2,2'-[(1-methylethylidene)bis(cyclohexane-4,1-diyloxymethylene)]bisoxirane

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:

16 June 2004 to 24 June 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Reference 2

Reference Type:

other: Amendment

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

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:

2,2'-[(1-methylethylidene)bis(cyclohexane-4,1-diyloxymethylene)]bisoxirane

EC Number:

236-502-3

EC Name:

2,2'-[(1-methylethylidene)bis(cyclohexane-4,1-diyloxymethylene)]bisoxirane

Cas Number:

13410-58-7

Molecular formula:

C21H36O4

IUPAC Name:

2-({[4-(2-{4-[(oxiran-2-yl)methoxy]cyclohexyl}propan-2-yl)cyclohexyl]oxy}methyl)oxirane

Test material form:

liquid: viscous

Details on test material:

- Appearance: Liquid, viscous / colourless
- Storage: Room temperature

Method

Target gene:

- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).

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):

- Deep-frozen (-70 to -80 °C) bacterial cultures (Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98) were thawed at room temperature, and 0.1 mL of this bacterial suspension was inoculated in nutrient broth solution (8 g Difco nutrient broth + 5 g NaCI/litre) and incubated in the shaking water bath at 37 °C for about 12-16 hours. As a rule, a germ density of ≥ 10^8 bacteria/mL is reached. These cultures were grown overnight and kept in iced water from the beginning of the experiment until the end in order to prevent further growth.
- The use of the strains mentioned is in accordance with the current scientific recommendations for the conduct of this assay.
- The Salmonella strains were obtained from KNOLL Aktiengesellschaft on October 30, 1989.
- The Salmonella strains were checked for the following characteristics at regular intervals: deep rough character (rfa) ; UV sensitivity (n uvrB); ampicillin resistance (R factor plasmid).

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Deep-frozen (-70 to -80 °C) bacterial cultures (E . coli WP2 uvrA) were thawed at room temperature, and 0.1 mL of this bacterial suspension was inoculated in nutrient broth solution (8 g Difco nutrient broth + 5 g NaCI/litre) and incubated in the shaking water bath at 37°C for about 12-16 hours. As a rule, a germ density of ≥ 10^8 bacteria/mL is reached. These cultures were grown overnight and kept in iced water from the beginning of the experiment until the end in order to prevent further growth.
- The use of the strains mentioned is in accordance with the current scientific recommendations for the conduct of this assay.
- The Escherichia coli strain was obtained from Merck on September 9, 1991.
- E.coli WP2 uvrA is checked for UV sensitivity.

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

1st Experiment (all strains): 0; 32; 160; 800; 4 000 and 8 000 µg/plate
2nd Experiment (TA 1535 only): 0; 100 ; 200 ; 400; 800 and 1 000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test material in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene (2-AA) - 2.5 µg/plate: TA 1535, 100, 1537, 98 - 60 µg/plate: E.coli WP2 uvrA (+ S-9 mix) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) 5 µg/plate: TA 1535, TA 100 and 4-nitro-o-phenylendiamine (NOPD) 10 µg/plate: TA 98 (- S-9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation

NUMBER OF REPLICATIONS: 3 test plates per dose or per control

SALMONELLA TYPHIMURIUM
- Test tubes containing 2 mL portions of soft agar (overlay agar), which consisted of 100 mL agar (0.8% agar + 0.6% NaCI) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0 .5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were 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 were 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 agar and 20 g D-glucose, monohydrate.
- After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (his+ revertants) were counted.

ESCHERICHIA COLI
- Test tubes containing 2 mL portions of soft agar (overlay agar), which consisted of 100 mL agar (0 .8% agar + 0.6% NaCI) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were 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 were poured onto minimal agar plates within approx. 30 seconds.
- Composition of the minimal agar: The composition of the minimal agar (SA1 selective agar) was 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) and 10 mL solution D (casein solution).
- After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted.

TITRE DETERMINATION
- 0.1 mL of the overnight cultures was diluted to 10^-6 in each case. Test tubes containing 2 mL portions of soft agar containing maximal amino acid solution (5 mM tryptophan or 5 mM histidine + 0 .5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order: 0.1 mL vehicle (without and with test material), 0.1 mL fresh bacterial culture (dilution: 10^-6) and 0.5 mL S-9 mix.
- After mixing, the samples were poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies were counted.

EVALUATION
- Mutagenicity: Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments . In general, five doses of the test material were tested with a maximum of 5 mg/plate, and triplicate plating was used for all test groups at least in the 1st experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments were based on the findings of the 1st experiment.
- Titre: The titre was generally determined only in the experimental parts with S-9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.
- Toxicity: Toxicity detected by a decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his- or trp- background growth) or reduction in the titre, was recorded for all test groups both with and without S-9 mix in all experiments.
- Solubility: Precipitation of the test material was recorded. As long as precipitation did not interfere with the colony scoring, 5 mg/plate was generally selected and analysed (in cases of non-toxic compounds) as the maximum dose at least in the 1st experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate may also have been tested in repeat experiments for further clarification/substantiation.

ACCEPTANCE CRITERIA
Generally, the experiment is 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 or above.
- The titre of viable bacteria was ≥ 10^8/mL.

Evaluation criteria:

EVALUATION CRITERIA
- The test material is considered positive in this assay if: 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 metabolising system.
- A test material is generally considered non-mutagenic 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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA 98 and TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium, other: TA 100 and TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Mutagenicity was observed from about 100 µg/plate onward (factor 2 4) with an increase in the number of his+ revertants by a factor of 13.5 at 1 000 µg/plate

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Slight increase in the number of revertant colonies at 800 µg/plate (factor 2.2)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

MUTAGENICITY TESTS
- Tests without S-9 mix: No increase in the number of his+ or trp+ revertants in any strain.
- Tests with S-9 mix:
TA 1535 : Mutagenicity was observed from about 100 µg/plate onward (factor 2.4) with an increase in the number of his+ revertants by a factor of 13.5 at 1 000 µg/plate.
TA 100: Slight increase in the number of revertant colonies at 800 µg/plate (factor 2.2).
TA 1537, TA 98 and E. coli WP2 uvrA: No increase in the number of his+ or trp+ revertants.

TOXICITY
- A bacteriotoxic effect (reduced his- or trp- background growth and / or decrease in the number of his+ revertants, reduction in the titre) was observed depending on the strain and test conditions from about 4 000 µg/plate onward.

SOLUBILITY
- Test material precipitation was found from about 4 000 µg/plate onward.

DISCUSSION
- According to the results of the present study, the test material led to a dose-dependent increase in the number of his+ revertants with the strains TA 1535 and TA 100 with S-9 mix.

Any other information on results incl. tables

Table 1: Summary of Experiment 1

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 32 160 800 4000 8000	108 107 109 118 61 54	18 16 17 15 10 7	37 37 33 35 27 27	28 23 27 23 13 9	11 7 6 6 3 2
+	Solvent 32 160 800 4000 8000	110 124 150 242 40 19	17 26 63 182 39 20	39 38 35 32 36 37	40 37 43 33 25 14	12 10 11 8 2 2
Positive Controls
-	Name	MNNG	MNNG	4-NQO	NOPD	ACC
	Concentration (µg/plate)	5	5	5	10	100
	Mean no. colonies/plate	1036	962	701	717	384
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	2.5	2.5	60	2.5	2.5
	Mean no. colonies/plate	1103	151	262	911	166

MNNG = N-methyl-N’-nitro-N-nitrosoguanidine

4-NQO = 4 -nitroquinoline-1-oxide

ACC = 9-aminoacridine

2AA = 2-aminoanthracene

NOPD = 4-nitro-o-phenylendiamine

Table 2: Summary of Experiment 2

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		TA1535
-	Solvent 100 200 400 800 1000	18 22 20 19 20 15
+	Solvent 100 200 400 800 1000	18 44 61 127 212 247
-	Name	MNNG
	Concentration (µg/plate)	5.0
	Mean no. colonies/plate	1414
+	Name	2AA
	Concentration (µg/plate)	2.5
	Mean no. colonies/plate	124

MNNG = N-methyl-N’-nitro-N-nitrosoguanidine

2AA = 2-aminoanthracene

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study the test material was a mutagenic agent in the bacterial reverse mutation test.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471, EU Method B.13/B.14 and OPPTS 870.5100, under GLP conditions.

The test material was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

S. typhinurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA were treated at 32 - 8 000 µg/plate in a standard plate test both with and without metabolic activation (Aroclor-induced rat liver S-9 mix).

Precipitation of the test material and a bacteriotoxic effect was found from about 4 000 µg/plate onward.

No increase in the number of his+ or trp+ revertants was observed in any strain without S9 mix.

With S9 mix a positive reaction from about 100 µg/plate (factor 2.4) onward with an increase in the number of mutant colonies by a factor of 13.5 at 1 000 µg/plate was observed in TA 1535. In TA 100 with S9 mix, a slight increase in the number of revertant colonies at 800 µg/plate (factor 2.2) was observed. No increase in the number of his+ or trp+ revertants were seen in TA 1537, TA 98 or E.coli WP2 uvrA with S9 mix.

Under the conditions of this study the test material was a mutagenic agent in the bacterial reverse mutation test.