cellulose columns, containing 50 mg/mL equal mix of type 50 and α-cellulose. Once the majority of the 2 mL had passed through the column a further 4 mL of serum was added to the sample tubes and loaded onto the columns.
Once filtered, the bone marrow cells were pelleted by centrifugation (200 g, 5 minutes, 15-25°C) and the supernatant aspirated and discarded. A further 3 mL of foetal bovine serum was added to the tubes followed by gentle resuspension of the cell pellet. The cells were pelleted again (as described above) and the supernatant aspirated to leave one or two drops and the cell pellet.

DETAILS OF SLIDE PREPARATION:
The pellet was mixed into this small volume of serum in each tube by using a Pasteur pipette, and from each tube one drop of suspension was placed on the end of each of two (Range-Finder Experiment) or three (Main Experiment) uniquely labelled slides. A smear was made from the drop by drawing the end of a clean slide along the labelled slide.
Slides were air-dried, then fixed for 10 minutes in absolute methanol and rinsed several times in distilled water. Two slides per animal were immediately stained for 5 minutes in 12.5 µg/mL acridine orange made up in 0.1 M phosphate buffer pH 7.4. Slides were rinsed in phosphate buffer, then dried and stored protected from light at room temperature prior to analysis.

METHOD OF ANALYSIS:
The relative proportions of polychromatic erythrocytes (PCE), seen as bright orange enucleate cells, and normochromatic erythrocytes (NCE), seen as smaller dark green enucleate cells, were determined until a total of at least 500 cells (PCE plus NCE) had been analysed as an indication of potential bone marrow toxicity. For the Main Experiment. analysis continued until at least 4000 PCE/animal were examined for the presence of MN. All PCE containing MN observed during these two phases of counting were recorded.

The following criteria were used for analysis of slides:
1. Cells were of normal cell morphology
2. Areas where erythrocytes overlapped were ignored
3. A MN was round or oval in shape
4. A cell containing more than one MN was scored as a single micronucleated cell
5. MN that were refractive, improperly stained or not in the focal plane of the cell were judged to be artefacts and were not scored.

Evaluation criteria:

ACCEPTANCE CRITERIA (STUDY VALIDITY)
The data were considered valid if the following criteria were met:
1. The vehicle control data were comparable to the laboratory historical control data for each tissue
2. The positive control induced responses that were compatible with the laboratory historical control data and that produced a statistically significant increase compared to the concurrent vehicle control
3. Adequate numbers of cells and doses were analysed
4. The high dose was considered to be the MTD, the maximum recommended dose, the maximum practicable dose or one that demonstrated cytotoxicity to the target cells.

CRITERIA FOR DNA DAMAGE
For valid data, the test article was considered to induce clastogenic/aneugenic damage if:
1. A statistically significant increase in the frequency of MN PCE occurred at one or more dose levels
2. The incidence and distribution of MN PCE exceeded the laboratory’s historical vehicle control data
3. A dose-response trend in the proportion of MN PCE (where more than two dose levels are analysed) was observed.

Statistics:

After completion of microscopic analysis and decoding of the data the following were calculated:
1. %PCE for each animal and the mean for each group. The group mean %PCE values were examined to see if there was any decrease in groups of treated animals that could be taken as evidence of bone marrow toxicity
2. Frequency of MN PCE (i.e. MN per number of PCE scored) and %MN PCE for each animal and the group mean %MN PCE (standard deviation).
The numbers of MN PCE in the vehicle control animals were compared with the laboratory's historical control data to determine whether the assay was acceptable. The numbers of MN PCE in each treated group were compared with the numbers in vehicle control groups by use of the Wilcoxon rank sum test (Lehmann, 1975). The tests were interpreted with one-sided risk for increased frequency with increasing dose. A Terpstra-Jonckheere test was conducted to evaluate dose response (Jonckheere, 1954). Probability values of p≤0.05 were accepted as significant.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

A reduction of 13% was observed in group mean %PCE at 1400 mg/kg/day.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
See above in the section "Any other information on materials and methods incl. tables".
A preliminary experiment was conducted at 2000 and 1400 mg/kg bw/day.
On Day 3, there were significant observations in both sexes for animals administered 2000 mg/kg (mouth rubbing, ataxia, decreased activity, excessive salivation, diarrhoea, raised hair, semi-closed eyes, red/brown mouth and/or snout, audible breathing, paddling, vocalisation, hunched posture and/or prone; two females were found dead 2 hours post dose). Therefore, 2000 mg/kg/day clearly exceeded an appropriate maximum tolerated dose in both sexes. As such, bone marrow toxicity was not assessed in surviving animals.
From these results, 1400 mg/kg/day was considered to be an appropriate estimate of the MTD and was therefore selected as the maximum dose for the Main Experiment. Two lower doses of 350 (25% MTD) and 700 mg/kg/day (50% MTD) were selected.

RESULTS OF MAIN STUDY
> Post dose observation
There were no clinical observations of toxicity on Day 1 or Day 2 for any animal dosed in the Main Experiment. On Day 3, clinical observations at 1400 mg/kg/day included liquid or soft faeces, mouth rubbing, mild decreased activity, raised hair, ataxia and low carriage. Transient observation of mouth rubbing was noted for one animal at 700 mg/kg/day on Day 3.

> Toxicity - Ratio PCE/NCE
Animals treated with Trimellitic anhydride chloride (TMAC) at 266 or 700 mg/kg/day exhibited group mean %PCE that were similar to the vehicle control group mean and which were comparable with the laboratory’s historical vehicle.
A reduction of 13% was observed in group mean %PCE at 1400 mg/kg/day. This reduction was slight and within the laboratory’s historical vehicle control data. However, as it was observed in 5 out of 6 animals, it was considered potential evidence of bone marrow toxicity.

> Genotoxicity - Induction of micronuclei
Animals treated with Trimellitic anhydride chloride (TMAC) at 266, 700 or 1400 mg/kg/day exhibited group mean %MN PCE frequencies that were similar to the vehicle control group mean and which fell within the 95% reference range of the laboratory's historical vehicle control data. There were no statistically significant increases in micronucleus frequency for any of the groups receiving the test article, compared to the concurrent vehicle control with no evidence of a dose response. Individual animal MN PCE frequencies were considered consistent with the laboratory's historical vehicle control animal distribution data. It was noted that there was an isolated animal with a %MN PCE value (0.50%) that exceeded the 95% reference range (0-0.33%); however, as this elevated value was not reproduced in any other animal in the high dose group and the group data was not statistically increased compared to the concurrent vehicle control group, this value was considered of no biological relevance.

> Results of formulations analysis
Test article formulations at 35, 70 and 140 mg/mL were prepared once for dosing in the Main Experiment. The analytical data confirmed that dosing formulations at 70 and 140 mg/mL were homogenous (0.83-1.29% RSD; target ≤5% RSD) and achieved target nominal concentration, mean values of 88-91% (within 100±15%). However, while the formulation at 35 mg/mL was also homogenous (1.43% RSD), it fell outside acceptance criteria of 85-115% with a mean value of 76% and therefore an actual dose level of 266 mg/kg/day.
As there were no analytical errors found upon investigation, the analytical data were considered accurate for reporting. Although the dose level of 350 mg/kg/day was not achieved, there is no impact to the validity of the study as the formulation was homogenous such that there were no concerns over the actual dose provided (266 mg/kg/day), which is within acceptable dose level intervals per OECD guidelines (i.e. within 4-fold of 700 mg/kg/day).
The formulations were therefore considered acceptable. No test article was detected in the vehicle sample.

Trimellitic anhydride chloride (TMAC): Summary of Micronucleus Data

Group	% PCE	% MN PCE	Statistic analysis WRS
	Mean  (SD)	Mean (SD)	P-value Significance
1 - Vehicle 0 mg/kg bw/day	49.00  (1.88)	0.10 (0.06)	N/A N/A
2- TMAC 266 mg/kg bw/day	49.70 (4.14)	0.13 (0.06)	0.1147 NS
3- TMAC 700 mg/kg bw/day	50.13 (6.21)	0.16 (0.08)	0.0996 NS
4- TMAC 1 400 mg/kg bw/day	42.70 (3.17)	0.12 (0.19)	0.8647 NS
5- Positive (EMS) 150 mg/kg bw/day	47.00 (2.82)	1.53 (0.35)	0.0119 *

EMS: Ethyl methanesulfonate / SD: Standard Deviation / WRS: Wilcoxon Rank Sum

PCE: Polychromatic erythrocytes / MN PCE: Micronucleated polychromatic erythrocytes

Conclusions:

When tested in the micronucleus test conducted according to the OECD 474 guideline in rats exposed up to 1400 mg/kg bw/day (maximum tolerated dose), Trimellitic anhydride chloride (TMAC), did not induce an increase in micronucleated polychromatic erythrocytes of the bone marrow.

Executive summary:

Trimellitic anhydride chloride (TMAC) was tested for its potential to induce micronuclei (MN) in the polychromatic erythrocytes (PCE) of the bone marrow of rats. The study was conducted according to the guideline OECD 474 and combined with the comet assay (according to the guideline OECD 489).

Male Sprague Dawley rats were treated by gavage once daily, for 3 days, at the actual dose levels of 266, 700 and 1400 mg/kg bw/day. Based on a range-finder experiment, the dose level of 1400 mg/kg bw/day was considered as being the MTD (maximum tolerated dose). The vehicle control group received the vehicle (corn oil) and the positive control group received Ethyl methanesulfonate (EMS) at 150 mg/kg bw/day.

Bone marrows from one femur were sampled at necropsy (Day 3, 48 hours, i.e. 3 hours after the last dosing).

The group mean vehicle control data (%MN PCE) was within the 95% reference ranges of the laboratory’s historical vehicle control data. The EMS positive control induced statistically significant increases in %MN PCE (over the current vehicle control group) that was comparable with the laboratory’s historical positive control data ranges. The assay was therefore accepted as valid.

There was a small reduction in group mean %PCE at 1400 mg/kg/day (-13%) that was observed in five animals. While the reduction was within the laboratory's historical control data, as it was observed in 5 out of 6 animals, it was considered potential evidence of bone marrow toxicity.

Animals treated with Trimellitic anhydride chloride (TMAC) at 266, 700 or 1400 mg/kg/day exhibited group mean %MN PCE frequencies that were similar to the vehicle control group mean and which fell within the 95% reference range of the laboratory's historical vehicle control data. There were no statistically significant increases in micronucleus frequency for any of the groups receiving the test article, compared to the concurrent vehicle control with no evidence of a dose response. Individual animal MN PCE frequencies were considered consistent with the laboratory's historical vehicle control animal distribution data. An isolated animal showed a %MN PCE value (0.50%) that exceeded the 95% reference range (0-0.33%); however, as this elevated value was not reproduced in any other animal in the high dose group and the group data was not statistically increased compared to the concurrent vehicle control group, this value was considered of no biological relevance.

It is concluded that under the conditions of this study, Trimellitic anhydride chloride (TMAC), did not induce an increase in micronucleated polychromatic erythrocytes of the bone marrow when tested up to 1400 mg/kg/day (an estimate of the maximum tolerated dose for this study).

 

Reference 2

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: 27 June to 18 August 2022

Reliability:

1 (reliable without restriction)

Reason / purpose for cross-reference:

reference to same study

Remarks:

/ in vivo Micronucleus test combined with in vivo Comet assay

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

2016

Deviations:

no

Principles of method if other than guideline:

This comet assay was combined with micronucleus test (according to the guideline OECD 474).

GLP compliance:

yes

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo, Blackthorn, UK.
- Age at study initiation: 6 to 8 weeks old for the range-finder experiment and 7 to 8 weeks old for the main experiment
- Weight at study initiation: 182-197 g (males) or 162-173 g (females) for the range-finder experiment and 215-256 g (males) for the main experiment
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: in wire topped, solid bottomed cages, with three animals of the same sex per cage.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Animals were acclimatised for at least 5 days and a health inspection was performed before the start of dosing to ensure their suitability for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 40-70%
- Air changes (per hr): 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours dark and 12 hours light

IN-LIFE DATES:
Study Initiation Date: 20 June 2022
Experimental Start Date: 27 June 2022
In-life Start Date: 27 June 2022
In-life End Date: 14 July 2022
Experimental Completion Date: 18 August 2022

Route of administration:

oral: gavage

Vehicle:

- Vehicle: corn oil
- Justification for choice of solvent/vehicle: common organic solvent used for oral dosing in rodents and has been used in previous in vivo toxicity studies with compounds that were close analogues of the test article.
- Concentration of test material in vehicle: 35, 70 and 140 mg/mL
- Dose volume: 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article was weighed out into a pre-labelled container and vehicle added to achieve the final volume. The mixture was stirred vigorously and heated to approximately 75°C (77°C, 78°C or 88°C for Main Experiment formulations) until visibly homogenous, then allowed to cool to room temperature whilst stirring. Formulations were then aliquoted as required.

STABILITY OF DOSING FORMULATIONS
Prior to the Main Experiment, Trimellitic anhydride chloride (TMAC) formulations in the vehicle at 10, 100 and 200 mg/mL were confirmed as stable and homogenous for 24 hours when stored at 15 to 25⁰C, and for 4 days when stored refrigerated (2 to 8°C) and protected from light.
All formulations used for animal dosing in the Main Experiment were protected from light and stored 2-8°C when not required for dosing. On each day of dosing of the Main Experiment, an aliquot of each formulation was held at room temperature in the animal facility and used on the day of removal from refrigerated conditions.
All formulations were used within 3 days of preparation.

Duration of treatment / exposure:

once daily administration, for 3 days (at 0, 24 and 45h)

Frequency of treatment:

daily

Post exposure period:

not applicable

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle control group

Dose / conc.:

350 mg/kg bw/day (nominal)

Remarks:

measured dose (actual dose) = 266 mg/kg bw/day

Dose / conc.:

700 mg/kg bw/day (nominal)

Remarks:

measured dose (actual dose) = 700 mg/kg bw/day

Dose / conc.:

1 400 mg/kg bw/day (nominal)

Remarks:

measured dose (actual dose) = 1400 mg/kg bw/day

No. of animals per sex per dose:

6 males/group

Control animals:

yes, concurrent vehicle

Positive control(s):

ethyl methanesulfonate
- Route of administration: oral (gavage)
- Doses / concentrations: 150 mg/kg bw/day
- Prepared in water at the concentration of 15 mg/mL

Tissues and cell types examined:

Liver, glandular stomach and duodenum
Germs cells were collected but not analyzed as results in somatic cells are negative.

Details of tissue and slide preparation:

PREPARATION OF COMET SLIDES SUSPENSIONS
Tissues were placed in pots appropriately covered in Merchants + DMSO (hereafter known as ‘Merchants’) solution in necropsy and held on ice until processed by Genetic Toxicology.

> The comet liver samples were cut into small pieces and washed thoroughly in Merchants solution. The pieces of liver were minced into smaller pieces and then pushed through bolting cloth (pore size of 150 µm) to produce single cell suspensions.
> The comet glandular stomach samples were incubated on ice for 15 minutes prior to processing. After incubation the stomach samples were removed from the Merchants solution and the inner surface gently scraped twice using the back of a scalpel blade. Cells were gently scraped from the inside surface of the stomach using the back of a scalpel blade in 200 μL of fresh Merchants solution to produce single cell suspensions.
> The comet duodenum samples were vortexed in ice cold Merchants solution for approximately 20 seconds. The tissue was removed from the Merchants solution and the inner surface gently scraped using the back of a scalpel blade. The tissue was vortexed in ice cold Merchants solution for a further 20 seconds prior to gently scraping the inside of the duodenum with the back of a scalpel blade in 150 μL of fresh Merchants solution to produce single cell suspensions.
> The comet gonad samples were prepared by making an incision along the length of a single gonad, removing the contents from the membrane and discarding the membrane. The remaining tissue was cut into small pieces and gently pushed through bolting cloth (pore size of 150 μm) with approximately 10 mL of Merchants solution to produce single cell suspensions.

All cell suspensions were held on ice prior to slide preparation.

PREPARATION OF COMET SLIDES
Three slides were prepared per single cell suspension per tissue. Slides were dipped in molten normal melting point agarose such that all of the clear area of the slide and at least part of the frosted area was coated. The underside of the slides was wiped clean and the slides allowed to dry. 40 µL of each single cell suspension was added to 400 µL of 0.7% low melting point agarose at approximately 37°C. 100 µL of cell suspension/agarose mix was placed on to each slide. The slides were then coverslipped and allowed to gel on ice.

CELL LYSIS
Once gelled the coverslips were removed and all slides placed in lysis buffer overnight at 2-8°C, protected from light.

UNWINDING AND ELECTROPHORESIS
Following lysis, slides were washed in purified water for 5 minutes, transferred to electrophoresis buffer at 2-8°C and the DNA unwound for 20 minutes (glandular stomach and duodenum) or 30 minutes (liver and gonad). At the end of the unwinding period the slides were electrophoresed in the same buffer at 0.7 V/cm for 20 minutes (glandular stomach and duodenum) or 40 minutes (liver and gonad). As not all slides could be processed at the same time a block design was employed for the unwinding and electrophoretic steps in order to avoid excessive variation across the groups for each electrophoretic run; i.e. for all animals the same number of triplicate slides was processed at a time.

NEUTRALISATION
At the end of the electrophoresis period, slides were neutralised in 0.4 M Tris, pH 7.0. After neutralisation the slides were dried and stored at room temperature prior to scoring.

STAINING
Prior to scoring, the slides were stained with 100 µL of 2 µg/mL ethidium bromide and coverslipped.

SLIDES ANALYSIS
Scoring was carried out using fluorescence microscopy at an appropriate magnification and with suitable filters for the stains used.

SCORING OF COMETS
Measurements of tail intensity (%DNA in tail) were obtained from 150 cells/animal. In general, this was evenly split over three slides.
The number of ‘hedgehogs’ observed during comet scoring was recorded for each slide. To avoid the risk of false positive results ‘hedgehogs’ were not used for comet analysis. Each slide was scanned starting to the left of the centre of the slide.
The following criteria were used for analysis of slides:
1. Only clearly defined non overlapping cells were scored
2. Hedgehogs were not scored
3. Cells with unusual staining artefacts were not scored.

Evaluation criteria:

ACCEPTANCE CRITERIA (STUDY VALIDITY)
The data were considered valid if the following criteria were met:
1. The vehicle control data were comparable to the laboratory historical control data for each tissue
2. The positive control induced responses that were compatible with the laboratory historical control data and that produced a statistically significant increase compared to the concurrent vehicle control
3. Adequate numbers of cells and doses were analyzed
4. The high dose was considered to be the MTD, the maximum recommended dose, the maximum practicable dose or one that demonstrated cytotoxicity to the target cells.

ACCEPTANCE CRIETRIA FOR DNA DAMAGE
For valid data, the test article was considered to induce DNA damage if:
1. A least one of the test doses exhibited a statistically significant increase in tail intensity, in any tissue, compared with the concurrent vehicle control
2. The increase was dose related in any tissue
3. The increase exceeded the laboratory’s historical control data for that tissue.
The test article was considered positive in this study if all the criteria listed were met.
The test article was considered negative in this study if for both end points, none of the following criteria were met and target tissue exposure was confirmed.

Statistics:

After completion of microscopic analysis and decoding of the data the percentage tail intensity (i.e. %DNA in the tail) was calculated.
Data were treated as follows:
1. The median value per slide was calculated
2. The mean of the slide medians was calculated to give the mean animal value
3. The mean of the animal means and standard error of the mean was calculated for each group.
Tail intensity data for each slide were supplied for statistical analysis. The median of the log-transformed tail intensities from each slide was averaged to give an animal summary statistic. Where the median value on a slide was zero, a small constant (0.0001) was added before taking the logarithm and calculating the average for the animal. This animal average was used in the statistical analysis.
Data was analysed using one-way analysis of variance (ANOVA) with the fixed factor for treatment group. The positive control group was excluded from this analysis. Levene’s test was used to test for equality of variances among groups. This showed no evidence of heterogeneity (P>0.01). Comparisons between each treated group and control were made using Dunnett’s test. The test was one-sided looking for an increase in response with increasing dose. The back-transformed difference and p value are reported. In addition, a linear contrast was used to test for an increasing dose response.
The positive control group was compared to the control group using a two-sample t test. Levene’s test was used to test for equality of variances between the groups. This showed no evidence of heterogeneity (P>0.01). The test was one-sided looking for an increase in response with increasing dose. The back-transformed difference and p-value are reported.

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

GLANDULAR STOMACH

Toxicity:

yes

Remarks:

Microscopic findings in the stomach were reported (erosion, inflammation and vesicle formation in the forestomach / minimal to moderate degeneration/atrophy in the glandular stomach).

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

DUODENUM

Toxicity:

no effects

Remarks:

no macroscopic or microscopic effects were reported on the duodenum.

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

LIVER

Toxicity:

yes

Remarks:

Decrease of glycogen and variations in liver enzymes activity were reported.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
See above in the section "Any other information on materials and methods incl. tables".
A preliminary experiment was conducted at 2000 and 1400 mg/kg bw/day.
On Day 3, there were significant observations in both sexes for animals administered 2000 mg/kg (mouth rubbing, ataxia, decreased activity, excessive salivation, diarrhoea, raised hair, semi-closed eyes, red/brown mouth and/or snout, audible breathing, paddling, vocalisation, hunched posture and/or prone; two females were found dead 2 hours post dose). Therefore, 2000 mg/kg/day clearly exceeded an appropriate maximum tolerated dose in both sexes. As such, bone marrow toxicity was not assessed in surviving animals.
From these results, 1400 mg/kg/day was considered to be an appropriate estimate of the MTD and was therefore selected as the maximum dose for the Main Experiment. Two lower doses of 350 (25% MTD) and 700 mg/kg/day (50% MTD) were selected.


RESULTS OF THE MAIN EXPERIMENT
- POST DOSE OBSERVATIONS
There were no clinical observations of toxicity on Day 1 or Day 2 for any animal dosed in the Main Experiment. On Day 3, clinical observations at 1400 mg/kg/day included liquid or soft faeces, mouth rubbing, mild decreased activity, raised hair, ataxia and low carriage. Transient observation of mouth rubbing was noted for animal R0206 at 700 mg/kg/day on Day 3.

- BODY WEIGHTS
There was a test article-related effect on animal body weight between Day 1 – Day 3 with group mean body weight change values of +5.0%, +4.4% and -1.1% at 266, 700 and 1400 mg/kg/day, respectively, compared to +4.2% in the concurrent vehicle control group.

- CLINICAL CHEMISTRY
Increased alanine aminotransferase (ALT) activity and decreased alkaline phosphatase (ALP) activity were observed for animals administered 700 or 1400 mg/kg/day. Increased cholesterol was recorded for animals administered 1400 mg/kg/day.
Decreased albumin and increased globulins were noted for animals administered 1400 mg/kg/day, with a decreased albumin:globulin ratio.
Decreased sodium and increased potassium were noted for animals administered 1400 mg/kg/day. Increased chloride was noted for animals administered 700 or 1400 mg/kg/day.
Increased calcium and phosphate were noted for two animals administered 1400 mg/kg/day.
Increased creatinine was noted for animals administered 1400 mg/kg/day. Increased urea was noted for animals administered 700 or 1400 mg/kg/day.
Increased glucose was noted for animals in all groups administered TMAC, with a dose relationship. Glucose was particularly high for Animal R0306 (34.7 mmol/L). This was considered indicative of a stress response.

- HISTOPATHOLOGY
> On macroscopic examination, cysts were noted in the cardia of the stomach of two animals administered 700 mg/kg/day, which correlated with microscopic vesicles. Red foci in the stomach of one animal administered 1400 mg/kg/day correlated with microscopic erosions/ulcers.
> On microscopic examination, TMAC-related changes were recorded for the liver and stomach.
Decreased hepatocyte glycogen was recorded for animals from all groups administered Trimellitic anhydride chloride (TMAC), with a dose relationship.
In the stomach, erosion/ulcer, inflammation, and vesicle formation of the forestomach were noted in animals from all groups administered Trimellitic anhydride chloride (TMAC) generally with a dose relationship. More vesicles were noted in animals administered 700 mg/kg/day, as ulceration in the high-dose group effaced the epithelial surface of the forestomach. In the glandular stomach, minimal to moderate degeneration/atrophy was noted in all groups administered TMAC. Occasional erosions/ulcers were noted in animals administered 700 or
1400 mg/kg/day.
> In absence of any genotoxic effect in somatic cells, germ cells were not analyzed.

- CONCLUSION ON TOXICITY
Findings in the stomach are consistent with an irritant or even corrosive effect of the test article.
In conclusion, TMAC was associated with increased ALT and decreased ALP activities; increased cholesterol, globulins, potassium, chloride, calcium, phosphate, urea, and creatinine; decreased albumin and sodium; macroscopic cysts in the stomach; erosions/ulcers, inflammation, vesicles, and degeneration atrophy in the stomach; and decreased glycogen in the liver.

- CONCLUSION ON VALIDITY OF THE STUDY AND EXPOSURE OF TARGET TISSUES
The data generated in this study confirm that:
1. The vehicle control data were comparable to laboratory historical control data for each tissue
2. The positive control induced responses that were compatible with the laboratory historical control data and are statistically significant compared to the concurrent vehicle control
3. Adequate numbers of cells and doses were analysed
4. The high dose was considered to be the MTD.

As dosing was via oral gavage, exposure to the glandular stomach and duodenum was assured. Furthermore, microscopic findings were observed in the stomach in response to test article exposure. With regards to the liver, decreased hepatocyte glycogen indicated liver perturbations following dosing, which was attributed directly or indirectly to the effects of the test article. Furthermore, liver enzyme (ALT and ALP) activity was impacted at the
intermediate and high dose group providing evidence of a toxicological response. The clinical chemistry, histopathology, clinical observations and body weight effects combined provide indirect and direct evidence of systemic test article exposure.
The assay data were therefore considered valid.

- GENETIC TOXICITY - COMET ANALYSIS
There were no marked increases in %hedgehogs in the liver, glandular stomach or duodenum that were considered indicative that treatment with Trimellitic anhydride chloride (TMAC) induced excessive DNA damage that could have interfered with comet analysis.
Animals treated with Trimellitic anhydride chloride (TMAC) at 266, 700 or 1400 mg/kg/day exhibited group mean percentage tail intensities in the liver, glandular stomach and duodenum that were similar to the concurrent vehicle control group mean and that fell within the 95% reference range of the laboratory's historical vehicle control data. There were no statistically significant increases in tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control, with no evidence of a dose response.
It was noted that there were elevated individual animal values in the liver above the 95% reference range in the vehicle control group and each test article dosed group. As these values were observed in each group and not replicated across the majority of animals in each group, they were considered not related to Trimellitic anhydride chloride (TMAC) treatment and therefore of no biological relevance.
In absence of any genotoxic effect in somatic cells, germ cells were not analyzed.

- FORMULATIONS ANALYSIS
Test article formulations at 35, 70 and 140 mg/mL were prepared once for dosing in the Main Experiment. The analytical data confirmed that dosing formulations at 70 and 140 mg/mL were homogenous (0.83-1.29% RSD; target ≤5% RSD) and achieved target nominal concentration, mean values of 88-91% (within 100±15%). However, while the formulation at 35 mg/mL was also homogenous (1.43% RSD), it fell outside acceptance criteria of 85-115% with a mean value of 76% and therefore an actual dose level of 266 mg/kg/day.

As there were no analytical errors found upon investigation, the analytical data were considered accurate for reporting. Although the dose level of 350 mg/kg/day was not achieved, there is no impact to the validity of the study as the formulation was homogenous such that there were no concerns over the actual dose provided (266 mg/kg/day), which is within acceptable dose level intervals per OECD guidelines (i.e. within 4-fold of 700 mg/kg/day).

The formulations were therefore considered acceptable. No test article was detected in the vehicle sample.

TMAC - Summary of Group Mean Comet Data – Liver

Group	Mean (SEM)	Back-Transformed Difference from Vehicle	Ranked	P-Value	Significance	Mean % Hedgehogs
1/ Vehicle 0 mg/kg bw/day	1.45 (0.39)	-	-	-	-	1.43
2/ TMAC 266 mg/kg bw/day	1.37 (0.54)	0.74	U	0.9209	NS	1.36
3/ TMAC 700 mg/kg bw/day	1.43 (0.57)	0.77	U	0.9096	NS	0.98
4/ TMAC 1400 mg/kg bw/day	1.74 (0.43)	1.08	U	0.6834	NS	2.38
5/ Positive (EMS) 150 mg/kg bw/day	46.81 (1.96)	39.18	U	< 0.0001	***	3.50
Dose response (groups 1, 2, 3, 4)			U	0.4270	NS	NA

 

TMAC - Summary of Group Mean Comet Data – Glandular stomach

Group	Mean (SEM)	Back-Transformed Difference from Vehicle	Ranked	P-Value	Significance	Mean % Hedgehogs
1/ Vehicle 0 mg/kg bw/day	0.85 (0.23)	-	-	-	-	7.24
2/ TMAC 266 mg/kg bw/day	0.90 (0.41)	0.77	U	0.8901	NS	10.94
3/ TMAC 700 mg/kg bw/day	0.78 (0.19)	0.87	U	0.8330	NS	8.45
4/ TMAC 1400 mg/kg bw/day	0.87 (0.33)	0.85	U	0.8466	NS	10.62
5/ Positive (EMS) 150 mg/kg bw/day	14.26 (2.22)	21.83	U	< 0.0001	***	10.62
Dose response (groups 1, 2, 3, 4)			U	0.5854	NS	NA

 

TMAC - Summary of Group Mean Comet Data – Duodenum

Group	Mean (SEM)	Back-Transformed Difference from Vehicle	Ranked	P-Value	Significance	Mean % Hedgehogs
1/ Vehicle 0 mg/kg bw/day	0.88 (0.20)	-	-	-	-	4.48
2/ TMAC 266 mg/kg bw/day	0.36 (0.07)	0.36	U	0.9983	NS	3.12
3/ TMAC 700 mg/kg bw/day	0.56 (0.11)	0.60	U	0.9686	NS	2.65
4/ TMAC 1400 mg/kg bw/day	0.77 (0.49)	0.40	U	0.9964	NS	5.54
5/ Positive (EMS) 150 mg/kg bw/day	12.99 (1.34)	18.03	U	< 0.0001	***	8.98
Dose response (groups 1, 2, 3, 4)			U	0.9190	NS	NA

NA: not applicable / SEM: Standard Error of Mean / EMS: Ethyl Methanesulfonate

NS: Not significant (P>0.05) / U: Unranked / ***: P <= 0.001

 

TMAC - Summary of Body Weight Percentage Change

Group	N	Mean % change (SD)
1/ Vehicle 0 mg/kg bw/day	6	4.2 (1.36)
2/ TMAC 266 mg/kg bw/day	6	5.00 (1.11)
3/ TMAC 700 mg/kg bw/day	6	4.4 (1.89)
4/ TMAC 1400 mg/kg bw/day	6	-1.1 (3.56)
5/ Positive (EMS) 150 mg/kg bw/day	6	-7.4 (2.31)

SD: standard deviation

N: number of individual values (animals)

Conclusions:

When tested in the Comet assay conducted according to the OECD 489 guideline in rats exposed up to 1400 mg/kg bw/day (maximum tolerated dose), Trimellitic anhydride chloride (TMAC), did not induce DNA strand breaks in the liver, glandular stomach or duodenum. In absence of any effect in somatic cells, germ cells were not analyzed.

Executive summary:

Trimellitic anhydride chloride (TMAC) was tested for its potential to induce DNA strand breaks in the liver, stomach and duodenum of rats. The study was conducted according to the guideline OECD 489 and combined with the micronucleus test (according to the guidelien OECD 474).

Male Sprague Dawley rats were treated by gavage once daily, for 3 days, at the actual dose levels of 266, 700 and 1400 mg/kg bw/day. Based on a range-finder experiment, the dose level of 1400 mg/kg bw/day was considered as being the MTD (maximum tolerated dose). The vehicle control group received the vehicle (corn oil) and the positive control group received Ethyl methanesulfonate (EMS) at 150 mg/kg bw/day.

Tissues (liver, glandular stomach, duodenum and germ cells) were collected at necropsy (Day 3, 48 hours, i.e. 3 hours after the last dosing).

There were no clinical observations of toxicity on Day 1 or Day 2 for any dosed animal. On Day 3, clinical observations at 1400 mg/kg/day included liquid or soft
faeces, mouth rubbing, mild decreased activity, raised hair, ataxia and low carriage. 

There was a test article-related effect on animal body weight between Day 1 – Day 3 with group mean body weight change values of +5.0%, +4.4% and -1.1% at 266, 700 and 1400 mg/kg/day, respectively, compared to +4.2% in the concurrent vehicle control group.

Liver enzyme (ALT and ALP) activity was impacted at the intermediate and high dose group providing evidence of a toxicological response. Variations were also observed for cholesterol, albumin, sodium and potassium, calcium, phosphate and glucose mainly for animals administered 1400 mg/kg bw/day.

On microscopic examination, TMAC-related changes were recorded for the liver and stomach. Decreased hepatocyte glycogen was recorded for animals from all groups administered TMAC, with a dose relationship. In the stomach, erosion/ulcer, inflammation, and vesicle formation of the forestomach were reported generally with a dose relationship. In the glandular stomach, minimal to moderate degeneration/atrophy was noted in all groups administered TMAC.

All these findings combined (clinical chemistry, histopathology, clinical observations and body weight effects) provide indirect and direct evidence of systemic test article exposure.

There were no marked increases in %hedgehogs in the liver, glandular stomach or duodenum that were considered indicative that treatment with TMAC induced excessive DNA damage that could have interfered with comet analysis.

Animals treated with TMAC at 266, 700 or 1400 mg/kg/day exhibited group mean percentage tail intensities in the liver, glandular stomach and duodenum that were similar to the concurrent vehicle control group mean and that fell within the 95% reference range of the laboratory's historical vehicle control data. There were no statistically significant increases in tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control, with no evidence of a dose response. As no genotoxic effects were observed in somatic cells, the analysis of germ cells was not conducted.

It is concluded that under the conditions of this study, Trimellitic anhydride chloride (TMAC), did not induce DNA strand breaks in the liver, glandular stomach or duodenum when tested up to 1400 mg/kg/day (an estimate of the maximum tolerated dose for this study).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Summary of the information available on trimellitic anhydride monochloride

Table: Summary of genetic toxicity tests

Test n°	Test guideline / reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1 (WIL Research, 2015)	Ames Test (OECD 471, EU Method B.13/14) KS, rel. 1	Gene mutation	S. Typhimurium TA 98, TA 100, TA 1535, TA 1537 E. coli WP2 uvr A	with/without S9	Up to the limit concentration	Mutagenic in S. Typhimurium TA 100 only (with and without S9 -mix)
2 (WIL Research, 2016)	MLA (OECD 490, EU Method B.17) KS, rel. 1	Gene mutation	Mouse lymphoma L5178Y cells	with/without S9	Up to the precipitating concentrations	Mutagenic in the mouse lymphoma L5178Y  (with and without S9 -mix)
3 (Charles River, 2016)	MNT (OECD 487) KS, rel. 1	Chromosome aberration	Peripheral human lymphocytes	with/without S9	Up to the precipitating concentrations	Mutagenic in the peripheral human lymphocytes after 3 hour exposure time without S9 -mix only
4 (Morinaga, 2011)	Ames Test (OECD 471) SS, rel. 2	Gene mutation	S. Typhimurium TA 98, TA 100, TA 1535, TA 1537 E. coli  WP2 uvr A	with/without S9	Up to the precipitating concentrations	Mutagenic in all strains (with and without S9 -mix)

(KS: Key Study; SS: Support study; rel.: reliability according to Klimisch criteria)

 

Gene mutation Assays (Tests n° 1, 2 and 4):

- Two Bacterial Reverse mutation Assays (Ames test) were performed according to OECD guideline No. 471 with the substance, Trimellitic anhydride mono-chloride. Significant increases in the frequency of revertant colonies were recorded for only one bacterial strain (TA 100) in the first Ames and for all strains in the second Ames test, in the presence or absence of metabolic activation. The substance induces gene mutations in bacteria. Therefore, the substance is considered as mutagenic according to the Ames test.

- Ability to produce gene mutation was confirmed in mammalian cells using an in vitro gene mutation assay in Mouse lymphoma L5178Y cells (Test n°2). The substance induced significant mutant frequency increases either in the presence or absence of metabolic activation. The substance is therefore considered as positive for inducing forward mutations at the Thymidine kinase (TK) locus in the mouse lymphoma L5178Y cells under activation and non-activation conditions. This result confirms the results of the Ames tests and extends the in vitro mutagenic effect of the substance to mammalian cells.

 

Chromosomal aberration (test n°3)

- An in vitro micronucleus test was performed on cultured peripheral human lymphocytes according to OECD Guideline 487 and in compliance with GLP. In a first cytogenetic assay, in the absence of S9-mix, after 3 hours exposure time, Trimellitic anhydride chloride induced a statistically significant increase in the number of binucleated cells with micronuclei at the highest dose level. The number of micronucleated cells was outside the 95% control limits of the distribution of the historical negative control database. In addition, a statistical significant dose related trend was observed in the absence of S9-mix (binucleated cells). These results indicate that Trimellitic anhydride chloride is positive in the in vitro micronucleus study and might be considered a clastogenic compound. The number of mononucleated cells with micronuclei was within the 95% control limits of the distribution of the historical negative control database. In contrast, in the presence of S9-mix, Trimellitic anhydride chloride did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei. A second cytogenetic assay with a 24 hours continuous exposure time showed the same results. Since Trimellitic anhydride chloride induces the micronuclei frequency, it may be considered as an in vitro clastogenic compound.

 

Summary of the information available on trimellitic anhydride

Table: Summary of genetic toxicity tests

Test n°	Test guideline / reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1 (San & Wagner, 1991)	Ames Test (OECD 471) WoE, rel. 2	Gene mutation	S. Typhimurium TA 98, TA 100, TA 1535 TA 1537 TA 1538	with/without S9	Up to 10000 µg/plate	Negative in all strains  (with and without S9 -mix)
2 (San & Olson, 1991)	Ames test (OECD 471) WoE, rel. 2	Gene mutation	S. Typhimurium TA 98, TA 100, TA 1535 TA 1537 TA 1538	with/without S9	Up to 10000 µg/plate	Negative in all strains  (with and without S9 -mix)
3 (Putman & Morris, 1991)	(OECD 473) WoE, rel. 2	Chromosome aberration	Chinese Hamster Ovary (CHO)	with/without S9	Up to the precipitating concentrations	Negative in all strains  (with and without S9 -mix)
4  (Putman & Morris, 1990)	(OECD 473) WoE, rel. 2	Chromosome aberration	Chinese Hamster Ovary (CHO)	with/without S9	Up to 5000 µg/plate	Negative in all strains  (with and without S9 -mix)
5  (Jacobson-Kram & Sigler, 1990)	(OECD 476) WoE, rel. 2	Gene mutation	Chinese Hamster Ovary (CHO)	with/without S9	Up to the precipitating concentrations	Negative in all strains  (with and without S9 -mix)
6 (Bigler & Sigler, 1991	(OECD 476) WoE, rel. 2	Gene mutation	Chinese Hamster Ovary (CHO)	with/without S9	Up to the precipitating concentration	Negative in all strains  (with and without S9 -mix)

(WoE: Weight-of-Evidence; rel.: reliability according to Klimisch criteria)

 

 

Summary of in vivo studies available on trimellitic anhydride monochloride

Table: Summary of genetic toxicity tests

Test n°	Test guideline / reliability	Focus	Specie	Tissues analyzed	Test concentration	Statement
1 (G. Watters, 2022)	Micronucleus test in rats (OECD 474) KS, rel. 1	Chromosome aberration	Sprague Dawley rats	Bone marrow	Up to 1400 mg/kg bw/day	Negative
2 (G. Watters, 2022)	Comet assay in rats (OECD 489) KS, rel. 1	Gene mutation	Sprague Dawley rats	Liver Glandular stomach Duodenum	Up to 1400 mg/kg bw/day	Negative

(KS: Key Study; rel.: reliability according to Klimisch criteria)

 

> In vivo micronucleus test:

Trimellitic anhydride chloride (TMAC) was tested for its potential to induce micronuclei (MN) in the polychromatic erythrocytes (PCE) of the bone marrow of rats (according to the guideline OECD 474, and combined with the comet assay according to the guideline OECD 489). Male Sprague Dawley rats were treated by gavage once daily, for 3 days, at the actual dose levels of 266, 700 and 1400 mg/kg bw/day. Based on a range-finder experiment, the dose level of 1400 mg/kg bw/day was considered as being the MTD (maximum tolerated dose). The vehicle control group received the vehicle (corn oil) and the positive control group received Ethyl methanesulfonate (EMS) at 150 mg/kg bw/day.

Bone marrows from one femur were sampled at necropsy (Day 3, 48 hours, i.e. 3 hours after the last dosing).

There was a small reduction in group mean %PCE at 1400 mg/kg/day (-13%) that was observed in five animals. While the reduction was within the laboratory's historical control data, as it was observed in 5 out of 6 animals, it was considered potential evidence of bone marrow toxicity.

Animals treated with Trimellitic anhydride chloride (TMAC) at 266, 700 or 1400 mg/kg/day exhibited group mean %MN PCE frequencies that were similar to the vehicle control group mean and which fell within the 95% reference range of the laboratory's historical vehicle control data. There were no statistically significant increases in micronucleus frequency for any of the groups receiving the test article. 

It is concluded that Trimellitic anhydride chloride (TMAC), did not induce an increase in micronucleated polychromatic erythrocytes of the bone marrow when tested up to 1400 mg/kg/day (an estimate of the maximum tolerated dose for this study).

 

- In vivo comet assay in rats

Trimellitic anhydride chloride (TMAC) was tested for its potential to induce DNA strand breaks in the liver, stomach and duodenum of rats (according to the guideline OECD 489, and combined with the micronucleus test according to the guideline OECD 474). Male Sprague Dawley rats were treated by gavage once daily, for 3 days, at the actual dose levels of 266, 700 and 1400 mg/kg bw/day. Based on a range-finder experiment, the dose level of 1400 mg/kg bw/day was considered as being the MTD (maximum tolerated dose). The vehicle control group received the vehicle (corn oil) and the positive control group received Ethyl methanesulfonate (EMS) at 150 mg/kg bw/day.

Tissues (liver, glandular stomach, duodenum and germ cells) were collected at necropsy (Day 3, 48 hours, i.e. 3 hours after the last dosing).

Clinical observations were only noted on day 3, at 1400 mg/kg/day included liquid or soft faeces, mouth rubbing, mild decreased activity, raised hair, ataxia and low carriage. 

There was a test article-related effect on animal body weight change between Day 1 – Day 3 at 1400 mg/kg/day (-1,1% compared to +4.2% in the concurrent vehicle control group).

Liver enzyme (ALT and ALP) activity was impacted at the intermediate and high dose group providing evidence of a toxicological response. Variations were also observed for cholesterol, albumin, sodium and potassium, calcium, phosphate and glucose mainly for animals administered 1400 mg/kg bw/day.

On microscopic examination, TMAC-related changes were recorded for the liver and stomach (decreased hepatocyte glycogen in the liver, erosion/ulcer, inflammation, and vesicle formation of the forestomach and degeneration/atrophy in the glandular stomach).

All these findings combined (clinical chemistry, histopathology, clinical observations and body weight effects) provide indirect and direct evidence of systemic test article exposure.

Animals treated with TMAC exhibited group mean percentage tail intensities in the liver, glandular stomach and duodenum that were similar to the concurrent vehicle control group mean and that fell within the 95% reference range of the laboratory's historical vehicle control data. There were no statistically significant increases in tail intensity for any of the groups receiving the test article. As no genotoxic effect were observed in somatic cells, the analysis of the germ cells was not conducted.

It is concluded that under the conditions of this study, Trimellitic anhydride chloride (TMAC), did not induce DNA strand breaks in the liver, glandular stomach or duodenum when tested up to 1400 mg/kg/day (an estimate of the maximum tolerated dose for this study).

 

Discusion and conclusion

Trimellitic anhydride mono-chloride (TMAC) showed positive results in the in vitro experiments: Ames test according to the guideline OECD 471, Gene mutation in mammalian cells according to the guideline OECD 476 and in vitro Micronucleus test according to the guideline 0ECD 487. 

In order to check if the in vitro effects on TMAC are observed in vivo, a micronucleus test combined with a comet assay was conducted in rats (according to the guidelines OECD 474 and 489). These studies were conducted up to 1400 mg/kg bw/day (maximum tolerated dose according to a preliminary experiment), and cover all the in vitro endpoints showing positive effects. No genotoxic effects were observed . The in vitro findings are not reproduced in the in vivo conditions. 

On the basis of the in vivo results, it can be concluded that 4-chloroformylphthalic anhydride (TMAC) has not genotoxic potential. 

Justification for classification or non-classification

Harmonized classification:

The test material does not have an harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

 

Self-classification:

Trimellitic anhydride mono-chloride showed positive results in the in vitro experiments (Ames, gene mutation in mamallian cells and micronucleus test). However, when tested in the in vivo micronucleus test combined with the comet assay, no genotoxic effects were observed. This combined in vivo test covers the in vitro endpoints showing positive effects (gene mutation and chromosome aberrations). The in vitro findings are not reproduced in vivo. Therefore it can be concluded that TMAC is not genotoxic according to the criteria defined in the CLP and ECHA regulations.

Based on the in vivo data and according to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) and ECHA guidance on the Application of the CLP criteria (version 5, 2017), no classification is required for genetic toxicity.