Octamethylenediamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-05-13 - 1997-07-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Remarks:

BASF AG, Department of Toxicology, Ludwigshafen/Rhein

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Octamethylenediamine

EC Number:

206-764-3

EC Name:

Octamethylenediamine

Cas Number:

373-44-4

Molecular formula:

C8H20N2

IUPAC Name:

octane-1,8-diamine

Details on test material:

- Name of test material (as cited in study report): 1,8-Oktamethylendiamin
- Physical state: white solidified melt
- Storage condition of test material: room temperature
- Test substance number: 96/668
- Analytical purity: 99.9 area % (GC)
- Lot/batch No.: 66-0280
- Date of manufacture: December 1994

Method

Target gene:

histidine (his-) and tryptophan (trp-)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver post-mitochondrial fraction (S-9) was prepared from 5 male Sprague-Dawley rats with a single intraperitoneal injection of Aroclor 1254 according to Ames et.al.

Test concentrations with justification for top dose:

Standard plate test (SPT): 0, 20, 100, 500, 2500 and 5000 µg/plate
Preincubation test (PIT): 0, 4, 20, 100, 500 and 1500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: Water was chosen due to the good solubility of the test substance

Controls

Untreated negative controls:

yes

Remarks:

Sterility control (soft agar, S9-mix, buffer, vehicle/test substance, but without test strains)

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle control

True negative controls:

no

Positive controls:

yes

Remarks:

different for with and without metabolic activation test strains

Positive control substance:

other: With S-9 mix: 2-aminoanthracene (2-AA) / Without S-9 mix: TA 1535, TA 100: N-methyl-N´-nitro-N-nitrosoguanidine (MNNG); TA 98: 4-nitro-o-phenylendiamine (NOPD); TA 1537: 9-aminoacridine (AAC); E.coli WP2 uvrA: N-ethyl-N´-nitro-N-nitrosoquanidine (ENNG)

Details on test system and experimental conditions:

SALMONELLA TYPHIMURIUM
The experimental procedure of the standard plate test (plate incorporation method) is based on the method of Ames et. al. The experimental procedure of the standard plate test (plate incorporation method) is based on the Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.6 % agar + 0.6 % NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants : 0 .5 mM histidine + 0 .5 mM biotin) are kept in a water bath at 45 °C, and the remaining components are added in the following order :
0.1 mL test solution or vehicle
0.1 mL fresh bacterial culture
0.5 mL S-9 mix (in tests with metabolic activation) or 0 .5 mL phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx . 30 seconds. Composition of the minimal glucose agar :
980 mL aqua dest .
20 mL Vogel-Bonner E medium
15 g Difco bacto aga r
20 g D-glucose, monohydrate .

ESCHERICHIA COLI
The experimental procedure is based on the method of Ames et. al. Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mLagar (0.6 % agar + 0.6 % NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants : 0.5 mM tryptophan) are kept in a water bath at 45 °C, and the remaining components are added in the following order :
0.1 mL test solution or vehicle
0.1 mL fresh bacterial culture
0.5 mL S-9 mix (in tests with metabolic activation) or 0.5 mL phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds.
Composition of the minimal agar: The composition of the minimal agar (SA1 selective agar) is based on the description of Green, M.H.L. and Muriel, W.J., with the exception of solution E (tryptophan solution), which has previously been added to the soft agar:
300 mL solution B (agar )
100 mL solution A(saline solution)
8 mL solution C (glucose solution)
10 mL solution D(casein solution )

DURATION
- Exposure duration: the plates incubated at 37 °C in the dark for at 48 - 72 h

Evaluation criteria:

ACCEPTANCE CRITERIA
Generally, the experiment is to be considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control articles both with and without S-9 mix induced a significant increase in the number of revertant colonies within the range of the historical control data.
- The titer of viable bacteria was >= 10^9/mL.

EVALUATION CRITERIA
- The test chemical is considered positive in this assay if the following criteria are met: A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.
- A test substance is generally considered nonmutagenic in this test if: The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Statistics:

No details available.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: A bacteriotoxic effect was observed under all test conditions.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TOXICITY
A bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed in the standard plate test from about 2,500 μg/plate onward. In the preincubation assay bacteriotoxicity was observed depending on the strain and test conditions from about 100 μg - 500 μg/plate onward.

SOLUBILITY
No test substance precipitation was found.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Standard Plate Test and Precincubation Test

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The study is considered as valid OECD Guideline 471 and 472 study without deviations and was performed under certificated Good Laboratory Praxis (GLP) and therefore considered to be of the highest quality (reliability Klimisch 1). The test material did not induce an increases in the frequency of revertant colonies in any of the bacterial strains, therefore the test material can be considered as non-mutagenic under these test conditions.

Executive summary:

The test substance octamethylenediamine was investigated according to OECD TG471 and 472 for its potential to cause gene mutation in Salmonella typhimurium strains (TA98, TA100, TA1537, TA1535 and TA1538) and Escherichia coli WP2 uvrA (Engelhardt and Hoffmann, 1999). The Standard plate test (SPT) was performed with test concentrations of 20 - 5000 µg/plate and concentrations of 4 - 1500 µg/plate were used in the Preincubation test (PIT). Due to the good water solubility of the substance, auqa dest. was used as vehicle. The test was performed with and without a metabolic activation system. For this purpose, the mammalian liver post-mitochondrial fraction (S-9) was prepared from 5 male Sprague-Dawley rats with a single intraperitoneal injection of Aroclor 1254 according to Ames et.al. A bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed in the standard plate test from about 2,500 μg/plate onward. In the preincubation assay bacteriotoxicity was observed depending on the strain and test conditions from about 100 μg - 500 μg/plate onward. Up to the highest investigated dose, no relevant increase of the revertant colony numbers was obtained in any Salmonella typhimurium strain as well as in the E.coli strain in comparison with the corresponding controls. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test material caused neither base-pair substitutions, nor frameshift mutations. Therefore these test results revealed no indication of gene mutagenic activity.