Benzene-1,2,4,5-tetracarboxylic acid, compound with 4,5-dihydro-2-phenyl-1H-imidazole (1:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An in vitro gene mutation study in bacteria (OECD 471), an in vitro mammalian chromosome aberration test (OECD 473) and an in vitro mammalian cell gene mutation test (OECD 490) have been performed on the substance.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix based on S9 fraction from Aroclor 1254 induced male Sprague Dawley rat liver

Test concentrations with justification for top dose:

0; 600; 3000; 5000 mg/l (+ S9) / 0; 50; 200; 400 mg/l (- S9)

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

Additional information on results:

CONTROLS:
The positive controls were functional.
The negative controls revealed low chromosomal aberration frequencies
(excluding gaps: 0 to 0.5 %; normal: 0 to 5 %).
PRECIPITATION CONCENTRATION: > 5000 mg/l
-------------------------------------------
MITOTIC INDEX AND CHROMOSOMAL ABERRATIONS:
-------------------------------------------
Concentration % M.I. % C.A.
-------------------------------------------
- Experiment # 1, without S9 (18 hours)
neg. control 7.0 0.0
50 mg/l 6.1 0.0
200 mg/l 7.0 0.0
400 mg/l 3.5 1.5
600 mg/l 2.2
0.03 mg/l MMC 5.2 13.0 *
-------------------------------------------
- Experiment # 1, with S9 (18 hours)
neg. control 6.8 0.5
600 mg/l 7.1 0.5
3000 mg/l 9.2 2.0
5000 mg/l 9.3 1.5
3 mg/l CP 2.5 38.0 *
-------------------------------------------
- Experiment # 2, without S9, 18 hours
neg. control 5.0 0.0
50 mg/l 4.7 1.5
200 mg/l 4.0 1.0
400 mg/l 4.8 1.0
0.03 mg/l MMC 5.4 20.5 *
-------------------------------------------
Experiment # 2, without S9, 28 hours
neg. control 7.6 0.0
400 mg/l 4.4 0.5
0.03 mg/l MMC 5.6 33.0 *
-------------------------------------------
- Experiment # 2, with S9, 18 hours
neg. control 5.3 0.5
600 mg/l 6.2 1.0
3000 mg/l 7.0 2.0
5000 mg/l 5.5 1.0
3 mg/l CP 2.0 26.5 *
-------------------------------------------
- Experiment # 2, with S9, 28 hours
neg. control 5.9 0.0
5000 mg/l 6.8 0.5
3 mg/l CP 8.2 19.5 *
-------------------------------------------
* indicates significance

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

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

Specific details on test material used for the study:

Batch No.: M(S)201170
Appearance/Color/State: Solid fine powder (whitish)
Content or purity: ≥99% (Nominal value)
99.50% (Measured result)

Target gene:

Thymidine Kinase Gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

In this test, the L5178Y (TK+/--3.7.2C) mouse lymphoma cell, generally called L5178Y were exposed at 6 exposure concentrations including 2000, 1000, 500, 250, 125, and 62.5μg/ml cell cultures in three treatment conditions .

Vehicle / solvent:

The solvent (DMSO) and positive controls were included in each condition at the same time.

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

According to the results of the preliminary test and OECD Guideline for the Testing of Chemicals No.490 (July 29, 2016)”, the L5178Y cells will be treated at 6 doses (exposure concentration), including 2000, 1000, 500, 250, 125 and 62.5μg/ml in the treatment conditions of exposure for 3 hours with and without metabolic activation respectively and for 24 hours without metabolic activation. The concurrent solvent control (DMSO) and positive controls will be included in each treatment condition

Evaluation criteria:

1) When IMF(s) in one or several doses are more than GEF* (GEF= 126×10–6), and the increase is concentration- related and/or replicated, the result is evaluated as positive.
2) The result is evaluated as negative if none of the above criteria is met.
3) In case the criteria above are not met at the same time, the repeated test should be performed using modified experimental conditions, and the biological relevance of the result should be considered. If the result is still not clear, the result is concluded to be equivocal.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Additional information on results:

In this test, the results of the solvent controls and positive controls met all criteria of the test validity, so the sensitivity of the test and the efficacy of the S9 mix were validated.
The results of test item precipitate: there was no test item precipitate was observed in the cell cultures of all exposure concentrationsat at the beginning and end of the test of all the treatment condition.
The results of mutant frequency showed that the induced mutant frequencies (IMF) of all cultures exposed for 3h and 24h were consistently less than the average background mutant frequency of 126×10–6.

Conclusions:

The result of this study is negative, so it is concluded that the test item, Benzene-1,2,4,5-tetracarboxylic acid, compound with 4,5-dihydro-2-phenyl-1H-imidazole(1:1) using the thymidine kinase gene, is non-mutagenic to the L5178Ymouse lymphoma cells under the test conditions of this study.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch No.: M(S)201170
Appearance/Color/State: Solid fine powder (whitish)
Content or purity: ≥99% (Nominal value); 99.50% (Measured result)

Target gene:

histidine

Species / strain / cell type:

S. typhimurium TA 97a

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 102

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Based on the results of the preliminary test that had been performed in this lab before, six doses were selected in the first test, including 5000, 1500, 500, 150, 50 and 15 g/plate with and without metabolic activation. The validation test was conducted using the same dose levels and solvent as the first test.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO) was used as solvent.

Positive controls:

yes

Remarks:

-CAS 140-56-7 Used in TA97a, TA98 and TA102 in S9-. -CAS 153-78-6 Used in TA97a, TA98 and TA100 in S9+ -CAS 117-10-2 Used in TA102 in S9+ -CAS 613-13-8 Used in TA1535 in S9+.

Positive control substance:

sodium azide

Details on test system and experimental conditions:

Standard plate incorporation method and the pre-incubation method at six dose levels, in triplicate, with the solvent control and positive controls, both in the presence and absence of the cofactor-supplemented S9 (S9 mix).

Evaluation criteria:

The test item is considered cytotoxicity if one of the below criteria is met.
1) Compared with the concurrent solvent control, the number of the revertant colonies has an obvious decrease or is absent;
2) Compared with the concurrent solvent control, the sign of the background lawn is thin or clearing.
CRITERIA OF POSITIVE RESULT
1) When there is a concentration-related increase over the range (equal to or greater than two times of that of the concurrent solvent control in TA97a, TA98, TA100, TA102 and equal to or greater than three times of that of the concurrent solvent control in TA1535) in the mean number of revertant colonies in at least one strain with or without metabolic activation system, the test item should be evaluated as positive.
2) When there is a reproducible increase (two times equal to or greater than two times of that of the concurrent solvent control in TA97a, TA98, TA100, TA102 and/or equal to or greater than three times of that of the concurrent solvent control in TA1535) at one or more concentrations in the mean number of revertant colonies in at least one strain with or without metabolic activation system, the test item should be evaluated as positive.

CRITERIA OF NEGATIVE RESULT
The test item should be evaluated as negative if none of the above criteria (See 11.7.2) is met.

CRITERIA OF EQUIVOCAL RESULT
Although most tests give clear positive or negative results, in some instances, the generated data prohibit test item from making a definite judgment. Results of this type should be reported as equivocal.

Species / strain:

S. typhimurium, other: all tested strains

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see "Additional information on results"

Additional information on results:

In two tests, the results of the viable count showed that the density of living bacteria in the cultures of each tester strain was within the acceptable range of 0.9~9×109 colony forming units (CFU)/ml. At the same time, all results of solvent control and positive controls met the requirements of this test, so the sensitivity of the test system and the efficacy of the S9 mix were validated.
In the first test, no precipitate was observed on the surface of MGA plate inoculated with TA98 at all dose level before and after incubation, with and without metabolic activation system. In the validation test, the same results of the precipitate were observed as those in the first test.
In the first test, cytotoxicity were detected by a reduction of the backgroud lawn at the doses of 5000 and 1500g/plate without the metabolic activation in all the bacteria strains, and at the doses of 5000g/plate with the metabolic activation in TA98 and TA1535. In the validation test, the same results of the cytotoxicity were observed as those in the first test.
In the first test, with and without metabolic activation, the mean number of revertant colonies at each dose level was less than two times of that of the concurrent solvent control in TA97a, TA98, TA100, TA102 and less than three times of that of the concurrent solvent control in TA1535. In the validation test, the similar mutagenic results were obtained as those in the first test.

Conclusions:

Under the conditions of this study, the results of the first test and the validation test were negative. Thus, the test item, Benzene-1,2,4,5-tetracarboxylic acid, compound with 4,5-dihydro-2-phenyl-1H-imidazole(1:1), was considered to be non-mutagenic in the bacterial reverse mutation test using histidine requiring tester strains of Salmonella typhimurium.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

 

Justification for classification or non-classification

The substance did not show genotoxic effects in an Ames tests with Salmonella typhimurium TA 97a, TA98, TA 100, TA102, TA 1535 and TA 1537 (50 -5000 microgram/plate), nor in the V79 Chinese hamster lung cell test (0 -5000 mg/l), all with and without metabolic activation.

The substance showed no genotoxic effects and there is no requirement for classification.