2-tetradecyloxirane, reaction products with boric acid

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:

2012-07-19 to 2012-09-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: OPPTS harmonised guidelines

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-tetradecyloxirane, reaction products with boric acid

EC Number:

701-392-2

IUPAC Name:

2-tetradecyloxirane, reaction products with boric acid

Test material form:

not specified

Details on test material:

- Physical state: Waxy off-white solid
- Analytical purity:100 % multiconstituent substance with 93 % main product
- Lot/batch No.: 0101033612
- Date of receipt: 02 July 2012
- Expiration date of the lot/batch: Not supplied
- Storage condition of test material: Room temperature in the dark

Method

Target gene:

Not required

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):

Non-mammalian study

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Non-mammalian study

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity test: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9 mix in TA100 and WP2 uvr A (plate incorporation method)

Mutation test (plate incorporation method)
Experiment 1 (Range-finding test):
- Salmonella strains TA100 and TA98 and E. coli strain WP2uvrA (with and without S9-mix): 50, 150, 500, 1500 and 5000 μg/plate.
- Salmonella strains TA1537 and TA1535 (with and without S9-mix): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
Experiment 2 (main test):
Salmonella strains TA100, TA1535 and TA1537 and E. coli strain WP2uvrA (with and without S9-mix): 50, 150, 500, 1500 and 5000 μg/plate.
Salmonella strain TA98 (with and without S9-mix): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tetrahydrofuran
- Justification for choice of solvent/vehicle: Test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL and acetone at 100 mg/mL but was fully soluble in tetrahydrofuran at 200 mg/mL in solubility test. Tetrahydrofuran was therefore selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene - 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537 and 10 µg/plate for WP2uvrA; Benzo(a)pyrene - 5 µg/plate for TA98

Remarks:

With metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N-nitro-N-nitrosoguanidine - 2 µg/plate for WP2uvrA, 3 µg/plate for TA100 and 5 µg/plate for TA1535; 9-Aminoacridine - 80 µg/plate for TA1537; 4-Nitroquinoline-1-oxide - 0.2 µg/plate for TA98

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) at multiple dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors).

The assay was performed by mixing 0.1 mL of bacterial culture (TAI00 or WP2uvrA-), 0.025 mL of the substance formulation, 0.5 ml of S9-mix or phosphate buffer and 2 mL of molten, trace histidine or tryptophan supplemented, top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). In addition, 0.025 mL of the maximum concentration of the test material and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 h incubation at 37 °C the plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

DURATION
- Preincubation period: N/A
- Exposure duration: Approximately 48 hours
- Expression time (cells in growth medium): N/A
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A

SELECTION AGENT (mutation assays): N/A
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: 3 replicates of each strain at each concentration both in the presence and absence of S9

NUMBER OF CELLS EVALUATED:
Cell viability at the end of pre-culture
RANGE FINDING TEST
S. typhimurium TA 100 = 2.4 x 10^9/mL
S. typhimurium TA1535 = 2.9 x 10^9/mL
S. typhimurium TA98 = 4.1 x 10^9/mL
S. typhimurium TA1537 = 3.7 x 10^9/mL
E. coli WP2uvrA- = 4.7 x 10^9/mL

MAIN TEST
S. typhimurium TA 100 = 1.2 x 10^9/mL
S. typhimurium TA1535 = 1.1 x 10^9/mL
S. typhimurium TA98 = 2.2 x 10^9/mL
S. typhimurium TA1537 = 1.2 x 10^9/mL
E. coli WP2uvrA- = 3.1 x 10^9/mL

DETERMINATION OF CYTOTOXICITY
- Method: N/A

OTHER EXAMINATIONS:
N/A

OTHER:
Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.

In order to select appropriate dose levels for use in the main test, a preliminary assay was carried out to determine the toxicity of the test material.

Evaluation criteria:

There are several criteria for determining a positive result. Any, following can be used to determine the overall result of the study:
- A dose-related increase in mutant frequency over the dose range tested (DeSerres and Shelby (1979)).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
- Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

- The substance would be considered non-mutagenic (negative) in the test system if the above criteria are not met.
- Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

None

Results and discussion

Test results

Test results 1

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 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
- Test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA).

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were comparable with historical solvent and positive control data (2010 and 2011) of laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Test item caused a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the frequency of revertant colonies of all of the Salmonella strains dosed in the absence of S9-mix and to TA98 and TA1537 dosed in the presence of S9-mix at the upper dose levels. The toxic response was generally noted at 5000 μg/plate and therefore, the test item was tested up to the maximum recommended dose level. A test item precipitate (white and particulate in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/1: Mean revertant frequencies

Strains	Doses (µg/plate)	Mean revertants per plate
		Range finding test				Main test
		-S9		+S9		-S9		+S9
		Mean	SD	Mean	SD	Mean	SD	Mean	SD
TA100	Solvent control (THF)	86	13.2	117	11.7	81	5.5	77	2.1
	5	NT		NT		NT		NT
	15	NT		NT		NT		NT
	50	87	12.5	100	20.7	79	5.1	76	14.0
	150	65	0.6	88	6.1	49	6.1	73	5.2
	500	67	4.7	100	17.3	56	5.9	68	6.2
	1500	74	8.0	113	11.1	37	3.8	65	3.0
	5000	45	6.6	75	2.5	36	3.0	69	2.1
	PC	487	17.3	726	39.0	556	32.1	919	113.2
TA1535	Solvent control (THF)	14	3.1	14	0.0	16	0.6	13	4.2
	5	17	6.1	14	2.5	NT		NT
	15	17	4.6	13	1.7	NT		NT
	50	16	4.5	17	5.0	15	1.7	11	1.0
	150	14	3.0	12	2.5	10	1.5	13	0.6
	500	16	0.6	14	5.8	10	2.1	8	0.6
	1500	14	2.0	11	3.5	8	1.5	8	0.6
	5000	6	2.0	9	1.5	5	3.0	8	0.0
	PC	229	29.1	202	15.3	414	38.0	186	4.5
WP2 uvrA	Solvent control (THF)	30	3.5	28	10.0	25	2.6	20	5.0
	5	NT		NT		NT		NT
	15	NT		NT		NT		NT
	50	31	6.7	36	4.7	19	0.6	21	5.6
	150	25	3.6	25	3.0	20	1.5	25	1.5
	500	23	4.0	26	5.6	22	9.3	24	2.0
	1500	31	6.0	34	4.4	25	3.6	19	5.8
	5000	24	1.2	31	2.5	15	4.9	21	7.0
	PC	615	21.7	275	10.6	636	25.2	302	28.4
TA 98	Solvent control (THF)	15	8.1	28	8.7	27	2.3	30	4.4
	5	NT		NT		26	1.0	23	5.2
	15	NT		NT		21	6.4	28	2.6
	50	15	3.1	20	1.7	26	1.5	32	7.2
	150	9	0.6	19	4.2	18	5.8	26	4.0
	500	10	2.9	20	5.5	15	3.6	25	1.2
	1500	8	2.9	21	1.5	15	2.5	28	2.1
	5000	5	2.1	13	2.1	12	0.0	20	5.5
	PC	138	6.4	228	7.4	166	7.0	210	14.6
TA1537	Solvent control (THF)	12	2.3	11	1.0	6	3.8	3	1.0
	5	7	4.0	6	1.7	NT		NT
	15	7	2.6	9	5.5	NT		NT
	50	8	1.2	8	0.6	7	7.8	3	1.7
	150	7	1.5	6	3.2	4	0.0	3	1.2
	500	4	2.0	7	2.1	5	1.2	4	0.6
	1500	3	1.7	5	1.2	4	3.8	3	0.6
	5000	1	1.2	3	1.7	0	0.0	0	0.0
	PC	563	33.9	218	30.0	1062	234.6	246	27.0

NT: Not tested; SD: Standard Deviation

Applicant's summary and conclusion

Conclusions:

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

The test substance was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Test Guidance

Reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP.

Method and materials

Strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and Escherichia coli WP2 uvr A were exposed to test material at the following concentrations:

Preliminary toxicity test: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9 mix in TA100 and WP2 uvr A (plate incorporation method)

Mutation test (plate incorporation method)

Experiment 1 (Range-finding test):

- Salmonella strains TA100 and TA98 and E. coli strain WP2uvrA (with and without S9-mix): 50, 150, 500, 1500 and 5000 μg/plate.

- Salmonella strains TA1537 and TA1535 (with and without S9-mix): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.

Experiment 2 (main test):

Salmonella strains TA100, TA1535 and TA1537 and E. coli strain WP2uvrA (with and without S9-mix): 50, 150, 500, 1500 and 5000 μg/plate.

Salmonella strain TA98 (with and without S9-mix): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.

Metabolic activation system used in this test was10 % S9 mix. S9 fraction was prepared from liver homogenates of rats induced with phenobarbitone/β-naphthoflavone. Vehicle control and positive control groups were also included in mutagenicity tests.

Results

The test item caused a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the frequency of revertant colonies of all of the Salmonella strains dosed in the absence of S9-mix and to TA98 and TA1537 dosed in the presence of S9-mix at the upper dose levels. The toxic response was generally noted at 5000 μg/plate and therefore, the test item was tested up to the maximum recommended dose level. A test item precipitate (white and particulate in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies. The positive and vehicle controls induced the appropriate responses in the corresponding strains. No significant increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations in either presence or absence of metabolic activation.

Conclusion

Under the test conditions, test material is not considered as mutagenic in this bacterial system.