Fatty acids, C16-18 (even numbered), reaction products with tetraethylenepentamine, acetates (salts)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 21, 2013 - March 04, 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:

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

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C16-18 (even numbered), reaction products with tetraethylenepentamine, acetates (salts)

EC Number:

939-688-0

Molecular formula:

Not applicable

IUPAC Name:

Fatty acids, C16-18 (even numbered), reaction products with tetraethylenepentamine, acetates (salts)

Test material form:

other: solid

Details on test material:

- Name of test material: Fatty acids, C16-18, reaction products with tetraethylenepentamine, acetates (salts)

Specific details on test material used for the study:

- Name of test material: Fatty acids, C16-18, reaction products with tetraethylenepentamine, acetates (salts)

Method

Target gene:

Thymidine Kinase Locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment I:
without S9 mix: 6.9; 13.8; 27.5; 55.0; 110.0 (p); 165.0 (p) µg/mL
with S9 mix: 6.9; 13.8; 27.5; 55.0; 110.0 (p); 165.0 (p) µg/mL
Experiment II:
without S9 mix: 0.9; 1.7; 3.4; 6.9; 13.8; 27.5; 55.0; 110.0 µg/mL
with S9 mix: 6.9; 13.8; 27.5; 55.0; 82.5; 110.0 µg/mL
(p) = precipitation visible to the unaided eye

Vehicle / solvent:

deionised water

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1E07 (3E06 during 24 h exposure) cells/flask
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration: 4 h / 24 h

FOR GENE MUTATION:
- Expression time: 48 h
- Selection time: 10 - 15 days
- Method used: microwell plates
- selective agent: trifluorothymidine, 5 μg/mL
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 4E03 cells in selective medium
- Criteria for small (slow growing) and large (fast growing) colonies: Criteria to determine colony size were the absolute size of the colony (more than 1/3 of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the optical density of the large ones).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- viability (cloning efficiency), relative total growth (RTG)

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 1E06 cells above the corresponding solvent control.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
A test item is considered equivocal in this assay if the threshold is reproducibly exceeded but the increase of the mutation frequency is not dose dependent.
However, in the evaluation of the test results the historical variability of the mutation rates in the solvent controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth is less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
A test item is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 1E06 cells above the corresponding solvent control.
A test item not meeting the conditions for a classification as mutagenic or non-mutagenic will be considered equivocal in this assay and may be considered for further investigation.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Summary Table

			relative	mutant		relative	mutant
	conc. µg	S9	total	colonies/		total	colonies/
	per mL	mix	growth	10^6cells	threshold	growth	10^6cells	threshold
Column	1	2	3	4	5	6	7	8
Experiment I / 4 h treatment			culture I			culture II
Solv. control with water		-	100.0	40	166	100.0	53	179
Pos. control with MMS	19.5	-	28.5	240	166	27.9	305	179
Test item	6.9	-	112.8	24	166	69.0	24	179
Test item	13.8	-	65.1	26	166	38.1	94	179
Test item	27.5	-	36.5	21	166	22.6	43	179
Test item	55.0	-	4.2	28	166	1.9	55	179
Test item	110.0 (P)	-	culture was not continued^#			culture was not continued^#
Test item	165.0 (P)	-	culture was not continued^#			culture was not continued^#
Experiment I / 4 h treatment			culture I			culture II
Solv. control with water		+	100.0	69	195	100.0	95	221
Pos. control with CPA	3.0	+	31.3	331	195	65.9	207	221
Pos. control with CPA	4.5	+	33.3	475	195	59.6	279	221
Test item	6.9	+	90.4	81	195	84.8	58	221
Test item	13.8	+	72.7	83	195	114.7	66	221
Test item	27.5	+	69.0	90	195	123.4	42	221
Test item	55.0	+	99.6	34	195	106.8	56	221
Test item	110.0 (P)	+	20.9	44	195	11.3	43	221
Test item	165.0 (P)	+	culture was not continued^#			culture was not continued^#
Experiment II / 24 h treatment			culture I			culture II
Solv. control with water		-	100.0	72	198	100.0	91	217
Pos. control with MMS	13.0	-	9.1	491	198	12.9	571	217
Test item	0.9	-	culture was not continued^##			culture was not continued^##
Test item	1.7	-	culture was not continued^##			culture was not continued^##
Test item	3.4	-	71.1	39	198	92.8	32	217
Test item	6.9	-	62.6	54	198	49.8	130	217
Test item	13.8	-	40.7	68	198	53.8	60	217
Test item	27.5	-	21.2	43	198	20.0	58	217
Test item	55.0	-	0.0	72	198	0.0	39	217
Test item	110.0 (P)	-	culture was not continued^#			culture was not continued^#
Experiment II / 4 h treatment			culture I			culture II
Solv. control with water		+	100.0	45	171	100.0	53	179
Pos. control with CPA	3.0	+	52.7	163	171	61.2	154	179
Pos. control with CPA	4.5	+	45.9	239	171	31.6	315	179
Test item	6.9	+	culture was not continued^##			culture was not continued^##
Test item	13.8	+	155.1	44	171	157.4	48	179
Test item	27.5	+	98.2	50	171	134.2	24	179
Test item	55.0	+	70.4	68	171	99.5	33	179
Test item	82.5 (P)	+	43.0	44	171	42.7	37	179
Test item	110.0 (P)	+	19.5	50	171	18.2	31	179

Threshold = number of mutant colonies per 1E06 cells of each solvent control plus 126

(p)  precipitation visible to the unaided eye

#    culture was not continued due to exceedingly severe cytotoxic effects

##   culture was not continued as a minimum of four concentrations is required by the guidelines

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

The test substance C16-18FA-TEPA-compound was concluded to be not mutagenic with and without metabolic activation in the L5178Y/TK Mouse Lymphoma Mutagenesis Assay when tested up to cytotoxic concentrations.

Executive summary:

In a mammalian cell gene mutation assay thymidine kinase locus according to OECD guideline 476, L5178Y mouse lymphoma cells cultured in vitro were exposed to C16-18FA-TEPA-compound at the following concentrations in the presence and absence of mammalian metabolic activation (S9- mix):

Experiment I:

without S9 mix: 6.9; 13.8; 27.5; and 55.0 µg/mL
with S9 mix: 6.9; 13.8; 27.5; 55.0; and 110.0 µg/mL

Experiment II:

without S9 mix: 3.4; 6.9; 13.8; 27.5; and 55.0 µg/mL
with S9 mix: 13.8; 27.5; 55.0; 82.5; and 110.0 µg/mL

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed in the absence of metabolic activation with a treatment period of 24 hours and in the presence of metabolic activation with a treatment time of 4 hours.The experimental part of the second experiment without metabolic activation was terminated prior to the generation of any data on mutagenicity as strong cytotoxic effects completely inhibited cell growth down to low concentrations. This experimental part was repeated in a lower concentration range. The data are reported as experiment II without metabolic activation.

The test medium was checked for precipitation visible to the naked eye at the end of the 4 hours treatment just before the test item was removed. Precipitation was observed at 110.0 and 165.0 µg/mL in experiment I with and without metabolic activation. In experiment II precipitation occurred at 110.0 µg/mL without metabolic activation and at 82.5 and 110.0 µg/mL with metabolic activation.

Relevant toxic effects indicated by a relative total growth of less than 50% of survival in both parallel cultures were observed in experiment I at 27.5 µg/mL and above without metabolic activation and at 110.0 µg/mL with metabolic activation following 4 hours of treatment. In experiment II cytotoxic effects as described above occurred at 27.5 µg/mL and above without metabolic activation (24 hours treatment) and at 82.5 µg/mL and above with metabolic activation (4 hours treatment). The recommended cytotoxic range of approximately 10-20% RTG was covered with and without metabolic activation.

No substantial and reproducible dose dependent increase of the mutation frequency was observed with and without metabolic activation up to cytotoxic concentrations. The positive control substances induced the appropriate responses.

There was no evidence of induced mutant colonies over background. Under the conditions of the study, the test substance was negative for mutagenic potential.