2-tetradecyloxirane, reaction products with boric acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test

Under the test conditions, test material is considered as non-mutagenic in this bacterial system ( OECD 471, EU Method B.13/14, OPPTS harmonised guidelines and relevant Japanese guidelines).

 

Chromosome aberration test

The test material was considered to be non-clastogenic to human lymphocytes in vitro ( OECD 473, EU Method B.10 and relevant Japanese guidelines).

 

Mouse Lymphoma assay

The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test (OECD 476).

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

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

Target gene:

Not required

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 / 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.

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

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

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

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

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

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

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'.

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012/08/01 to 2013/02/19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Principles of method if other than guideline:

N/A

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

N/A

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

- Type and identity of media: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 15% foetal calf serum,
- Properly maintained: NDA
- Periodically checked for Mycoplasma contamination: NDA
- Periodically checked for karyotype stability: NDA
- Periodically "cleansed" against high spontaneous background: NDA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1
20, 40*, 80*, 160*, 320, 640 µg/ml without S9 mix
20, 40, 80*, 160*, 320*, 640 µg/ml with S9 mix
* = Dose levels selected for metaphase analysis

Experiment 2
20, 40, 80*, 120*, 160**, 240 µg/ml without S9 mix
40, 80, 120, 160*, 240*, 320* µg/ml with S9 mix
* = Dose levels selected for metaphase analysis

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle:The test substance was insoluble in water, DMSO and Acetone, but was soluble in THF at 250 mg/ml.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.2 and 0.4 ug/ml without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

5.0 ug/ml with S9

Details on test system and experimental conditions:

- Type and identity of media: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 15% foetal calf serum,
- Properly maintained: NDA
- Periodically checked for Mycoplasma contamination: NDA
- Periodically checked for karyotype stability: NDA
- Periodically "cleansed" against high spontaneous background: NDA

Experiment 1
20, 40*, 80*, 160*, 320, 640 µg/ml without S9 mix
20, 40, 80*, 160*, 320*, 640 µg/ml with S9 mix
* = Dose levels selected for metaphase analysis

Experiment 2
20, 40, 80*, 120*, 160**, 240 µg/ml without S9 mix
40, 80, 120, 160*, 240*, 320* µg/ml with S9 mix
* = Dose levels selected for metaphase analysis

METHOD OF APPLICATION: in medium;
With Metabolic Activtion
Cultures were established approximately 48 hours prior to treatment. Cultures were incubated at 37 °C for 4 hours in the presence of the test material prior to washing.

Without Metabolic Activation
Cultures were established approximately 48 hours prior to treatment. In Experiment 1, cultures were incubated at 37 °C for 4 hours in the presence of the test material prior to washing. In Experiment 2, the cultures were incubated in the presence of the substance at 37 °C for 24 hours

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 24 hours
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): Mitosis was arrested by addition of democolcine two hours prior to the required harvest time and the cells were harvested and fixed

SELECTION AGENT (mutation assays): N/A
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): 5% Gurrs Giemsa

NUMBER OF REPLICATIONS: Treatments performed in duplicate.

NUMBER OF CELLS EVALUATED: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Where possible the first 100 consecutive well-spread metaphases from each culture were counted.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes in comparison to controls
- Determination of endoreplication: If the chromosomes are arranged in closely apposed pairs, ie. 4 chromatids instead of 2, the cell is scored as endoreduplicated

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 4.88, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 µg/ml.

The maximum dose was based on precipitate in the 4 hour exposure groups and cytoxicity and precipitate in the 24 hour exposure group.

* Precipitate was observed at and above 78.13 and 156.25 µg/ml in the 4-hour exposure group in the presence and absence of metabolic activation respectively and at and above 39.06 ug/ml the continuous exposure group.

* Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present at all dose levels except 625 and 1250 ug/ml in the contninuous exposure group.

The selection of the maximum dose level was based on precipitate for the 4 hour exposure groups and on the basis of cytotoxicity for the 24 hour exposure group.

Experiment 1
20, 40*, 80*, 160*, 320, 640 µg/ml without S9 mix
20, 40, 80*, 160*, 320*, 640 µg/ml with S9 mix
* = Dose levels selected for metaphase analysis

Experiment 2
20, 40, 80*, 120*, 160**, 240 µg/ml without S9 mix
40, 80, 120, 160*, 240*, 320* µg/ml with S9 mix
* = Dose levels selected for metaphase analysis

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

A small but statistically significant increase in the frequency of aberrant cells was observed in Experiment 1, in the presence of S9 only, at the two highest concentrations analysed. The responses observed were fairly even, not dose-related and not reproducible in a repeat experiment. They were also accompanied by large increases in the mitotic indeces, which is indicative of toxicity. The majority of the aberrations observed in Experiment 1 were break-type aberrations and not chromatid exchanges. It was therefore concluded that the clastogenic activity was a consequence of a toxicity driven mechanism giving some doubt to its biological relevance and, therefore, was considered to be of no toxicolgical significance.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

See attached background material.

Conclusions:

Interpretation of results (migrated information):
negative

The substance was considered to be was considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction. This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1997) No. 473"Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008 and the US EPA OPPTS 870.5375 Guideline and is acceptable to the Japanese New Chemical Substance Law (METI).

 

Methods.Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4-hour exposure with addition of S9 was repeated with 2% S9, whilst in the absence of metabolic activation, the exposure time was increased to 24 hours.

The dose levels used in the main experiments were selected using data from thepreliminary toxicity test and were as follows:

Group	Final concentration of Test Item (ug/ml)
4(20)-hour without S9	20,	40,	80,	160,	320,	640
4(20)-hour with S9	20,	40,	80,	160,	320,	640
24-hour without S9	20,	40,	80,	120,	160,	240
4(20)-hour with S9	40,	80	120,	160,	240,	320

 

Results. All vehicle (tetrahydrofuran) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

In Experiment 1, the test item induced small but statistically significant increases in the frequency of cells with aberrations in the presence of metabolic activation, using a dose range that generally included a precipitating dose level. However, in the absence of S9,the test item did not induce any statistically significant increases in the frequency of cells with aberrations. In Experiment 2,

the test item did not induce any statistically significant increases in the frequency of cells with aberrations using a dose range that included a precipitating dose level.

Discussion. A small but statistically significant increase in the frequency of aberrant cells was observed in the first experiment, in the presence of S9 only, at the two greatestconcentrations analysed. The responses observed were fairly even, not dose-relatedand not reproducible in a repeat experiment. They were also accompanied by large increases in the Mitotic Indices which is indicative of toxicity. The majority of the aberrations observed in Experiment 1 were break type aberrations. It was therefore concluded that the clastogenic activity was a consequence of a toxicity driven mechanism giving some doubt to its biological relevance and, therefore, considered to be of no toxicological significance.

Conclusion. The test item was considered to be non-clastogenic to human lymphocytes in vitro.

Reference 3

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:

2012/08/08 to 2012/12/18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

The thymidine kinase, TK +1-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 ug/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 ug/ml) and 10% donor horse serum (giving R10 media) at 37 oC with 5% CO2 in air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 was prepared in-house from the livers of male Wistar Han™ rats weighing -200g. These had each received, orally, three consecutive daily doses of phenobarbitall~-naphthoflavone(80/100 mg per kg per day) prior to S9 preparation on the fourth day.

Test concentrations with justification for top dose:

Experiment 1 (ug/ml) without S9: 39.06, 78.13, 156.25, 312.5, 468.75, 625
Experiment 1 (ug/ml) with S9: 39.06, 78.13, 156.25, 312.5, 468.75, 625
Experiment 2 (ug/ml) without S9: 1.25, 2.5, 5, 10, 20, 40
Experiment 2 (ug/ml) with S9:5, 10, 20, 40, 60, 80

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:Tetrohydrofuran (THF)
- Justification for choice of solvent/vehicle: Based on solubility checks done in parallel chromosome aberration study Project No. 41203363

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

400 ug/ml in Ex. 1, 150 ug/ml in Ex. 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

2 ug/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 ~g/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 ~g/ml) and 10% donor horse serum (giving R10 media). Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10^6 cells/ml in 10 ml aliquots in R10 medium in sterile plastic universals. The cells were exposed to doses of the test material, vehicle and positive control, both with and without metabolic activation. Cultures were maintained at 37 °C in a humidified atmosphere of 5 % CO2 in air.
The treatment regimes were as follows:

EXPERIMENT 1:
a). Without metabolic activation: 4-hour exposure, dose levels 39.06, 78.13, 156.25, 312.5, 468.75, 625 ug/ml
b). With metabolic activation (2% S9): 4-hour exposure groups, dose levels 39.06, 78.13, 156.25, 312.5, 468.75, 625 ug/ml

EXPERIMENT 2:
a). Without metabolic activation: 24-hour exposure, dose levels 1.25, 2.5, 5, 10, 20, 40 ug/ml
b). With metabolic activation (1% S9):4-hour exposure, dose levels 5, 10, 20, 40, 60, 80 ug/ml

DURATION
- Preincubation period: Not applicable.
- Exposure duration: 4 h (Experiment 1 and 2), or 4 and 24 h (Experiment 2).
- Expression time (cells in growth medium): 2 days (viability test)
- Selection time (if incubation with a selection agent): 10~14 days (plate scoring for colony formation)
- Fixation time (start of exposure up to fixation or harvest of cells): ~ 2 h

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable.
STAIN (for mutant colony visualisation): MTT vital stain

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: seeded 2000 cells/well for mutant frequency; 2 cells/well for viability.

DETERMINATION OF CYTOTOXICITY
- Method: other: Suspension Growth values (SG)

OTHER EXAMINATIONS:
- Determination of polyploidy: No.
- Determination of endoreplication: No.
- Other: Evaluated for small and large mutant colony size

OTHER: Calculation of Day 2 Viability (%V), Calculation of Relative Total Growth (RTG), Calculation of Mutation Frequency (MF) were performed, and the experimental data was analyzed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS.

Evaluation criteria:

The normal range for mutant frequency per survivor is 50-170 x 10^-6 for the TK+/- locus in L5178Y cells at this laboratory. Vehicle controls results should be within this range, experiments where the vehicle control values are markedly > 200 x 10^-6 mutant frequency per survivor will be repeated. Positive control chemicals should induce at least 3~5 fold increases in mutant frequency greater than the corresponding vehicle control.
The optimum toxicity is approximately 20% survival, but no less than 10% surviva. Both %RSG and RTG values are used either individually or combined to designate the level of toxicity achieved by the test material for any individual dose level. Dose levels that have survival values less than 10% are excluded from any statistical analysis.
For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. The Global Evaluation Factor (GEF) value was set at 126 x 10^-6 for the microwell method. Therefore any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10^-6 will be considered positive. However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgment will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.

Statistics:

The experimental data was analyzed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS statistical package. Dose levels that have survival values less than 10% are excluded from any statistical analysis, as any response they give would be considered to have no biological or toxicological relevance.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Precipitate also observed

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: mouse lymphoma L5178Y cells

Remarks:

Migrated from field 'Test system'.

See attached results summary table.

Conclusions:

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

Introduction. The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.

 

Methods. Two independent experiments were performed. In Experiment 1, L5178Y TK /- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, induplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9 final concentration). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9 final concentration) and a 24-hour exposure group in the absence of metabolic activation.

 

The dose range of test item was selected following the results of a preliminary toxicity test and was 39.06 to 625 µg/ml in both the absence and presence of metabolic activation for Experiment 1. In Experiment 2 the dose range was 1.25 to 80 µg/ml in both the absence and presence of metabolic activation.

 

Results. The maximum dose levels used initially in the Mutagenicity Test was the maximum achievable dose level of 625 µg/ml. However, due to the toxicity observed in Experiment 1, the maximum dose level in Experiment 2 was limited by a combination of toxicity and the presence of precipitate effectively reducing exposure of the test item to the cells. Overall, precipitate of test item was observed at and 20 µg/ml in the Mutagenicity Test. The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test item did not induce any reproducible toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

 

Conclusion. The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Negative results were obtained during investigation of in vitro gene mutation in bacteria (Ames test), in vitro cytogenicity in mammalian cells (chromosome aberration study) and in vitro gene mutation in mammalian cells (mouse lymphoma assay). As a result, and in accordance with ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance (Version 6.0; July 2017), the substance is not considered to be genotoxic and no further testing is required.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test

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).

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 was 10 % 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.

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. Under the test conditions, test material is considered as non-mutagenic in this bacterial system.

Chromosome aberration

The method used in this key study was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1997) No. 473"Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008 and the US EPA OPPTS 870.5375 Guideline and is acceptable to the Japanese New Chemical Substance Law (METI).

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used in the study, i.e. in Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 2% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material was either toxic or tested to the limit of precipitation and did not induce any toxicologically significant increases in the frequency of cells with aberrations, in either of two seperate experiments. The test material was considered to be non-clastogenic to human lymphocytes in vitro.

Mouse Lymphoma Assay

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and to be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4‑hour exposure group in the presence of metabolic activation (1% S9) and a 24‑hour exposure group in the absence of metabolic activation.

The dose range of test item was selected following the results of a preliminary toxicity test, and was 39.06 to 625 µg/ml in the four hour exposure both in the absence of metabolic activation, and in the presence of metabolic activation. The dose range was 1.25 to 80 µg/ml in the absence of metabolic activation for the 24 hour exposure group.

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. Precipitate of test item was also observed at several dose levels in the Mutagenicity Test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment. The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Justification for classification or non-classification

Three in vitro assays, an Ames test, a chromosome aberration test and a mammalian cell assay all showed negative results both in the presence and absence of a metabolising activation system (S9). Therefore classification for mutagenicity is not required.