Hexamethylene diacrylate

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted in accordance with the OECD guideline 471. The rate of induced back mutations was tested in the S. typhimurium indicator organisms TA1535, TA1537, TA98 and TA100. However, the mutagenic potential of the test substance was not tested in an additional strain, as recommended in the latest version of OECD guideline 471 (July 1997).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Principles of method if other than guideline:

The rate of induced back mutations was tested in the S. typhimurium indicator organisms TA1535, TA1537, TA98 and TA100 according to OECD guideline 471. However, the mutagenic potential of the test substance was not tested in an additional E. coli strain or in S. typhimurium TA102, as recommended in the latest version of OECD guideline 471 (July 1997).

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Hexamethylene diacrylate

EC Number:

235-921-9

EC Name:

Hexamethylene diacrylate

Cas Number:

13048-33-4

Molecular formula:

C12H18O4

IUPAC Name:

hexane-1,6-diyl bisacrylate

Details on test material:

- Name of test material (as cited in study report): Hexandioldiacrylat
- Test substance number: 88/1005
- Analytical purity: > 85 %
- Impurities (identity and concentrations): not specified
- Storage condition of test material: + 4 °C to + 6 °C

Method

Target gene:

Determination of the rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+).

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from the liver of male Sprague-Dawley rats (treated with a single dose of 500 mg/kg bw Aroclor 1254 five days before sacrifice) and mixed with a series of cofactors (MgCl2, KCl, glucose-6-phosphate, NADP, phosphate buffer pH 7.4).

Test concentrations with justification for top dose:

Standard plate test:
1st experiment:
0, 20, 100, 500, 2500 and 5000 µg/plate (tested in the strains S. typhimurium T98 and TA100)
2nd experiment:
0, 4, 20, 100, 500 and 1500 µg/plate without S9-mix; 0, 4, 20, 100, 500 and 2500 µg/plate with S9-mix (tested in the strains S. typhimurium T98, TA100, TA1535 and TA1537).

Preincubation test:
0, 4, 20, 100, 500 and 1000 µg/plate (tested in the strains S. typhimurium T98, TA100, TA1535 and TA1537).

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see details on test system

Details on test system and experimental conditions:

METHOD OF APPLICATION:
A standard plate test and a preincubation test were conducted, both with and without metabolic activation (S9 mix). Each test was conducted in triplicates.

STANDARD PLATE TEST:
The experimental procedure is based on the method of Ames et al. (Mut. Res. 31: 347-364, 1975):
Test tubes containing 2 ml portions of soft 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
0.1 ml bacterial suspension
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.

PREINCUBATION TEST:
The experimental procedure is based on the method described by Yahagi et al. (Mut. Res. 48: 121-130, 1977) and Matsushima et al. (Factors modulating mutagenicity in microbial tests. In: Norpoth, K.H. and R.C. Garner, Short-Term Test Systems for Detecting Carcinogens. Springer Verlag Berlin, Heidelberg, New York, 1980):
0.1 ml test solution, 0.1 ml bacterial suspension and 0.5 ml S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the Vogel-Bonner 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 agar
20 g D-glucose monohydrate.
After incubation at 37 °C for 48 hours in the dark, the bacterial colonies (his+ revertants) are counted.

CONTROLS:
- Negative control:
Parallel with each experiment with and without S-9 mix, a negative control (with vehicle DMSO) is carried out for each tester strain in order to determine the spontaneous mutation rate.
- Positive controls:
The following positive control substances are used to check the mutability of the bacteria and the activity of the S-9 mix:
with S-9 mix: 10 µg 2-aminoanthracene (dissolved in DMSO) for the strains TA 100, TA 98, TA 1537 and TA 1535.
without S-9 mix: 5 µg N-methyl-N´-nitro-N-nitrosoguanidine (MNNG) (dissolved in DMSO) for the strains TA 100 and TA 1535; 10 µg 4-nitro-o-phenylendiamine (dissolved in DMSO) for the strain TA 98; 100 µg 9-aminoacridine chloride monohydrate (dissolved in DMSO) for the strain TA 1537.

TITER DETERMINATION:
In general, the titer is determined only in the experiments with S-9 mix both without test substance (solvent only) and after adding the two highest amounts of substance. For this purpose, 0.1 ml of the overnight cultures is diluted to 1 x 10exp-6 in each case. Test tubes containing 2 ml portions of soft agar containing maximal amino acid solution (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 solvent (without and with test substance)
0.1 ml bacterial suspension (dilution: 1 x 10exp-6)
0.5 ml S-9 mix
After mixing, the samples are poured onto the Vogel-Bonner agar plates within approx. 30 seconds. After incubation at 37°C for 48 hours in the dark, the bacterial colonies are counted.

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>500 µg/plate and higher, depending on test strain.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MUTAGENICITY:
No increase in the number of his+ revertants was observed, neither in the standard plate test nor in the preincubation test, with or without the addition of S9 mix.
The results of the negative and positive controls were as expected and confirmed the validity and sensitivity of the test system used.

SOLUBILITY:
The test substance was completely soluble in DMSO.

TOXICITY:
A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ revertants) was observed depending on the strain and test conditions from about 500-2500 µg/plate onward in the standard plate test and at doses >=500 µg/plate in the preincubation test.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: in standard plate test and preincubation test

Any other information on results incl. tables

Table 1: Maximum revertants/plate and corresponding test concentrations in the standard plate test:
Strain	Tested compound	Maximum revertants/plate [corresponding dose unit in µg/plate]
		without S9-mix	with S9-mix
S. typhimurium TA1535	DMSO	18 ± 2	21 ± 2
	Test substance	19 ± 2 [100]	34 ± 1 [500]
	Positive Control	1817 ± 236 [5; MNNG]	171 ± 17 [10; 2 -AA]
S. typhimurium TA100	DMSO	96 ± 7	116 ± 15
	Test substance	102 ± 5 [4]	129 ± 7 [500]
	Positive Control	1550 ± 132 [5; MNNG]	1493 ± 129 [10; 2 -AA]
S. typhimurium TA98	DMSO	22 ± 1	35 ± 5
	Test substance	24 ± 3 [4]	37 ± 7 [4]
	Positive Control	855 ± 35 [10; NOPD]	1110 ± 26 [10; 2 -AA]
S. typhimurium TA1537	DMSO	6 ± 1	13 ± 2
	Test substance	7 ± 1 [4]	12 ± 4 [4]
	Positive Control	418 ± 50 [100; AAC]	148 ± 17 [10; 2 -AA]
2-AA = 2-aminoanthracene
MNNG = N-methyl-N'-nitro-N-nitrosoguanidine
NOPD = 4-nitro-o-phenylenediamine
AAC = 9-aminoacridine

Table 2: Maximum revertants/plate and corresponding test concentrations in the preincubation test:
Strain	Tested compound	Maximum revertants/plate [corresponding dose unit in µg/plate]
		without S9-mix	with S9-mix
S. typhimurium TA1535	DMSO	17 ± 2	21 ± 2
	Test substance	15 ± 3 [4]	19 ± 3 [500]
	Positive Control	1343 ± 21 [5; MNNG]	149 ± 20 [10; 2 -AA]
S. typhimurium TA100	DMSO	101 ± 3	110 ± 13
	Test substance	97 ± 5 [4]	92 ± 6 [20]
	Positive Control	1091 ± 113 [5; MNNG]	1353 ± 220 [10; 2 -AA]
S. typhimurium TA98	DMSO	19 ± 2	31 ± 3
	Test substance	22 ± 1 [100]	31 ± 3 [100]
	Positive Control	754 ± 113 [10; NOPD]	1283 ± 257 [10; 2 -AA]
S. typhimurium TA1537	DMSO	7 ± 1	8 ± 1
	Test substance	8 ± 3 [4]	7 ± 2 [100]
	Positive Control	268 ± 91 [100; AAC]	189 ± 33 [10; 2 -AA]
2-AA = 2-aminoanthracene
MNNG = N-methyl-N'-nitro-N-nitrosoguanidine
NOPD = 4-nitro-o-phenylenediamine
AAC = 9-aminoacridine