Bicyclo[2.2.1]heptanebis(methylamine)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 November 2015 - 05 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to OECD and EC guidelines and in compliance with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Health and Welfare. Evaluation and Licensing Division, Pharmaceutical and Medical Safety Bureau, Notification No. 1604, 1 November 1999.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bicyclo[2.2.1]heptanebis(methylamine)

EC Number:

260-280-7

EC Name:

Bicyclo[2.2.1]heptanebis(methylamine)

Cas Number:

56602-77-8

Molecular formula:

C9H18N2

IUPAC Name:

1-[3-(aminomethyl)bicyclo[2.2.1]heptan-2-yl]methanamine

Test material form:

other: Liquid

Details on test material:

- Name of test material (as cited in study report): NBDA
- Chemical name: Bicyclo[2.2.1]heptanebis (methylamine)
- Physical state: Clear liquid
- Analytical purity: 99.4%
- Lot/batch No.: K14B0001
- Expiration date of the lot/batch: 30 June 2016
- Storage condition of test material: Room Temperature in the dark, under nitrogen

Method

Target gene:

Thymidine kinase

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Purchased from a reputable supplier
- Cells are heterozygous at the thymidine kinase locus, TK +/-.
- Spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24-hour incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196°C, in heat-inactivated donor horse serum (HiDHS) containing 10% DMSO. Cultures were used within ten days of recovery from frozen stock
- Cell stocks are periodically checked for freedom from mycoplasma contamination.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was stored at ca -80°C.

Test concentrations with justification for top dose:

Preliminary toxicity test: 3.01, 6.03, 12.05, 24.1, 48.2, 96.41, 192.81 µg/mL

Mutation test: 50.54, 63.18, 78.97, 98.72, 123.4, 154.25, 192.81 µg/mL - With and without S9 mix (3 hours)
50.54, 63.18, 78.97, 98.72, 123.4, 154.25, 192.81 µg/mL - Without S9 mix (24 hours)

Vehicle / solvent:

Purified water (purified by reverse-osmosis)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

Preliminary toxicity test procedure
Cells were exposed to the test substance for 3 hours in the absence and presence of S9 mix and for 24 hours in the absence of S9 mix.
For 3-hour exposures, cultures contained a total of 6 x 106 cells. The final volume of the cultures was 5 mL and the final concentration of the S9 fraction was 2% v/v, if present.
For 24-hour exposures, cultures contained a total of 1.5 x 106 cells in a total volume of 5 mL. One culture was prepared for each concentration of the test substance for each test condition.
Vehicle controls were tested in duplicate for each test condition.

The test substance was formulated and serially diluted in the solvent. Aliquots of 50 µL of test substance dilution (at 100 times the desired final concentration) or vehicle were added to each culture prior to incubation for 3 hours (continuous shaking at 37°C) or 24 hours (static incubator, at 37°C, 5% (v/v) CO2). At the end of the 3-hour exposure period, the cells were washed once, resuspended in R10p to nominally 2 x 10E5 cells/mL (assuming no cell loss), incubated (at 37°C, 5% (v/v) CO2) and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10E5 cells/mL with R10p where necessary. At the end of the 24-hour exposure period, the cells were
washed once, resuspended in 5 mL R10p and counted, to ascertain treatment growth. The cultures were then diluted to 2 x 10E5 cells/mL with R10p as appropriate, incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10E5 cells/mL with R10p where necessary.
The RSG was used to determine the concentrations of test substance used in the main test; ideally the maximum concentration should reduce RTG to approximately 10 to 20% of the concurrent vehicle control value. There was some evidence of toxicity in the preliminary toxicity test, so the maximumconcentration tested in the main mutation test in the 3-hour exposure in the absence and presence of S9 mix and in the 24-hour exposure in the absence of S9 mix was 192.81 g/mL. The formulations were added at 1% final volume in medium.

Mutation test procedure
3-hour treatment in the absence and presence of S9 mix
The procedure for preparing the cell suspension was the same as for the preliminary toxicity test. Cultures contained a total of 1.2 x 10E7 cells in a final volume of 10 mL. The final concentration of the S9 fraction was 2% v/v, if present. Duplicate cultures were prepared throughout for each concentration of test substance and positive control. Quadruplicate cultures were prepared for vehicle controls. Aliquots of 100 µL of test substance dilution
(at 100 times the desired final concentration), vehicle or positive control were added, then all cultures were incubated, with continuous shaking, for 3 hours at 37°C. At least four serial dilutions of the test substance were tested.
Following the 3-hour treatment, the cells were washed once, resuspended in R10p to nominally 2 x 10E5 cells/mL (assuming no cell loss) and incubated for a further 48 hours to allow for expression of mutant phenotype. The cultures were sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10E5 cells/mL with R10p where necessary. After 48 hours cultures with a density of more than 1 x 105 cells/mL were assessed for cloning efficiency (viability) and mutant potential by plating in 96-well plates. Cloning efficiency was assessed by plating 1.6 cells/well in R20p, two plates being prepared per culture. Mutant potential was assessed by plating 2 x 103 cells/well in
selective medium, two plates being prepared per culture. The plates were placed in a humidified incubator at 37C in an atmosphere of 5% CO2 in air.
After the plates had been incubated for at least 7 days for viability plates and approximately 10 to 14 days for mutant plates, the number of empty wells was assessed for each 96-well plate (P0). P0 was used to calculate the cloning efficiency (CE) and mutant frequency (MF). The colony size distribution in the vehicle and positive controls was examined to ensure that there was an adequate recovery of small colony mutants. The maximum concentration assessed for mutant frequency in the main test was 192.81 g/mL in the absence and presence of S9 mix respectively.

24-hour treatment in the absence of S9 mix
A test with a 24-hour treatment in the absence of S9 mix was carried out. Duplicate 10 mL cultures containing 3 x 10E6 cells were treated for 24 hours with 100 µL of test substance or positive control. Quadruplicate cultures were prepared for vehicle controls. At the end of the exposure period, the cells were washed once, resuspended in 10 mL R10p and counted to ascertain treatment growth. The cultures were then diluted to 2 x 105 cells/mL with R10p as appropriate, incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10E5 cells/mL with R10p where necessary. Following this, the procedure was the same as in the 3-hour treatment. The maximum concentration assessed for mutant frequency in the main test was 192.81 g/mL.

Evaluation criteria:

GEF = Global Evaluation Factor. For microwell assays this is 126 x 10-6
The assay was considered valid in accordance with the assay acceptance criteria.

The test agent was regarded as negative if:
The mean mutant frequency of all test concentrations was less than the sum of the mean concurrent vehicle control mutant frequency and the GEF.

If the mutant frequency of any test concentrations exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, a linear trend test was applied:
If the linear trend test was negative, the result was regarded as negative.
If the linear trend test was positive, this indicated a positive, biologically relevant response.

Where appropriate, other factors were considered in the interpretation of the results, for example, the reproducibility within and between tests, the overall number of mutant colonies (as opposed to mutation frequency) and the nature of any concentration-related effect(s).

Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. In cases where the results were inconclusive, further testing and/or a test modification may have been required to better define the assay response.

Statistics:

Statistical analysis performed using: Fluctuation application SAFEStat (SAS statistical applications for end users)

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

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:

On completion of each main mutagenicity test, data were examined for cell growth parameters, cytotoxicity, plating efficiencies, spontaneous and positive control MF, and percent small colonies in positive control cultures.
The criteria used to assess whether an assay was valid are based on the recommendations of the Plymouth and Aberdeen papers (Robinson et al., 1989; Moore et al., 2000; Moore et al., 2002; Moore et al., 2003; Moore et al., 2006 and Moore et al., 2007).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

It was concluded that NBDA did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Executive summary:

The test material was assessed for mutagenic potential in an in vitro mammalian cell mutation assay. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 476, EU Method B.17 and EPA OPPTS 870.5300.

This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-).

The study consisted of a preliminary toxicity test and three independent mutagenicity assays. The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix.

The test material was soluble at 154.25 mg/mL in water. A final concentration of 192.81 µg/mL, dosed at 1%v/v, was the highest concentration assessed in the preliminary toxicity test as higher concentrations altered the pH of the media by more than 1.0 unit.

Toxicity was observed in the preliminary toxicity test. Following a 3-hour exposure to the test material at concentrations from 3.01 to 192.81 µg/mL, relative suspension growth (RSG) was reduced from 134 to 6% and from 101 to 57% in the absence and presence of S9 mix respectively. Following a 24-hour exposure in the absence of S9 mix RSG was reduced from 93 to 68%. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess exposure up to the highest concentration at which the pH of the media was altered by less than 1.0 unit.

Following 3-hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentration assessed for mutant frequency in the 3-hour treatment in both the absence and presence of S9 mix was 192.81 µg/mL, there were no significant reductions in RTG at any concentration tested.

In the 24-hour treatment, the maximum concentration assessed for mutant frequency was 192.81 µg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF. There were no significant reductions in RTG.

In all tests the concurrent vehicle and positive control were within acceptable ranges.

It was concluded that the test material did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.