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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

Referenceopen allclose all

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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Adopted July 21, 1997
Deviations:
no
Qualifier:
according to
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.

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:
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:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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.

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:
reference to other study
Qualifier:
according to
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 micronuclei1) Number of binucleated cells with micronuclei1) 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*** 12) 22) 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.

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:
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

Trimellitic anhydride mono-chloride showed positive results in in vitro experiments. No in vivo somatic mutagenicity / genotoxicity data is currently available. Chemical structural related substance Trimellitic anhydride is not mutagenic/genotoxic.

According to REACH regulation, appropiated in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or AVIII. A testing proposal for an in vivo study is therefore included in this dossier.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study planned
Remarks:
Following ECHA approval
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: trimellitic anhydride monochloride

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies:
VERSPEEK-RIP (2015), "Evaluation of the mutagenic activity of trimellitic anhydride chloride in the Salmonella Typhimurium reverse mutation assay and the Escherichia Coli reverse mutation assay (Plate incorporation and pre-incubation methods)" (OECD 471 and EU Method B.13/14, GLP): positive in S. Typhimurium TA 100 only (with and without S9 -mix). Negative in other S. Typhimurium strains and in E. Coli, both with and without metabolic activation.
MORINAGA (2011), "The mutagenicity test on the microbe using trimellitic anhydride chloride" (OECD 471, GLP): positive in all strains with and without metabolic activation.
VERSPEEK-RIP (2016), "Evaluation of the mutagenicity activity of trimellitic anhydride chloride in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells" (OECD 490 and EU Method B.17, GLP, klimisch 1): positive with and without metabolic activation
VERBAAN (2016) "An in vitro Micronucleus Assay with Trimellitic anhydride chloride in Cultured Peripheral Human Lymphocytes" (OECD 487, GLP, Key, klimisch 1): positive with and without metabolic activation
- Historical human data: No substance-specific data identified.
- (Q)SAR: It is acknowledged that the results of QSAR modelling are of very limited applicability for substances that rapidely hydrolyse.
- In vitro methods: The available in vitro methods have been considered as part of the tiered approach to testing. See "Available GLP studies"
- Weight of evidence: Trimellitic anhydride chloride showed positive results in in vitro experiements. No in vivo somatic mutagenicity / genotoxicity data has been identified.
Chemical structural related substance Trimellitic anhydride is not mutagenic/genotoxic (OECD SIDS Initial Assessment Report for 15th SIAM (Boston, USA, 22 -25, October 2002) on Trimellitic anhydride and Trimellitic acid. Although Trimellitic anhydride chloride and Trimellitic anhydride are considered to fall within the scope of read-across analogy and due to the difference observed in the results for this specific endpoint, an in vivo test is proposed with trimellitic anhydride chloride in order to substantiate the dataset.
- Grouping and read-across: see weight-of-evidence.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Column 2 "Specific rules for adaptation from column 1 in Annex VII of the REACH Regulation (consolidated version of 11 october 2016)", point 8.4 is not applicable as no adequate in vivo information is available. The substance is not classified to be carcinogenic or mutagenic, therefore genetic toxicity testing cannot be waived.
- According to Column 2 "Specific rules for adaptation from column 1 in Annex VIII of the REACH Regulation (consolidated version of 11 october 2016)", point 8.4, "appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII. This is the case for trimellitic anhydride chloride.
- Column 2 "Specific rules for adaptation from column 1 in Annex IX of the REACH Regulation (consolidated version of 11 october 2016) point 8.4 is not applicable: there positive result in in vitro genotoxicity studies

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed:

In order to address positive results detected in the battery of in vitro tests and save on ressources and numbers of animals used, it is proposed (if feasible by the specialised laboratories), an alkaline comet assay (OECD Test Guideline 489) after oral exposure in rodent, with a concomitant micronucleus assay. Some modification of treatment and sampling times maybe required (ECHA, 2017). The purpose of the comet assay is to detect DNA strand breaks and alkali labile DNA lesions. On the other hand, the micronucleus test has the advantage of detecting not only structural chromosomal aberrations (clastogenicity) but also numerical chromosomal aberrations (aneuploidy).

No target organ has been identified for trimellitic anhydride chloride sofar. It is therefore proposed to investigate liver, kidney and bone marrow in the in vivo Comet assay. Liver and kidney are recognised to be key organs for metabolism, and they have been extensively studied in Comet assay. Lung is not proposed as the substance is a hygroscopic solid and therefore exposure to Trimellitic anhydride chloride by inhalation is unlikely.
Bone marrow is proposed because it is a rapidly dividing tissue. Bone marrow is also proposed as the target tissue for micronuclei assessment.

Inclusion of a targeted investigation of toxicokinetics is proposed for the purpose of confirming that absorption occurs. It is also intended to demonstrate that adequate target tissue exposure to the test substance has been achieved. However, as Trimellitic anhydride chloride is rapidly hydrolysed into Trimellitic anhydride, some difficulties to develop an analytical method are forseen.

REFERENCES

OECD SIDS Initial Assessment Report for 15th SIAM (Boston, USA, 22 -25, October 2002) on Trimellitic anhydride and Trimellitic acid.

REACH Regulation (consolidated version 02006R1907 — EN — 11.10.2016 — 031.001)

OECD guideline 489

ECHA (2017), Chapter R.7a: Endpoint specific guidance Version 6.0.
Specific details on test material used for the study:
4-chloroformylphthalic anhydride
CAS 1204-28-0
Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

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)

(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)

Discusion and conclusion

Trimellitic anhydride mono-chloride showed positive results in in vitro experiments. No in vivo somatic mutagenicity / genotoxicity data is currently available.

Chemical structural related substance Trimellitic anhydride is not mutagenic/genotoxic. This is the conclusion of OECD SIDS Initial Assessment Report for 15th SIAM (Boston, USA, 22 -25, October 2002) on Trimellitic anhydride and Trimellitic acid.

According to REACH regulation, appropiated in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or AVIII. A testing proposal for an in vivo study is therefore included in this dossier.

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 in vitro experiments. No in vivo somatic mutagenicity / genotoxicity data is currently available. Chemical structural related substance Trimellitic anhydride is not mutagenic/genotoxic.

Based on the currently available 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), the information is not sufficient for classification.

According to REACH regulation, appropiated in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or AVIII. A testing proposal for an in vivo study is therefore included in this dossier.