Reaction mass of 1-(4-aminophenyl)-1,3,3-trimethylindan-5-amine and 3-(4-aminophenyl)-1,1,3-trimethyl-2,3-dihydro-1H-inden-5-amine

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:

9th October - 30th October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

Diamino Trimethylphenylindane

IUPAC Name:

Diamino Trimethylphenylindane

Test material form:

solid: particulate/powder

Details on test material:

Purity:
97.6% (49.9% 6-4’ isomer, 47.7 5-4’ isomer; per Protocol)

Specific details on test material used for the study:

Identification:
Diamino Trimethylphenylindane
Synonym:
DAPI
Batch No.:
AEF0030300
Purity:
97.6%(49.9% 6-4’ isomer, 47.7 5-4’ isomer) (per Protocol)
Molecular Weight:
266.38 g/mol
Description:
Off-white powder
Storage Conditions:
Room temperetaure, protected from light
Receipt Date:
07 June 2017

Method

Target gene:

WP2 uvrA

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot No. 3841, Exp. Date: 08 Aug 2019) was purchased commercially from MolTox (Boone, NC).
BioReliance Study No. AE95CF.502REACH.BTL 11
Upon arrival at BioReliance, the S9 was stored at -60°C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and 2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.
The S9 mix was prepared on the day of use as indicated below:
Component
Final Concentration
β-nicotinamide-adenine dinucleotide phosphate
4 mM
Glucose-6-phosphate
5 mM
Potassium chloride
33 mM
Magnesium chloride
8 mM
Phosphate Buffer (pH 7.4)
100 mM
S9 homogenate
10% (v/v)
The Sham mix, containing 100 mM phosphate buffer at pH 7.4, was also prepared on the day of use.

Test concentrations with justification for top dose:

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg per plate. Precipitate was observed beginning at 3333 or at 5000 μg per plate with all conditions. Toxicity was observed beginning at 667, 1000, 3333 or at 5000 μg per plate with all conditions. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.
In the mutagenicity assay, the dose levels tested were 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO) was used as the vehicle.

Controls

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976).
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).
Salmonella tester strains were derived from Dr. Bruce Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

Rationale for test conditions:

The test system was exposed to the test substance via the plate incorporation methodology originally described by Ames et al. (1975) and updated by Maron and Ames (1983).

Evaluation criteria:

The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using the codes shown in the following table. As appropriate, colonies were enumerated either by hand or by machine.
On the day of use in each assay, all tester strain cultures were checked for the appropriate genetic markers.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Positive controls validity:

valid

Additional information on results:

No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.Positive mutagenic responses were observed (2.6- and 4.3-fold, maximum increases) with tester strains TA98 and TA100 in the presence of S9 activation.

Applicant's summary and conclusion

Conclusions:

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, Diamino Trimethylphenylindane did cause a positive mutagenic response with tester strains TA98 and TA100 in the presence of S9 activation. The study was concluded to be positive without conducting a confirmatory (independent repeat) assay because the results were clearly positive; hence, no further testing was warranted.

Executive summary:

The test substance, Diamino Trimethylphenylindane, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg per plate. Precipitate was observed beginning at 3333 or at 5000 μg per plate with all conditions. Toxicity was observed beginning at 667, 1000, 3333 or at 5000 μg per plate with all conditions. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.

In the mutagenicity assay, the dose levels tested were 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. Precipitate was observed at 5000 μg per plate with all conditions. Toxicity was observed beginning at 1500 or at 5000 μg per plate with most conditions. Positive mutagenic responses were observed (2.6- and 4.3-fold, maximum increases) with tester strains TA98 and TA100 in the presence of S9 activation.

These results indicate Diamino Trimethylphenylindane was positive for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium (TA98 and TA100) in the presence of an exogenous metabolic activation system.