Fatty acids, C16-18 (even numbered), ammonium 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:

August 10, 2012 - November 5, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

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:

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

Test material

Test material information

Constituent 1

Reference substance name:

diammonium hexadecanoate octadecanoate

EC Number:

939-066-9

Molecular formula:

Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance).

IUPAC Name:

diammonium hexadecanoate octadecanoate

Test material form:

liquid

Method

Target gene:

Thymidine kinase, TK +/-

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:
With metabolic activation: 1.0, 2.0, 5.0, 10.0, 25.0, 50.0, 100.0 and 200.0 µg/mL (4h exposure period)
Without metabolic activation: 1.0, 2.0, 5.0, 10.0, 20.0, 40.0, 50.0 and 80.0 µg/mL. (4h exposure period)
Experiment II:
With metabolic activation: 3.0, 7.0, 15.0, 30.0, 60.0, 90.0, 150.0 and 180.0 µg/mL (4h exposure period)
Without metabolic activation: 0.2, 0.5, 1.0, 3.0, 8.0, 10.0, 15.0 and 20.0 µg/mL (24h exposure period)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI cell culture medium (RPMI + 5% HS)
- Justification for choice of solvent/vehicle: Based on the results of the solubility test.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

RPMI cell culture medium (RPMI + 5% HS)

Controls 2

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

ethylmethanesulphonate

Remarks:

300 µg/mL in medium

Controls 3

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

methylmethanesulfonate

Remarks:

8 and 10 µg/mL in 0.9% NaCl

Controls 4

Positive controls:

yes

Remarks:

With metabolic activation

Positive control substance:

benzo(a)pyrene

Remarks:

1.5 and 2.5 µg/Ml in DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment I: Short-term exposure (4h): 1x10E+07 cells were suspended in 11 mL RPMI medium with 5% horse serum (25 cm2 flasks) and exposed to designated concentrations of the test item either in the presence of absence of metabolic activation. After exposure, test item was removed by centrifugation (200 x g, 10 min) and the cells were washed twice with PBS. Subsequently the cells were suspended in 30 mL complete culture medium and incubated for an expression and growth period. The cell density was determined each day and adjusted to 3x10E+05 cells/mL in a total culture volume of 20 mL, if necessary.
Experiment II: Long-term exposure (24h): 5x10E+06 cells were suspended in 25 mL RPMI medium with 7.5% horse serum (75 cm2 flasks) and exposed to designated concentrations of the test item in the absence of metabolic activation. After exposure, test item was removed by centrifugation (200 x g, 10 min) and the cells were washed twice with PBS. Subsequently 3x10E+05 cells were suspended in 14 mL complete culture medium and incubated for an expression and growth period. The cell density was determined each day and adjusted to 3x10E+05 cells/mL in a total culture volume of 20 mL, if necessary.

DURATION
- Exposure duration:
Experiment 1: 4 hours (with and without metabolic activation)
Experiment II: 4 hours (with metabolic activation) and 24 hours (without metabolic activation)
- Expression time (cells in growth medium): 2 days, at 37 ºC in 5% Co2/95% humidified air.
- Selection time (if incubation with a selection agent): ~ 14 days at 37 ºC in 5% CO2/95% humidified air

SELECTION AGENT (mutation assays): Cells from each experimental group were seeded in four 96-well plates at a density of approximately 2000 cells/well in 200 µL selective medium with TFT.

NUMBER OF REPLICATIONS: 1 replicate per dose (negative control, 2 replicates).

NUMBER OF CELLS EVALUATED: 2000 cells/well. The mutant frequency was calculated by dividing the number of TFT resistant colonies by the number of cells plated for selection, corrected for the plating efficiency of cells from the same culture grown in the absence of TFT.

DETERMINATION OF CYTOTOXICITY:
The toxicity of the test item was determined in pre-experiments up to a maximum concentration of 5 mg/mL. For experiment I (4h exposure) six concentrations (50, 150, 500, 1000, 2000 and 5000 µg/ml) were tested with and without metabolic activation. For the 24h long-term exposure assay (Experiment II, without metabolic activation) six concentrations (0.1, 1.0, 5.0, 10.0, 40.0 and 100.0 µg/mL) were tested. The number of cells at 4h, 24h and 48h after treatment were measured and the relative suspension growths were calculated.

In the main test the following parameters were calculated:
- Mutant frequency (MF)
- Cloning efficiency (CE) and relative cloning efficiency (RCe)
- Suspension growth (SG) and relative suspension growth (RSG)

OTHER EXAMINATIONS:
Colony sizing was performed for the highest concentrations of the test item and for the negative and positive controls. Small colonies were defined by slow growth and/or morphological alteration of the cell close.

Evaluation criteria:

The test item is considered mutagenic if the following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation Factor (GEF) of 126 mutants per 1E+06 cells.
- A dose-dependent increase in mutant frequency is detected.

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (>= 40$ of total colonies) is an indication for potential clastogenic effect and/or chromosomal aberrations.

Statistics:

The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH-value of the test item suspension was adjusted with HCl to the physiological range.
- Precipitation: Precipitation of the test item was noted in the pre-experiment for experiment I with metabolic activation at concentrations of 500 µg/mL and higher and without metabolic activation at concentrations of 150 µg/mL and higher. No precipitations were noted in experiment I and II with and without metabolic activation and in pre-experiment II without metabolic activation.

RANGE-FINDING/SCREENING STUDIES: The selection of the concentrations used in the main experiments was based on data from the pre-experiments. In experiment I 200.0 µg/mL (with metabolic activation) and 80 µg/mL (without metabolic activation) were selected as the highest concentrations. In experimental II 180.0 µg/mL (with metabolic activation) and 20.0 µg/mL (without metabolic activation) were selected as the highest concentrations.

TOXICITY: Growth inhibition was observed in experiment I and II with and without metabolic activation.

MUTAGENICITY: In experiment I and II no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). The Global Evaluation Factor (GEF) was exceed by the induced mutant frequency at only one concentration (100 µg/mL) in experiment I with metabolic activation. The exceeded threshold of the GEF could not be verified in the second independent experiment with metabolic activation. Therefore, this effect was considered as not biologically relevant.

CLASTOGENICITY: In experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation).

CONTROLS: EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

Any other information on results incl. tables

Summary: Experiment I and II with metabolic activation:

	Test groups	Conc [µg/ml]	RCE [µg/ml]	RTG [%]	MF [mutants/10^6cells]	IMF [mutants/10^6cells]	GEF exceded	Statistical Significance	Precipitate
Exp I	C1	0	100.0	100.0	129.1	/	/	-	-
	C2					/	/	-	-
	1	1.0	94.8	97.9	114.7	-14.4	-	-	-
	2	2.0	79.9	86.2	116.1	-13.0	-	-	-
	3	5.0	90.4	78.8	148.6	19.5	-	-	-
	4	10.0	87.6	72.7	122.3	-6.8	-	-	-
	5	25.0	81.1	70.6	147.0	17.9	-	-	-
	6	50.0	78.7	51.2	155.2	26.1	-	-	-
	7	100.0	74.2	21.8	289.1	160.0	+	+	-
	10	200.0	72.1	12.7	225.7	96.9	-	+	-
	B[a]P	2.5	60.7	25.4	848.6	719.5	+	+	-
Exp II	C1	0	100.0	100.0	86.6	/	/	/	-
	C2					/	/	/	-
	3	3.0	99.7	91.5	86.6	4.9	-	-	-
	4	7.0	109.7	98.1	91.4	-14.0	-	-	-
	5	15.0	82.1	74.6	72.5	16.1	-	-	-
	6	30.0	88.3	81.9	102.7	1.4	-	-	-
	7	60.0	89.6	48.7	101.4	14.9	-	-	-
	8	90.0	84.5	33.0	119.1	32.5	-	+	-
	10	150.0	77.5	24.3	110.6	24.0	-	+	-
	11	180.0	64.4	11.9	151.2	64.7	-	+	-
	B[a]P	1.5	67.2	37.8	690.7	604.1	+	+	-

Summary: Experiment I and II without metabolic activation:

	Test groups	Conc [µg/ml]	RCE [µg/ml]	RTG [%]	MF [mutants/10^6cells]	IMF [mutants/10^6cells]	GEF exceded	Statistical Significance	Precipitate
Exp I	C1	0	100.0	100.0	121.4	/	/	-	-
	C2					/	/	-	-
	3	1.0	116.3	114.4	111.2	-10.3	-	-	-
	4	2.0	100.7	98.6	123.1	1.7	-	-	-
	5	5.0	88.4	81.6	180.2	58.8	-	-	-
	6	10.0	104.2	98.0	128.9	7.4	-	-	-
	7	20.0	84.4	42.4	216.3	94.8	-	+	-
	8	40.0	100.7	9.3	145.2	23.8	-	-	-
	9	50.0	78.3	15.1	180.2	58.8	-	+	-
	12	80.0	57.1	10.1	232.9	111.5	-	+	-
	EMS	300	77.1	64.9	687.4	566.0	+	+	-
	MMS	10	64.8	45.2	690.6	569.2	+	+	-
Exp II	C1	0	100.0	100.0	60.7	/	/	/	-
	C2					/	/	/	-
	2	0.2	105.0	95.4	67.2	6.6	-	-	-
	3	0.5	110.1	100.8	60.0	-0.7	-	-	-
	4	1.0	94.4	91.2	68.3	7.6	-	-	-
	5	3.0	85.1	76.6	83.2	22.5	-	-	-
	7	8.0	108.4	70.6	89.1	28.4	-	-	-
	8	10.1	95.8	52.2	67.2	6.5	-	-	-
	9	15.0	91.6	22.4	82.4	21.7	-	+	-
	10	20.0	80.4	7.7	83.9	23.2	-	-	-
	EMS	200	61.4	24.2	1617.7	1557.0	+	+	-
	MMS	8	65.0	17.0	725.8	665.1	+	+	-

C: Negative controls

RCE: Relative Cloning Efficiency

RTG: Relative Total Growth

MF: Mutant Frequency

IMF: Induced Mutant Frequency

GEF: Global Evaluation Factor

Statistical Significance: Mann Whitney test, p<0.05

B[a]P: Benzo[a]pyrene

EMS: Ethylmethanesulfonate

MMS: Methylmethanesulfonate

Applicant's summary and conclusion

Conclusions:

Test item Fatty acids, C16-18 (even numbered), ammonium salts was considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells, under test conditions (with and without metabolic activation.

Executive summary:

An in vitro gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells was performed on test item Fatty acids, C16 -18 (even numbered), ammonium salts in accordance with OECD Guideline 476. The selection of the concentrations used in the main experiment was based on the data from the pre-experiments. In experiment I 200.0 µg/mL (with metabolic activation, 4h short-term exposure) and 80.0 µg/mL (without metabolic activation, 4h short-term exposure) were selected as the highest concentrations. In experiment II 180.0 µg/mL (with metabolic activation, 4h short-term exposure) and 20.0 µg/mL (without metabolic activation, 24h long-term exposure) were selected as the highest concentrations. No precipitation was noted in the main experiment. Growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I and II no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). The Global Evaluation Factor (GEF) was exceed by the induced mutant frequency at only one concentration (100 µg/mL) in experiment I with metabolic activation. The exceeded threshold of the GEF could not be verified in the second independent experiment with metabolic activation. Therefore, this effect was considered as not biologically relevant. Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation). EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects. Based on these results, test item Fatty acids, C16-18 (even numbered), ammonium salts was considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells, under test conditions (with and without metabolic activation.