Octadecanoic acid, reaction products with diethylenetriamine and urea, acetates

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

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

Deviations:

no

Test guideline 4

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

Test material

Test material information

Constituent 1

Test material form:

other: waxy solid

Details on test material:

None

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.

Method

Target gene:

Histidine gene

Species / strain

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.

Controls

Controls 1

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

Controls 2

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)

Results and discussion

Test results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.