Fatty acids, C18-unsatd., compds. with triethanolamine

Genetic toxicity: in vitro

Administrative data

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:

2016-11-02 till 2016-11-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C18 unsatd., reaction products with triethanolamine

EC Number:

938-513-5

Cas Number:

1187872-27-0

IUPAC Name:

Fatty acids, C18 unsatd., reaction products with triethanolamine

Test material form:

liquid: viscous

Method

Species / strain

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II:
Strain TA 100: 1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate
The remaining strains: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Based on the observed toxic effects and the precipitation of the test item at least seven concentrations were tested in experiment II. 5000 µg/plate were chosen as maximal concentration.

Vehicle / solvent:

Solvent used: Ethanol
Justification for choice of solvent: best suitable solvent, because of its solubility properties

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (experiment I: plate incorporation assay; experiment II: pre-incubation assay
exposure duration: 72 hours
Number of Replications: 3 plates for each concentration incl. the controls
DETERMINATION OF CYTOTOXICITY: Evident as a reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA

Metabolic activation:

with and without

Additional information on results:

TEST SPECIFIC CONFOUNDING FACTORS
Effects of pH: none
Water solubility: not soluble
Precipitation: The test item precipitated in the overlay agar in the test tubes from 1000 up to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate. The undissolved particles had no influence on the data recording.
Other confounding effects: none

COMPARISON WIT HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):
Experiment I: TA 100 without S9 mix: 100 - 5000 µg/plate
TA 100 with S9 mix: 2500 - 5000 µg/plate
Experiment II: TA 1537 without S9 mix: 1000 - 5000 µg/plate
TA 100 without S9 mix: 333 - 2500 µg/plate

Remarks on result:

other: reverse mutation assay migrated from the field Test System

Any other information on results incl. tables

Summary of Experiment I

Date plated: 02.11.2016

Date counted: 09.11.2016

	Test Group	Dose Level (per plate)	TA 1535 Revertant Colony Counts (Mean ±SD)	TA 1537 Revertant Colony Counts (Mean ±SD)	TA 98 Revertant Colony Counts (Mean ±SD)	TA 100 Revertant Colony Counts (Mean ±SD)	WP2 uvrA Revertant Colony Counts (Mean ±SD)
Without	Ethanol		12 ± 0	10 ± 3	29 ± 6	135 ± 16	37 ± 7
Activation	Untreated		13 ± 6	11 ± 1	23 ± 3	147 ± 14	44 ± 11
	Emulsogen	3 µg	13 ± 6	11 ± 4	28 ± 5	139 ± 15	33 ± 7
	TO	10 µg	9 ± 2	10 ± 1	28 ± 5	144 ± 7	39 ± 11
		33 µg	11 ± 5	11 ± 1	36 ± 8	121 ± 17	38 ± 10
		100 µg	11 ± 3	9 ± 3	38 ± 2	59 ± 2	44 ± 7
		333 µg	9 ± 3	13 ± 3	31 ± 5	23 ± 2^R	37 ± 10
		1000 µg	9 ± 2	13 ± 3^R	38 ± 11^R	19 ± 2^R M	40 ± 4
		2500 µg	9 ± 2^P M	10 ± 1^P M R	20 ± 2^P M R	12 ± 2^P M R	26 ± 5^P M
		5000 µg	8 ± 2^P M	12 ± 4^P M R	21 ± 1^P M R	10 ± 2^P M R	27 ± 2^P M
	NaN3	10 µg	1549 ± 108			2669 ± 39
	4-NOPD	10 µg			552 ± 45
	4-NOPD	50 µg		96 ± 7
	MMS	2.0 µL					975 ± 104
With	Ethanol		19 ± 2	13 ± 3	45 ± 9	112 ± 8	42 ± 4
Activation	Untreated		13 ± 6	15 ± 3	40 ± 2	98 ± 19	40 ± 3
	Emulsogen	3 µg	17 ± 5	13 ± 6	53 ± 13	80 ± 20	39 ± 7
	TO	10 µg	13 ± 2	17 ± 6	52 ± 14	71 ± 24	46 ± 4
		33 µg	17 ± 3	13 ± 2	41 ± 9	75 ± 21	57 ± 5
		100 µg	15 ± 5	16 ± 3	40 ± 2	69 ± 16	49 ± 7
		333 µg	16 ± 5	16 ± 4	44 ± 4	105 ± 9	41 ± 8
		1000 µg	14 ± 4	15 ± 5	46 ± 3	103 ± 9	40 ± 5
		2500 µg	16 ± 2^P	17 ± 2^P	47 ± 10^P	43 ± 13^P	45 ± 9^P
		5000 µg	11 ± 4^P M	9 ± 2^P M	47 ± 13^P	39 ± 2^P	26 ± 5^P M
	2-AA	2.5 µg	425 ± 26	120 ± 10	4485 ± 452	2912 ± 106
	2-AA	10.0 µg					302 ± 93

                   

Summary of Experiment II

Date plated: 18.11.2016

Date counted: 21.11.2016

	Test Group	Dose Level (per plate)	TA 1535 Revertant Colony Counts (Mean ±SD)	TA 1537 Revertant Colony Counts (Mean ±SD)	TA 98 Revertant Colony Counts (Mean ±SD)	TA 100 Revertant Colony Counts (Mean ±SD)	WP2 uvrA Revertant Colony Counts (Mean ±SD)
Without	Ethanol		13 ± 2	15 ± 4	29 ± 4	194 ± 1	40 ± 7
Activation	Untreated		12 ± 3	14 ± 2	26 ± 1	202 ± 7	36 ± 4
	Emulsogen	1 µg				190 ± 20
	TO	3 µg				206 ± 21
		10 µg	11 ± 4	15 ± 5	39 ± 6	191 ± 18	42 ± 7
		33 µg	12 ± 5	12 ± 3	34 ± 6	176 ± 16	45 ± 3
		100 µg	14 ± 3	10 ± 3	30 ± 2	149 ± 8	43 ± 5
		333 µg	16 ± 2	13 ± 2	34 ± 2	80 ± 3	48 ± 11
		1000 µg	11 ± 5	6 ± 2	24 ± 10	43 ± 12	46 ± 3
		2500 µg	11 ± 3^P	6 ± 1^P M	30 ± 7^P	46 ± 6^P M	50 ± 7^P
		5000 µg	11 ± 2^P M	4 ± 1^P M	20 ± 4^P M		34 ± 9^P M
	NaN3	10 µg	1591 ± 43			2508 ± 102
	4-NOPD	10 µg			499 ± 18
	4-NOPD	50 µg		111 ± 9
	MMS	2.0 µL					830 ± 36
With	Ethanol		17 ± 3	15 ± 2	44 ± 6	203 ± 12	61 ± 8
Activation	Untreated		10 ± 0	13 ± 3	39 ± 9	210 ± 7	49 ± 3
	Emulsogen	1 µg				208 ± 13
	TO	3 µg				209 ± 16
		10 µg	16 ± 5	12 ± 4	39 ± 7	196 ± 12	57 ± 5
		33 µg	18 ± 3	13 ± 3	37 ± 9	181 ± 15	51 ± 4
		100 µg	16 ± 3	12 ± 2	37 ± 1	203 ± 18	55 ± 6
		333 µg	16 ± 1	14 ± 2	49 ± 11	192 ± 9	51 ± 12
		1000 µg	21 ± 4	14 ± 2	44 ± 5	201 ± 24	53 ± 9
		2500 µg	17 ± 2^P	17 ± 3^P	34 ± 5^P	174 ± 9^P	57 ± 8^P
		5000 µg	15 ± 1^P M	8 ± 2^P M	23 ± 3^P M		43 ± 3^P M
	2-AA	2.5 µg	447 ± 39	155 ± 30	5595 ± 259	5303 ± 330
	2-AA	10.0 µg					392 ± 102

Key to Plate Postfix Codes:              

P: Precipitate

M: Manuel Count

R:  Reduced Background growth

Key to positive controls:

NaN3: sodium azide

4 -NOPD: 4 -nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

2-AA: 2 -aminoanthracene

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The test item was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) usingSalmonellatyphimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.

The assay was performed in two independent experiments both with and without rat liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:            3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:

Strain TA 100:                                     1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate

The remaining strains:                          10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 1000 up to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed reduced back­ground growth at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	/	/	/	/
TA 1537	1000 – 5000	/	/	/
TA 98	1000 – 5000	/	/	/
TA 100	333 – 5000	/	/	/
WP2 uvrA	/	/	/	/

/ = normal background growth

 

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	/	/	/	/
TA 1537	/	/	1000 – 5000	/
TA 98	/	/	/	/
TA 100	100 – 5000	2500 – 5000	333 – 2500	/
WP2 uvrA	/	/	/	/

/ = no Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5)

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowled­ged border of biological relevance.

 

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.