2-methylpentane-1,5-diamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29 JUL 2003 to 10 OCT 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Pursuant to the Netherlands GLP Compliance Monitoring Programme and according to Directive 88/320/EEC

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-methylpentane-1,5-diamine

EC Number:

239-556-6

EC Name:

2-methylpentane-1,5-diamine

Cas Number:

15520-10-2

Molecular formula:

C6H16N2

IUPAC Name:

2-methylpentane-1,5-diamine

Details on test material:

- Name of test material (as cited in study report): Dytek A (MPMD)
- Substance type: colourless to slightly yellow, clear liquid
- Physical state: fluid
- Analytical purity: 98.88 %
- Lot/batch No.: M200170A
- Expiration date of the lot/batch: 2004-01-15
- Storage condition of test material: ambient temperature in the dark

Method

Target gene:

Thymidine kinase (TK)-locus on chromosome 11 of cultured mouse lymphoma L5178Y cells

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 (with HEPES and Glutamax-I) supplemented with heat-inactivated horse serum, sodium pyruvate and penicillin/streptomycin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

Experiment I (with and without metabolic activation): 10, 6.5, 4.2, 2.7, 1.8, 1.2, 0.58, 0.29, 0.15, 0.073, 0.036, 0.018, and 0.009 and 0.0 mmol/l
Experiment II (without metabolic activation): 6.3, 5.4, 4.6, 3.9, 2.7, 1.9, 1.3, and 0.0 mmol/l
in parallel: 6.3, 5.4, 4.6 mmol/l with pH adjusted solutions
Experiment II (with metabolic activation): 10, 8.5, 7.2, 6.1, 4.3, 3.0, and 2.1 mmol/l
in parallel: 10, 8.5, 7.2 mmol/l with pH adjusted solutions

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 h (experiment I), 4 h (experiment II)
- Expression time (cells in growth medium): 44-48 h
- Selection time (if incubation with a selection agent): 10-14 days

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: In experiment I single cultures (except for the vehicle control which had two cultures) were used. In experiment II duplicate cultures were used.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG)

OTHER: pH adjustment was done using 5N HCl (starting with stock solution of 25 mmol/l). pH was adjusted to 7.9 at a concentration of 10 mmol/l in the final culture medium.

Evaluation criteria:

A response was considered to be positive if the induced mutant frequency (mutant frequency of the test substance minus that of the vehicle negative control) was more than 100 mutants per 1,000,000 clonable cells (Aaron et al, 1994; Clive et al., 1995). A response was considered to be equivocal if the induced mutant frequency was more than 50 mutants per 1,000,000 clonable cells. Any apparent increase in mutant frequency at concentrations of the test substance causing more than 90% cytotoxicity was considered to be an artefact and not indicative of genotoxicity.

The test substance was considered to be mutagenic in the gene mutation test at the TK-locus if a concentration-related increase in mutant frequency was observed, or if a reproducible positive response for at least one of the test substance concen¬trations was observed.

The test substance was considered not to be mutagenic in the gene mutation test at the TK-locus if it produced neither a dose-related increase in the mutant frequency nor a reproducible positive response at any of the test points.

Statistics:

No statistical analysis was performed.

Results and discussion

Test results

Test results 1

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

details see below

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the presence of S9-mix the relative initial growth and the RTG were decreased above 4.3 mmol/l. The relative total growth at the highest concentration evaluated for mutagenicity was 11 ± 3%.

Vehicle controls validity:

not valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

details see below

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The relative total growth at the highest concentration was not decreased, but the initial cell yield was below 10%. After pH adjustment at the highest concentration (5.4 mmol/l) less toxicity was observed and no increase of the mutant frequency.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
Historical data on negative and positive controls are available.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Dytek A (MPMD) was cytotoxic in both the absence and presence of S9-mix. In the absence of S9-mix, the initial cell yield was decreased above a concentration of 2.7 mmol/l; the relative total growth (RTG) was not decreased in any culture that survived the treatment. The initial cell yield at the highest concentration evaluated for mutagenicity (5.4 mmol/l) was 7.9 ±3.6%. After pH-adjustment Dytek A (MPMD) was less cytotoxic; the initial cell yield at 5.4 mmol/l was 56 ±2%. In the presence of S9-mix, the initial cell yield and RTG were decreased above 4.3 mmol/l Dytek A (MPMD). The RTG at the highest concentration evaluated for mutagenicity (7.2 mmol/l) was 11 ± 3%. After pH-adjustment Dytek A (MPMD) was less cytotoxic. The RTG at 7.2 mmol/l was 64 ±13% and at 10 mmol/l it was 67 ±0.3%.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Mutagenicity

In the absence of S9-mix in the first assay the mutant frequency was increased by more than 50 mutants per 1,000,000 clonable cells compared to the negative control at two single concentrations of Dytek A (MPMD). At 4.2 and 1.8 mmol/l the mutant frequencies were increased by 51 and 76 mutants per 1,000,000 clonable cells, respectively, above the negative control. In the second assay, in which duplicate cultures were used, no increase of the mutant frequency was observed at any dose level. The RTG at the highest concentration was not decreased, but the initial cell yield was below 10%. After pH adjustment at the highest concentration (5.4 mmol/l) less toxicity was observed and no increase of the mutant frequency.

In the presence of S9-mix in the first assay in a single culture at 6.5 mmol/l the mutant frequency was increased by 72 mutants per 1,000,000 clonable cells above the negative control. In the second assay at 3.0 mmol/l the mutant frequency of one of the duplicate cultures was increased by 646 mutants per 1,000,000 clonable cells above the negative control. In the accompanying culture at 3.0 mmol/l and in all other cultures tested, no increase of the mutant frequency was observed. The RTG at the highest concentration was 11 ± 3%. No clear explanation could be found for the extreme high value of the mutant frequency in the single culture at 3.0 mmol/l. The results in the same concentration range considered, in both the first and second assay, the inexplicable value is regarded to be an artefact. After pH adjustment, the mutagenicity could be evaluated at higher concentrations. At 10 mmol/l no increase of the mutant frequency was observed.

The observed increases of the mutant frequencies in single cultures in the first assay were not confirmed in the second assay. Therefore these increases were

considered to be fortuitous and not indicative of mutagenicity.

Colony sizing

In both the absence and the presence of S9-mix the number of large and small colonies formed at concentrations causing an increase in mutant frequency was more or less equal.

Positive and negative controls

Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and in the presence of the S9-mix, respectively. Treatment with the positive controls yielded the expected significant increase in mutant frequency compared to the negative controls. The negative controls were within acceptable ranges.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test the test substance did not induce mammalian cell gene mutations in a mouse lymphoma cell line.

Executive summary:

In an OECD 476 guideline study induction of mammalian cell gene mutations in vitro has been investigated in mouse lymphoma L5178Y cells in the presence (rat liver S9) and absence of metabolic activation. Concentration tested were in Experiment I (with and without metabolic activation): 10, 6.5, 4.2, 2.7, 1.8, 1.2, 0.58, 0.29, 0.15, 0.073, 0.036, 0.018, 0.009 and 0.0 mmol/l in Experiment II (without metabolic activation): 6.3, 5.4, 4.6, 3.9, 2.7, 1.9, 1.3, and 0.0 mmol/l (in parallel: 6.3, 5.4, 4.6 mmol/l with pH adjusted solutions) and in Experiment II (with metabolic activation): 10, 8.5, 7.2, 6.1, 4.3, 3.0, and 2.1 mmol/l (in parallel: 10, 8.5, 7.2 mmol/l with pH adjusted solutions). The test item did not induce gene mutations in concentrations up to 10 mmol/l under the tested conditions. Treatment with the respective positive control substances yielded the expected significant increase in mutant frequency compared to negative controls. Negative controls were within the accepted range.