N,N'-bis(2,4,6-triisopropylphenyl)methanediimine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-07-10 - 2017-11-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for Testing of Chemicals No. 471. "Bacterial Reverse Mutation Test". Adopted: 21st July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Council Regulation No. 440/2008 of 30 May 2008. Official Journal of the European Communities of May 31, 2008, L 142/248 - L142/255. B.13/14. Mutagenicity - Reverse Mutation Test using Bacteria.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Health Effects Test Guidelines; United States Environmental Protection Agency; Prevention, Pesticides and Toxic Substances (7101); EPA712-C-98-247, August 1998. OPPTS 870.5100 - Bacterial Reverse Mutation Test.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-bis[2,4,6-tris(propan-2-yl)phenyl]methanediimine

EC Number:

815-171-4

Cas Number:

300382-79-0

Molecular formula:

C31H46N2

IUPAC Name:

N,N'-bis[2,4,6-tris(propan-2-yl)phenyl]methanediimine

Test material form:

liquid

Remarks:

yellowish

Details on test material:

Storage: Room temperature

Method

Target gene:

his-

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

0, 10, 25, 50, 160, 500, 1600, 5000 µg/plate, top dose as indicated in the guideline
The highest dose tested should cause toxicity or should correspond to the substance's precipitation range (visible precipitates on the plates) but should not exceed 5 mg/plate or 5 µL/plate. At least five doses were routinely used.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethylenglycoldimethylether (EGDME)
- Justification for choice of solvent/vehicle: The solvent used was chosen according to information given by the sponsor.

Controls

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

50 µl EDGME

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

cumene hydroperoxide

mitomycin C

other: 4-NPDA (4-Nitro-o-phenylenediamine) / 2-AA (Anthracene-2-amine)

Remarks:

Mitomycin C, cumene hydroperoxide and sodium azide were dissolved in phosphate buffer. The other positive controls were dissolved in DMSO.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) or preincubation
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: 20 min
- Exposure duration: ca. 48 hours (TA102) or 72 hours (TA1535, TA100, TA1537, TA98).

SELECTION AGENT (mutation assays): his-minimal agar

NUMBER OF REPLICATIONS: triplicate cultures, two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: gross appraisal of background growth / marked and dose-dependent reduction in the mutant count per plate

Rationale for test conditions:

The evaluation of the mutagenicity of the test substance was performed using the Salmonella/microsome test, also termed the Arnes test, as described by Maron and Arnes (1983), and Gatehouse et al. (1994).
The Salmonella/microsome test detects gene mutations caused by chemical agents in vitro. Auxotrophic mutants of Salmonella typhimurium are used to demonstrate this effect. For this purpose, the rate of reversion to prototrophy is evaluated in solvent control and treated groups. A mutagenic effect is assumed if this rate increases sufficiently in the treated groups.
Mammalian metabolism, which is of great significance in chemical mutagenesis, is simulated in this test by the 9000 g fraction of homogenized mammalian livers. Together with cofactors, this forms the "S9 mix" which represents the metabolic model in this test.

Evaluation criteria:

Assessment Criteria
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA1535, TA100, TA1537 and TA98 this increase should be about twice that of solvent controls. For TA102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

both plate incorporation and preincubation test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

and precipitation started at 1600 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

both plate incorporation and preincubation test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

and precipitation started at 1600 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

both plate incorporation and preincubation test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

and precipitation started at 1600 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

both plate incorporation and preincubation test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

and precipitation started at 1600 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Remarks:

both plate incorporation and preincubation test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

and precipitation started at 1600 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The positive controls sodium azide, 4-nitro-1,2-phenylene diamine, 2-nitrofluoren, mitomycin C, cumene hydroperoxide and 2-aminoanthracene increased mutant counts to well over those of the solvent controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.
None of the five strains used showed a dose-related and biologically relevant increase in mutant counts over those of the solvent controls in the plate incorporation test. This applied both to the tests with and without S9 mix and was confirmed by the results of the preincubation trials.

Applicant's summary and conclusion

Conclusions:

The study was conducted under GLP according to EU method B.13/14, OECD guideline 471, and US EPA OPPTS 870.5100 on the registered substance itself without deviations. The method is to be considered scientifically reasonable, the validity criteria are fulfilled. Hence, the results can be considered as sufficiently reliable to assess the ability of Bis(2,4,6-triisopropylphenyl)carbodiimide to induce gene mutations in bacteria.
None of the five strains used showed a dose-related and biologically relevant increase in mutant counts over those of the solvent controls in the plate incorporation test. This applied both to the tests with and without S9 mix and was confirmed by the results of the preincubation trials. In conclusion it can be stated that under the experimental conditions reported (direct plate incorporation procedure and preincubation modification) the test item did not induce gene mutations by base-pair changes or frame-shifts in the genome of the S. typhimurium strains used, when tested up to a maximum recommended dose of 5000 µg/plate in the absence and presence of S9 mix.

Executive summary:

Bis(2,4,6-triisopropylphenyl)carbodiimide was investigated for point mutagenic effects in the Salmonella/microsome test (plate incorporation test and preincubation method) in a study according to OECD 471 under GLP. The test item, dissolved in EGDME dried with a molecular sieve, was administered in doses of up to and including 5000 µg per plate without and with S9 mix on the five Salmonella typhimurium LT2 mutant strains TA1535, TA100, TA1537, TA98 and TA102. No bacteriotoxic effects were observed. Substance precipitation occurred at the dose of 1600 µg per plate and above.

In both experiments, the employed positive controls had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding solvent controls.

Evidence of mutagenic activity of the test item was not seen. No biologically relevant increase in the mutant count, in comparison to the solvent controls, was observed in any of the strains tested, without and with S9 mix, in the plate incorporation as well in the preincubation modification, under the experimental conditions applied.

Therefore, the test item was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.