[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

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:

31st August 2016 to 19th September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

EC Number:

289-348-4

EC Name:

[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

Cas Number:

87788-32-7

Molecular formula:

C10H22B2F6N2

IUPAC Name:

[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

impurity 1

Reference substance name:

{3-[(amino-N)methyl]-3,5,5-trimethylcyclohexanamine}(trifluoro)boron

Molecular formula:

C10H22BF3N2

IUPAC Name:

{3-[(amino-N)methyl]-3,5,5-trimethylcyclohexanamine}(trifluoro)boron

additive 1

Reference substance name:

Methanol

EC Number:

200-659-6

EC Name:

Methanol

Cas Number:

67-56-1

Molecular formula:

CH4O

IUPAC Name:

methanol

Test material form:

liquid

Specific details on test material used for the study:

Test Substance
Identification:
[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')] hexafluorodiboron
Batch No.:
AEF0009100
Description:
Clear colorless liquid
Storage Conditions:
Room temperature, protected from light
Receipt Date:
12 July 2016

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

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.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

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.

Metabolic activation:

with

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system.

Vehicle / solvent:

Water was used as the vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

Details on test system and experimental conditions:

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).

To confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective agar. To confirm the sterility of the test substance and the vehicle, all test substance dose levels and the vehicle used in each assay were plated on selective agar with an aliquot volume equal to that used in the assay. These plates were incubated under the same conditions as the assay.
One-half (0.5) milliliter of S9 or Sham mix, 100 μL of tester strain (cells seeded) and 100 μL of vehicle or test substance dilution were added to 2.0 mL of molten selective top agar at 45±2°C. When plating the positive controls, the test substance aliquot was replaced by a 50.0 μL aliquot of appropriate positive control. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

Evaluation criteria:

Evaluation of Test Results
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:
Strains TA1535 and TA1537
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value.
Strains TA98, TA100 and WP2 uvrA
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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, [μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')] hexafluorodiboron did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9.

Executive summary:

The test substance, [μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')] hexafluorodiboron, 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. Water was used as the vehicle.

In the initial toxicity-mutation assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. Neither precipitate nor toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

In the confirmatory mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. Neither precipitate nor toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

These results indicate [μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')] hexafluorodiboron was negative for the 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.