Trimethyl benzene-1,2,4-tricarboxylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay - No mutagenic effects observed in Salmonella typhimurium strains TA98, TA1535, and TA1537 in the absence and presence of metabolic activation.

Ambiguous/positive results in Salmonella typhimurium strain TA100 and Escherichia coli strain WP2uvrA in the absence and presence of metabolic activation.

Mammalian cell gene mutation assay - Dose-related increases of the mutant frequency observed in the presence and absence of metabolic activation.

In-vitro mammalian chromosome aberration test - Positive for the induction of structural and numerical chromosome aberrations.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006 - 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

5 - 2522 micrograms/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

The highest concentrations chosen for the chromosome aberration assay were 1000 micrograms/mL for the 4 -hour exposure assay without a metabolic activation system, 2522 micrograms/mL

for the 4 hour exposure period with a metabolic activation system and 750 micrograms/mL for the 20 hour exposure period without a metabolic activation system.

A visible precipitate was observed at the beginning and end of the treatment periods at concentrations of 1000 micrograms/mL and greater.

Substantial toxicity (greater than a 50% reduction in cell growth relative to the vehicle control) was observed at the highest concentration in each test condition. The selection of doses for analysis was based on these dose concentration levels as well as precipitation. Cytogenetic evaluations were conducted at 500, 750 and 1000 microgramsg/mL for the 4 hour exposure period both with and without metabolic activation and at 100, 250 and 500 micrograms/mL for the 20 hour exposure period without metabolic activation.

The percentage of cells with structural aberrations following 4 hours exposure with metabolic activation was significantly increased above that of the vehicle control at 500, 750 and 1000 micrograms/mL. The percentage of cells with structural aberrations following 20 hours exposure without metabolic activation was also significantly increased above that of the vehicle control at 500 micrograms/mL. The observed changes were outside of the historical control range for structural aberrations, were accompanied by a concentration-related increase, and were therefore considered biologically significant. The percentage of cells with numerical aberrations following 4 hours exposure without metabolic activation showed a significant trend as compared to the vehicle control at 750 and 1000 micrograms/mL. The percentage of cells with numerical aberrations following 4 hours exposure with metabolic activation was also significantly increased above that of the vehicle control at 500, 750 and 1000 micrograms/mL. The observed changes were outside of the historical control range for numerical aberrations, and were therefore considered biologically significant.

Conclusions:

A cytogenetic assay was positive for the induction of chromosome aberrations in Chinese hamster ovary cells.

Executive summary:

A cytogenetic assay in Chinese hamster ovary cells resulted in positive findings for the induction of chromosome aberrations.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006 - 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

3 hours exposure with S9: 600 - 1500 microgram/mL
3 hours exposure without S9: 200 - 900 micrograms/mL
24 hours exposure without S9: 300 - 700 micrograms/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Details on test system and experimental conditions:

Two assays conducted with 3 hours exposure in the presence of a metabolic activation system.
One assay conducted with 3 hours exposure in the absence of a metabolic activation system.
One assay conducted with 24 hours exposure in the absence of a metabolic activation system.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks on result:

other:

Remarks:

Statistically significant increases in mutation frequency observed at > 500 micrograms/mL

Conclusions:

Statistically significant increases in the mutant frequency in both the absence and presence of a metabolic activation system were observed. In the non-activated test systems, biologically significant increases were observed after 24 hours exposure at dose levels of > 500 micrograms/mL. In the presence of a metabolic activation system, increases were observed at dose levels of > 500 micrograms/mL in the first assay and > 600 micrograms/mL in the second assay.

Executive summary:

Gene mutation in a mammalian cell line has been investigated. Statistically significant increases in the mutant frequency in both the absence and presence of a metabolic activation system were observed. In the non-activated test systems, biologically significant increases were observed after 24 hours exposure at dose levels of > 500 micrograms/mL. In the presence of a metabolic activation system, increases were observed at dose levels of > 500 micrograms/mL in the first assay and > 600 micrograms/mL in the second assay.

Reference 3

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 2014 - 30 May 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (for S. typhimurium strains) and trp operon (for E. coli strains).

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: The salmonella strains were checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity (∆ uvrB); ampicillin resistance (R factor plasmid).

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: E. coli WP2 uvrA was checked for UV sensitivity.

Metabolic activation:

with and without

Metabolic activation system:

cofactors supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Wistar rats treated 80 mg/kg bw phenobarbital i.p. and β-naphthoflavone orally, each on 3 consecutive days

Test concentrations with justification for top dose:

1st Experiment (all strains, standard plate test with and without S-9 mix; 3 plates/dose): 0; 33; 100; 333; 1000; 26500and 5300 µg/plate
2nd Experiment because mutagenicity was observed in the standard plate test:
(TA100 and E.coli, standard plate test with and without S-9 mix; 3 plates/dose): 0; 33; 100; 333; 1000; 2650 and 5300 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in ultrapure water DMSO was used as vehicle.

Untreated negative controls:

yes

Remarks:

(sterility control)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: with S9-mix

Remarks:

2-aminoanthracene (2-AA): 2.5 µg/plate dissolved in DMSO for strains TA 1535, TA 100, TA 1537 and TA 98; 60 µg/plate dissolved in DMSO for Escherichia coli WP2 uvrA

Positive controls:

yes

Positive control substance:

other: without S9-mix

Remarks:

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG): 5 µg/plate (DMSO), TA1535, TA100; 4-nitro-o-phenylenediamine (NOPD): 10 µg/plate (DMSO), TA98; 9-aminoacridine (AAC): 100 µg/plate (DMSO), TA1537; 4-nitroquinoline-N-oxide (4-NQO): 5 µg/plate (DMSO), E.coli

Details on test system and experimental conditions:

TEST DESIGN
Standard plate test (SPT) with and without metabolic activation, were performed.

Standard plate test (SPT):
The experimental procedure was based on Ames et al. (Mut. Res. 31: 347-364, 1975) and Maron & Ames (Mut. Res. 113: 173-215, 1983).
Test tubes containing 2 mL portions of soft agar [100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin for S. typhimurium or 0.5 mM tryptophan for E.coli)] were kept in a water bath at about 42 - 45°C. An amount of 0.1 mL test solution or vehicle (negative control), 0.1 mL fresh bacterial culture and 0.5 mL S9 mix (in case of metabolic activation) or 0.5 mL phosphate buffer (in case of no metabolic activation) were added. After mixing, the samples were poured onto agar plates and incubated at 37°C for 48 to 72 hours in the dark. After incubation, the bacterial colonies were counted for revertants.

PARAMETERS EXAMINED

Mutagenicity:
Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, six doses of the test substance were tested with a maximum of 5 mg/plate, and triplicate plating was used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments were based on the findings of the 1st Experiment.


Cytotoxicity:
Toxicity was detected by (1) decrease in the number of revertants, (2) clearing or diminution of the background lawn (= reduced his- or trp- background growth) and (3) reduction in the titer. Cytotoxicity was recorded for all test groups both with and without S9 mix in all experiments.

Solubility:
Precipitation of the test item was recorded and indicated. As long as precipitation does not interfere with colony scoring, 5000 µg/plate is generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5000µg/plate might also be tested in repeat experiments for further clarification/substanstiation.

Evaluation criteria:

Acceptance criteria:
The experiment is considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls is within the range of the historical negative control data for each tester strain;
- The sterility controls reveales no indication of bacterial contamination;
- The positive control substances both with and without S9 mix induce a distinct increase in number of revertant colonies within the range of the historical positive control data or above;
- The titer of viable bacteria is egal to/greater than 10E+9/mL.

Assessment criteria:
The test substance was considered positive in this assay if the following criteria were met:
A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

The test item is generally nonmutagenic if the number of revertants for all tester strains is within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Species / strain:

S. typhimurium, other: TA 1535; TA1537; TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

increases in the number of his+ revertants at 1.000, 2.650 and 5.300 µg/plate (factors 2.6 up to 8.4 respectively).

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

1st. Exp.:increases in the number of trp+ revertants at 1.000, 2.650 and 5.300 µg/plate (factors 2.4 to 7 respectively)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: standard plate test (SPT)

Executive summary:

According to the results of the present study led to a evident and dose-dependent increase in the number of his+ and trp+ revertants with the strains TA 100 and E. coli WP2 uvrA both with and without S9 mix. The increase of revertants was reproducible in two experiments carried out independently of each other. Based on the recent assessment criteria the test substance has to be considered positive.

Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.

In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.

In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within or above the range of the historical positive control data or above.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Mammalian erythrocyte micronucleus test in mice - Negative

In-vivo/in-vitro UDS assay in rats - 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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

not specified

Sex:

male/female

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Conclusions:

No toxicity nor decrease in the ratio of polychromatic erythroctes were seen up to the maximum recommended dose of 2000 g/kg bw.

Executive summary:

A mammalian erythrocyte micronucleus test has been conducted in mice. No toxicity nor decrease in the ratio of polychromatic erythroctes were seen up to the maximum recommended dose of 2000 g/kg bw.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

GLP compliance:

not specified

Type of assay:

unscheduled DNA synthesis

Species:

rat

Sex:

male

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Conclusions:

No indication of genotoxicity observed.

Executive summary:

An unscheduled DNA synthesis assay has been conducted in rats. No indications of genotoxicity were observed at dose levels up to the maximum recommended for the assay, 2000 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The genotoxic effects observed in-vitro are not expressed in-vivo and the substance is considered to be non-genotoxic in vivo. Classification is therefore not indicated.