Isobutyl 4-chloro-3,5-diaminobenzoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1978-09-29 - 1978-10-17

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Well-documented bacterial reverse mutation assay, conducted on the registered subtance itself, similar to OECD 471. TA 1538 was used as fifth tester strain instead of E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102. However, as these strains were induced to primarily detect cross-linking mutagens, and this is not the expected mode of action for a potential genotoxicity of isobutyl 4-chloro-3,5-diaminobenzoate, this deviation is not expected to have any impact on the reliability of the results. Also, minor parts of the test report were not available.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1978

Report date:

1978

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not applicable

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Isobutyl 4-chloro-3,5-diaminobenzoate

EC Number:

251-311-5

EC Name:

Isobutyl 4-chloro-3,5-diaminobenzoate

Cas Number:

32961-44-7

Molecular formula:

C11H15ClN2O2

IUPAC Name:

2-methylpropyl 3,5-diamino-4-chlorobenzoate

Test material form:

other: crystalline powder

Details on test material:

- Storage condition of test material: room temperature

Method

Target gene:

his

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: see "any other information on materials and methods"

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: see "any other information on materials and methods"

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

10, 50, 100, 500, 1000 µg/plate

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Remarks:

bacteria only / S9 fraction

Negative solvent / vehicle controls:

yes

Remarks:

DMSO 100 µL

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

9-aminoacridine

2-nitrofluorene

benzo(a)pyrene

other: N-methyl-N-nitro-N-nitrosoguanidine (MNNG), 6-Aminochrysene (6-AC), 2- Aminoanthracene (2-AA), Aflatoxin B1, 2-Aminofluorene (2-AF)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

SELECTION AGENT (mutation assays): histidine-free media

NUMBER OF REPLICATIONS: triplicates

Evaluation criteria:

All 3 control plates must have approximately equal numbers of revertant colonies. The solvent controls must have approximately the same number of colonies as spontaneous reversion controls. Positive mutagens must give at least 3x the number of colonies as the controls for spontaneous reversion. Any test with a strain which does not meet these criteria must be repeated on a separate day.
All criteria noted above must be met before results with an unknown agent can be evaluated. To be considered positive, an unknown agent should exhibit a dose response effect. That is, there should be an increasing number of mutants with increased amounts of test agent. For strains TA1535, TA1537, TA1538 and TA98, the first positive dose must produce 3x the number of colonies as the negative control. For strain TA100, at least one dose tested must produce 3.5x the number of mutants as the spontaneous reversion control.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

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

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

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

Test results 5

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: none stated

Applicant's summary and conclusion

Conclusions:

Interpretation of results: ambiguous

The study was well-performed according a method similar to OECD 471, there is no indication given that the results are not reliable. Hence, they can be used to assess the potential of isobutyl 4-chloro-3,5-diaminobenzoate to induce gene mutations in bacteria. The test agent did not induce a significant increase in the number of point mutations in Salmonella typhimurium strains in the absence and presence of the activating system for strains TA1535, TA100. Hence, it should be regarded as negative is this test. On the other side, it did induce a significant increase in the number of point mutations in Salmonella typhimurium strains in the absence and presence of the activating enzyme system of strains TA1537, TA1538, TA98. The increase was dose-dependent and ≥ 3 times over control, hence, it should be regarded as positive in this test. So, only based on this data, no conclusions can be drawn for the genotoxic potential of isobutyl 4-chloro-3,5-diaminobenzoate. The results should be considered as ambiguous, and additional data is required for a definitive assessment.

Executive summary:

In a reverse gene mutation assay in bacteria (similar to OECD 471), strains TA1535, TA100, TA1537, TA1538, TA98 of S. typhimurium were exposed to of isobutyl 4-chloro-3,5-diaminobenzoate in DMSO at concentrations of 10, 50, 100, 500, 1000 µg/plate in the presence and absence of mammalian metabolic activation in plate incorporation.

The positive controls induced the appropriate responses in the corresponding strains. There was partial evidence and a concentration related positive response of induced mutant colonies over background.

The test agent did not induce a significant increase in the number of point mutations in Salmonella typhimurium strains in the absence and presence of the activating system for strains TA1535, TA100. It did induce a significant increase in the number of point mutations in Salmonella typhimurium strains in the absence and presence of the activating enzyme system of strains TA1537, TA1538, TA98. The increase was dose-dependent and ≥ 3 times over control. The results should be considered as ambiguous.

This study is classified as acceptable.