Octadecanoic acid, reaction products with diethylenetriamine and urea, acetates

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT 93580/A was found to be non-mutagenic in Ames test and in mouse lymphoma cells and not clastogenic in the Chromosome aberration test in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identification: FAT 93580/A
Batch: 0022357000
Purity: 94.3 %
Description: Extremely pale yellow waxy solid
Expiry Date: 15 December 2015
Storage Conditions: Room temperature in the dark.

Target gene:

Histidine gene

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally, due to the "deep rough" (rfa) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB-bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TAlOO, the R­ factor plasmid pKMlOl enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Pre-Incubation Method: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofuran
- Justification for choice of solvent/vehicle: The 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 checks performed in-house. Tetrahydrofuran was therefore selected as the vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2- Aminoanthracene, Benzo(a)pyrene

Remarks:

with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) - Experiment 1

NUMBER OF REPLICATIONS: 3

For each strain and dose level, including the controls, a minimum of three plates were used.
Experiment 1: The following materials were mixed in a test tube and poured onto the selective agar plates:
- 0.025 mL of test item or vehicle or 0.1 mL of positive control.
- 2 mL of trace amino-acid supplemented media (at approximately 45 °C) containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer.
- 0.5 mL of S9-mix (for experiments with metabolic activation).


METHOD OF APPLICATION: preincubation - Experiment 2

NUMBER OF REPLICATIONS: 3

For each strain and dose level, including the controls, a minimum of three plates were used.
- 0.1 mL of the appropriate bacterial strain culture
- 0.5 mL of phosphate buffer and 0.025 mL of the test item formulation or solvent or 0.1 mL of appropriate positive control
- 2 mL of amino-acid supplemented media
- 0.5 mL of S9-mix (for experiments with metabolic activation).

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

Evaluation criteria:

1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will 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 making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Acceptance Criteria:
All tester strain cultures should be in the range of 0.9 to 9x10^9 bacteria per mL.
There should be a minimum of four non-toxic test item dose levels.

Statistics:

Statistical analysis of the data as determined by UKEMS (Mahon et al. 1989)

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:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

at and above 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate (particulate in appearance) was observed above 1500 µg/plate (under an inverted microscope at 500 µg/plate in Experiment 2), this observation did not prevent the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. Small, statistically significant increases in TA98 revertant colony frequency were observed in Experiment 2 at 50, 150, 500, 1500 µg/plate (absence of S9-mix) and 1500 µg/plate (presence of S9-mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.6 times the concurrent vehicle controls.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table 1 Experiment 1 without metabolic activation

Test period		from 2013-04-04 (2013-04-11/16)				to 2013-04-07 (2013-04-14/19)
S9-Mix (-)	Dose level per plate (µg)	Number of revertants
		Base-pair substitutions strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
		mean	SD	mean	SD	mean	SD	mean	SD	mean	SD
	solvent control	93	17	18	3	32	10	25	9	10	2
	1.5	87	21	14	4	38	6	19	7	9	6
	5	81	7	12	2	32	7	21	6	11	6
	15	99	4	17	1	34	5	20	7	11	4
	50	81	17	18	2	32	6	20	8	5	2
	150	79	11	16	5	37	2	21	5	10	5
	500	76	9	20	6	37	0	18	2	15	6
	1500	101	2	21	4	34	7	23	6	11	5
	5000	73	11	19	4	31	7	19	6	12	2
	Positive control	ENNG		ENNG		ENNG		4NQO		9AA
	Dose level (µg)	3		5		2		0.2		80
	No. revertants	364	36	977	339	567	75	166	28	377	48

Table 2 Experiment 1 with metabolic activation

Test period		from 2013-04-04 (2013-04-11)				to 2013-04-07 (2013-04-14)
S9-Mix (+)	Dose level per plate (µg)	Number of revertants
		Base-pair substitutions strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
		mean	SD	mean	SD	mean	SD	mean	SD	mean	SD
	solvent control	105	15	16	3	33	6	29	8	15	1
	1.5	105	20	12	3	38	2	28	4	11	5
	5	99	14	12	5	35	5	21	5	15	4
	15	101	9	10	2	37	3	19	5	15	3
	50	97	10	15	2	31	12	32	2	14	6
	150	96	9	12	4	35	11	18	1	10	3
	500	95	3	15	2	31	6	25	5	13	4
	1500	97	2	13	4	31	6	24	8	11	2
	5000	102	7	14	3	38	4	21	4	16	4
	Positive control	2AA		2AA		2AA		BP		2AA
	Dose level (µg)	1		2		10		5		2
	No. revertants	644	110	178	10	306	27	299	3	231	25

Table 3 Experiment 2 without metabolic activation

Test period		from 2013-04-23				to 2013-04-26
S9-Mix (-)	Dose level per plate (µg)	Number of revertants
		Base-pair substitutions strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
		mean	SD	mean	SD	mean	SD	mean	SD	mean	SD
	solvent control	105	22	14	6	38	6	14	3	16	6
	50	111	18	18	3	34	10	23 **	2	16	8
	150	101	5	21	5	44	3	21 *	3	21	7
	500	109	8	18	8	34	6	21 *	4	14	6
	1500	106	13	17	2	31	5	23 **	2	13	5
	5000	80	8	11	2	30	4	20	3	13	8
	Positive control	ENNG		ENNG		ENNG		4NQO		9AA
	Dose level (µg)	3		5		2		0.2		80
	No. revertants	973	11	1298	37	910	59	232	6	983	226

* p<0.05; ** p<0.01

Table 4 Experiment 2 with metabolic activation

Test period		from 2013-04-23				to 2013-04-26
S9-Mix (+)	Dose level per plate (µg)	Number of revertants
		Base-pair substitutions strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
		mean	SD	mean	SD	mean	SD	mean	SD	mean	SD
	solvent control	88	9	9	2	39	9	20	3	18	6
	50	90	2	12	3	44	4	23	2	17	3
	150	88	8	12	4	44	15	25	3	14	2
	500	90	4	11	4	47	7	28	6	14	2
	1500	87	1	14	8	34	1	30 *	3	13	2
	5000	75	6	11	3	28	6	23	6	8	1
	Positive control	2AA		2AA		2AA		BP		2AA
	Dose level (µg)	1		2		10		5		2
	No. revertants	1166	34	267	7	207	30	158	6	261	51

* p<0.05

Conclusions:

FAT#: 93580/A was considered to be non-mutagenic under the conditions of this test.

Executive summary:

FAT 93580/A was tested according to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 44012008 of 30 May 2008 and the USA, EPA (TSCA) OCSPP harmonized guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and preincubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10 % liver S9 in standard co-factors).

The dose range for Experiment 1 was pre-determined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 50 to 5000 µg/plate.

The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate (particulate in appearance) was observed by eye at and above 1500 µg/plate (under an inverted microscope at 500 µg/plate in Experiment 2), this observation did not prevent the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. Small, statistically significant increases in TA98 revertant colony frequency were observed in Experiment 2 at 50, 150, 500, 1500 µg/plate (absence of S9-mix) and 1500 µg/plate (presence of S9-mix).

These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.6 times the concurrent vehicle controls. FAT #: 93580/A was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

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:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Identification: FAT 93580/A
Batch: 0022357000
Purity: 94.3 %
Description: Extremely pale yellow waxy solid
Expiry Date: 15 December 2015
Storage Conditions: Room temperature in the dark.

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air.

For lymphocytes
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The mean value of the AGT for the pool of regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment 1:
4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 50, 100, 200, 250, 300 and 400 μg/mL.

4-hour exposure to the test item with S9-mix (2 %), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 50, 100, 200, 250, 300 and 400 μg/mL.

Experiment 2:
24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 12.5, 25, 50, 100, 200 and 300 μg/mL.

4-hour exposure to the test item with S9-mix (1 %) followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 12.5, 25, 50, 100, 200 and 300 μg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: test item was soluble in tetrahydrofuran (THF) at 322.13 mg/mL. Due to the toxic effects of THF on the cells and the necessity of dosing with it at only 0.25 %, the maximum concentration that could be achieved was 805.32 μg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4-h (-S9 mix), 4-h (+S9 mix, 2 %); 24-h (-S9 mix), 4-h (+S9-mix)
- Expression time (cells in growth medium): 20 h
- Selection time (if incubation with a selection agent): no data
- Fixation time (start of exposure up to fixation or harvest of cells): 22 h

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (Colcemid 0.1 μg/mL)

STAIN (for cytogenetic assays): 5 % Giemsa for 5 min

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 50-100 cells

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

Negative control (vehicle)
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures will normally be within the laboratory historical control data range.

Positive control values
All the positive control chemicals must induce positive responses (p≤0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix.

A test item can be classified as non-genotoxic if:
1. The number of induced chromosome aberrations in all evaluated dose groups is within the range of our historical control data.
2. No toxicologically or statistically significant increase of the number of structural chromosome aberrations is observed following statistical analysis.

A test item can be classified as genotoxic if:
1. The number of induced structural chromosome aberrations is not in the range of our historical control data.
and
2. Either a concentration-related or a statistically significant increase of the number of structural chromosome aberrations is observed. Marked increases only observed in one dose level will be assessed on a case by case basis.

Biological relevance of the results will be considered first. Statistical methods will be used to analyze the increases in aberration data as recommended in the OECD 473 guidelines. However, statistical significance will not be the only determining factor for a positive response.
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

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: Human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: Human

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

25 % mitotic inhibition was achieved at 200 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: 200 μg/mL (Experiment 1), 100 μg/mL (Experiment 2)

RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 3.15, 6.29, 12.58, 25.17, 50.33, 100.66, 201.33, 402.66 and 805.22 μg/mL. The maximum dose was the maximum achievable dose level using THF as a solvent. A cloudy precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure period, at and above 25.17 μg/mL, in all three exposure groups. Precipitate was also observed at and above 201.33 μg/mL in the 4(20)-hour exposure group in the absence of S9 and in the 24-hour exposure group. In the 4 (20)-hour exposure group in the presence of S9 precipitate was seen at and above 100.66 μg/mL which became aggregated at 805.32 μg/mL. The precipitate was carried through onto the slides and was seen at and above 100.66 μg/mL in the 4(20)-hour exposure group in the presence of S9 and at and above 201.33 μg/mL in the 4(20)-hour exposure group in the absence of S9 and in the 24-hour exposure group.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 805.32 μg/mL in all three exposure groups. However, because of heavy precipitate on the slides it was considered that the dose levels of 402.66 μg/mL and 805.32 μg/mL could not be accurately scored. Therefore, the maximum dose level selected for mitotic index analysis was 201.33 μg/mL for all three exposure groups. The test item induced some evidence of toxicity in all three of the exposure groups, although the presence of precipitate on some of the slides may have affected the accuracy of the scoring. The maximum dose level selected for testing was limited by the presence of precipitate on the slides, affecting the ability to accurately score for mitotic index and chromosome aberrations and was 400 μg/mL for the 4(20)-hour exposure groups of Experiment 1 and was 300 μg/mL for both the exposure groups used in Experiment 2.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Remarks on result:

other: other: Experiments 1 (4-h exposure, ± S9 mix) and 2 (24-h exposure, - S9 mix)

Conclusions:

FAT #: 93580/A did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in absence or presence of a liver enzyme metabolizing system, in either of two separate experiments. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

FAT 93580/A was evaluated according to the GLP and procedures indicated by the following internationally accepted guidelines and recommendations:

* OECD Guidelines for Testing of Chemicals No. 473 "In Vitro Mammalian Chromosome Aberration Test"

* Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

* US EPA OPPTS 870.5375 Guideline.

* 40 CFR 799.9537 TSCA in vitro mammalian chromosome aberration test.

* Japanese Ministry of Economy, Trade and Industry (METI), Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. in

Experiment 1: 4 hours in the presence of an induced rat liver homogenate metabolizing 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: 4 hours exposure with addition of S9 was repeated (using a 1 % final S9 concentration), 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 the preliminary toxicity test.

All vehicle (THF) controls 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. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that was limited by precipitate occurring on the slides and affecting the ability to score accurately.

The test item did exhibit some toxicity but this was at dose levels where metaphase analysis was not possible due to the presence of precipitate on the slides.

FAT 93580/A did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system, in either of two separate experiments. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

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

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Sponsor's identification: FAT 93580/A
Purity: 94.3 %
Batch number: 0022357000
CAS number: 84962-05-0
Appearance: White-yellowish waxy solid
Date received: 23 January 2013
Expiration date: 15 December 2015
Storage conditions: Room temperature, in the dark

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

For cell lines:
- Absence of Mycoplasma contamination:Yes
- Methods for maintenance in cell culture: liquid nitrogen at approximately -196 °C
- Type and identity of media: RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 μg/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/ml) and 10 % donor horse serum (giving R10 media) at 37 °C with 5 % CO 2 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 mix

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, 200, 400 μg/mL
Experiment 1: 0, 9.38, 18.75, 37.5, 75, 150, 300, 450, 600 μg/mL
Experiment 2: 0, 25, 50, 100, 200, 300, 400, 500, 600 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: test item was soluble in tetrahydrofuran (THF) at 322.13 mg/mL. Due to the toxic effects of THF on the cells and the necessity of dosing with it at only 0.25 %, the maximum concentration that could be achieved was 805.32 μg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

-S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

+S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (±S9 mix) and 24 h (-S9 mix only) in preliminary toxicity test, 4 h (±S9 mix) in experiment 1, 24 h (-S9 mix) and 4 h (+S9 mix) in experiment 2
- Expression time (cells in growth medium): 2 days

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)

STAIN (for cytogenetic assays): thiazolyl blue tetrazolium bromide

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : duplicate
- Number of independent experiments : 2


NUMBER OF CELLS EVALUATED: 10E6 viable cells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

Evaluation criteria:

The normal range for mutant frequency per survivor is 50-170 x 10E-6 for the TK+/- locus in L5178Y cells at this laboratory. Vehicle controls results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 200 x 10E-6 mutant frequency per survivor are not normally acceptable and will be repeated.
Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.
Optimum toxicity is approximately 20 % survival (80 % toxicity), but no less than 10 % survival (90 % toxicity). RTG values are usually the primary factor to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, % RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values markedly less than 10 % are excluded from any statistical analysis, as any response they give would be considered to have no biological or toxicological relevance.
For a test item 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. Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 (Moore et al. 2003) it was felt that the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al. 2006) at 126 x 10^-6 for the microwell method.

Statistics:

The experimental data was analyzed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al, 1989).

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

ambiguous

Remarks:

at and above 450 μg/mL (only marginally exceeded mutant frequency values)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 450 μg/mL RTG 0.4 and less

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 600 μg/mL RTG 0.27

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 500 μg/mL RTG 0.25

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 500 μg/mL RTG 0.32 and less

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: at and above 18.75 μg/mL (Experiment 1), at and above 25 μg/mL (Experiment 2)

RANGE-FINDING/SCREENING STUDIES: The dose range of the test item used in the preliminary toxicity test was 1.56 to 400 µg/ml. Modest dose-related reductions in %RSG were observed in the 4-hour exposure groups, in both the absence and presence of metabolic activation, when compared to the concurrent vehicle control group. However, more marked dose-related reductions in %RSG were observed in the 24-hour exposure group in the absence of metabolic activation. Overall, precipitate of the test item was observed at and above 12.5 µg/ml. Based on the %RSG values observed, the maximum dose level in the subsequent Mutagenicity Test was increased to the maximum achievable dose level of 800 µg/ml to ensure that the test item had been adequately tested. However, this dose level proved unsuitable for use, the maximum achievable dose level was therefore reduced to 600 µg/ml.

Maximum dose levels were selected using the following criteria:
1) Maximum recommended dose level, 5000 μg/ml or 10 mM.
2) The presence of excessive precipitate where no test item-induced toxicity was observed.
3) Test item-induced toxicity, where the maximum dose level used should produce 10 to 20 % survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA
(Moore et al. 2002).

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Remarks on result:

other: other: Experiment 1

Remarks:

Migrated from field 'Test system'.

Preliminary Toxicity Test

Dose (µg/ml)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
1.56	99	97	99
3.13	100	98	96
6.25	100	101	85
12.5	80	86	87
25	103	94	107
50	105	103	94
100	102	95	80
200	80	88	78
400	51	51	23

Conclusions:

FAT 93580/A 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:

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.

Two independent experiments were performed.

In Experiment 1, L5178Y TK +/- 3.7.2 c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item FAT #: 93580/A 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 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 for Experiment 1 was 9.38 to 600 μg/ml in both the absence and presence of metabolic activation. In Experiment 2 the dose range was 25 to 600 μg/ml in both the absence and presence of metabolic activation.

The maximum dose level used in the Mutagenicity Test was limited by test item formulation difficulties. Overall, precipitate of the test item was observed at and above 18.75 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.

FAT 93580/A 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

no data available

Additional information

An in vitro chromosome aberration test in human lymphocytes was performed in 2013 according OECD TG 476 to assess the potential mutagenicity of the test item FAT 93580 on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. In this study 2 independent tests were performed. The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or second experiment. The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

An in vitro mouse lymphoma assay was performed in 2013 according to OECD TG 473 for the detection of structural chromosomal aberrations in cultured mammalian cells. Duplicate cultures of human lymphocytes, treated with the test item FAT 93580, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study. The test item did exhibit some toxicity but this was at dose levels where metaphase analysis was not possible due to the presence of precipitate on the slides. The test item, FAT 93580/A was considered to be non-clastogenic to human lymphocytes in vitro.

The third key study "Bacterial Reverse Mutation Test" was performed according to OECD TG 471, EU Method B13/14 and USA, EPA (TSCA) OCSPP and in accordance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and preincubation methods up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10 % liver S9 in standard co-factors). No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. FAT 93580/A was considered to be non-mutagenic under the conditions of this test.

Results of the three key studies indicate that the test item is not classified as genotoxic.

Justification for selection of genetic toxicity endpoint: The Key studies for determining Chromosome aberration test Human Lymphocytes, Bacterial Reverse Mutation Test and chromosomal aberrations in cultured mammalian cells were conducted in accordance to OECD TG 476, 471 and 473 respectively following the principles of GLP.

Short description of key information: Results of the three key studies performed in accordance to OECD TG 476, 471 and 473 indicate that the test item FAT 93580/A is Non-classified as gene toxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Test item FAT 93580 did not show any genetic toxic effects in in vitro tests conducted to determine its potency to cause gene toxicity. Hence, test substance is Non-classified as gene toxic.