Fatty acids, C16-18 (even numbered), esters with 1,2,3-propanetriol and oligomers

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 March 2017 to 03 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

In accordance with the OECD guideline for testing of chemicals No. 471 “Bacterial Reverse Mutation Test”, adopted on 21st July 1997.

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, C16-18 (even numbered), esters with 1,2,3-propanetriol and oligomers

IUPAC Name:

Fatty acids, C16-18 (even numbered), esters with 1,2,3-propanetriol and oligomers

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material:Clariant Produkte (Deutschland)
GmbH 84504 Burgkirchen, Germany
- Expiration date of the batch:04.10.2018
- Purity test date:96,9%

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Solubility of the test substance in the solvent/vehicle:Distilled water

Method

Target gene:

Hisitidine and tryptophan locus

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

1 mL of S9 homogenate was mixed with 9 mL of co-factor

Test concentrations with justification for top dose:

The test item was tested at the concentrations of 0.02, 0.06, 0.20, 0.63 and 2 mg/plate based on the results of solubility, precipitation and initial cytotoxicity test.

Vehicle / solvent:

Distilled water- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle:The test item at the given concentration of 50 mg/mL was soluble in distilled at 40 to 45º C

Controls

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

2-nitrofluorene

sodium azide

other: 2-aminoantracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation;

DURATION
- Preincubation period: 30 minutes
others: cytotoxicity by lawn evaluation and mutagenicity by evaluating revertant counts.

NUMBER OF REPLICATIONS: Triplicates

Rationale for test conditions:

not applicable

Evaluation criteria:

The test will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value in Salmonella typhimurium strains TA98, TA100 and Escherichia coli WP2 uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value in tester strains TA1535 and TA1537.vehicle control, in both the trials, in the presence and absence of metabolic activation. There was no appreciable increase in number of revertant colonies at any of the tested concentrations in both the trials.

Statistics:

not applicable

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: yes
- Precipitation: minimal precipitation at 2 mg/plate


HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
Plate in corporation method
- Positive historical control data:
Tester strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (With S9)
Mean 408.7 386.1 140.4 118.5 385.5
SD ±16.6 ±13.8 ±8.3 ±6.3 ±5.3
Min 280 306 118 100 370
Max 440 419 182 149 401
Tester strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (Without S9)
Mean 401.5 382.2 141.4 119.8 386.0
SD ±26.3 ±16.5 ±10.6 ±6.5 ±5.8
Min 310 313 112 103 371
Max 428 446 190 158 416


- Negative (solvent/vehicle) historical control data:
Tester strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (With S9)
Mean 20.7 102.5 21.4 9.3 171.8
SD ±1.7 ±2.2 ±2.0 ±1.5 ±5.8
Min 18 98 17 6 159
Max 26 108 25 12 184
Tester strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (Without S9)
Mean 20.9 102.2 21.6 9.3 171.4
SD ±1.8 ±2.6 ±1.9 ±1.5 ±4.9
Min 18 98 18 7 159
Max 25 110 26 13 183

Pre incubation method

- Positive historical control data:
Tester strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (With S9)
Mean 408.0 386.3 140.7 117.9 384.6
SD ±14.5 ±14.2 ±9.8 ±5.2 ±6.3
Min 290 298 103 102 363
Max 429 425 187 128 403
Tester strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (Without S9)
Mean 404.8 383.1 141.9 119.1 385.3
SD ±20.8 ±15.8 ±11.9 ±5.5 ±6.0
Min 314 317 110 105 371
Max 428 450 200 134 418

- Negative (solvent/vehicle) historical control data:
Tester strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (With S9)
Mean 20.6 102.2 22.0 9.5 171.5
SD ±1.5 ±2.5 ±1.9 ±1.4 ±4.5
Min 18 97 18 7 161
Max 24 107 25 12 182
Tester strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (Without S9)
Mean 20.9 102.4 21.5 9.3 170.6
SD ±1.7 ±2.4 ±2.2 ±1.4 ±4.6
Min 18 97 17 7 158
Max 24 110 25 12 181

Any other information on results incl. tables

 

 Table 1: SUMMARY OF INITIAL CYTOTOXICITY TEST-SALMONELLA TYPHIMURIUM TA100

Test Item Concentration (mg/plate)	No. of Revertants/plate
	With S9						Without S9
	R1	R2	R3	Average	±SD	Bacterial Lawn Intensity	R1	R2	R3	Average	±SD	Bacterial Lawn Intensity
Vehicle Control	99	87	106	97	9.6	4+	105	112	96	104	8.0	4+
2	102	107	103	104	2.6	4+	110	106	104	107	3.1	4+
1	106	96	102	101	5.0	4+	101	95	89	95	6.0	4+
0.9	88	105	101	98	8.9	4+	104	91	97	97	6.5	4+
0.8	93	97	84	91	6.7	4+	96	89	104	96	7.5	4+
0.7	103	96	91	97	6.0	4+	94	107	97	99	6.8	4+

Values of Revertants are in Mean±SD

Lawn intensity: 4+= Thick lawns: Distinguished by a healthy (Normal) background lawn comparable tovehiclecontrol plates.

Table 2: SUMMARY OF COLONY COUNTS OF REVERTANTS OF TRIAL-I, PLATE INCORPORATION METHOD

Treatment	Test Concentration  (mg/plate)		No. of Revertants (Mean of 3 Plates)
			With S9						Without S9
			Salmonella typhimurium				E.coliWP2 uvrA (pKM 101)		Salmonella typhimurium				E.coliWP2 uvrA (pKM 101)
			TA 98	TA 100	TA 1535	TA 1537			TA 98	TA 100	TA 1535	TA 1537
Vehicle Control	100 µL of Distilled Water	Mean	25.7	98.0	20.3	9.3	165.3		27.3	94.0	21.3	9.7	175.0
		±SD	5.7	9.5	3.5	4.2	7.8		1.5	9.8	2.1	1.5	10.1
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
Hostacerin DGMS	2	Mean	24.7	95.0	20.7	8.7	171.7		21.7	96.0	18.3	6.3	169.3
		±SD	4.7	7.9	2.5	1.5	7.4		3.5	8.9	3.5	2.5	11.0
	Fold Increase		1.0	1.0	1.0	0.9	1.0		0.8	1.0	0.9	0.7	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.63	Mean	23.3	94.3	17.3	7.3	173.0		21.0	97.3	20.0	9.7	175.3
		±SD	5.7	11.6	2.1	2.1	13.1		4.4	4.0	3.0	2.5	7.8
	Fold Increase		0.9	1.0	0.9	0.8	1.0		0.8	1.0	0.9	1.0	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.2	Mean	20.3	87.3	22.0	9.0	170.0		21.3	96.0	19.3	7.0	172.7
		±SD	4.2	8.0	1.0	2.6	4.6		4.9	15.1	3.2	2.6	9.3
	Fold Increase		0.8	0.9	1.1	1.0	1.0		0.8	1.0	0.9	0.7	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.06	Mean	21.3	92.7	19.7	8.0	170.7		21.3	95.3	20.7	7.7	179.0
		±SD	2.5	7.4	4.9	3.6	5.7		6.0	9.6	3.1	1.5	1.7
	Fold Increase		0.8	0.9	1.0	0.9	1.0		0.8	1.0	1.0	0.8	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.02	Mean	26.3	91.3	20.3	8.3	177.7		19.0	88.7	19.3	8.7	172.7
		±SD	4.7	9.1	1.5	4.9	6.8		3.0	5.5	4.7	3.1	6.0
	Fold Increase		1.0	0.9	1.0	0.9	1.1		0.7	0.9	0.9	0.9	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
Positive Control	100 µL of respective Positive Control	Mean	353.0	335.3	133.7	130.0	360.7		357.3	351.3	132.0	120.0	361.0
		±SD	31.1	20.5	5.0	8.0	35.3		38.5	20.4	15.1	28.4	20.7
	Fold Increase		13.8	3.4	6.6	13.9	2.2		13.1	3.7	6.2	12.4	2.1
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+

Values of Revertants are in Mean±SD

Positive controls:

For with S9:

ForSalmonella typhimuriumTA98, TA100, TA1535 and TA1537 = 4 µg/plate of 2-Aminoanthracene

ForEscherichia coliWP2 uvrA (pKM101)strain +S9 = 30µg/plate of 2-Aminoanthracene

For without S9:

For TA98: 2 µg/plate of 2-Nitrofluorene

For TA100 and TA1535: 1 µg/plate of Sodium azide.

For TA1537: 50 µg/plate of 9-Aminoacridine

ForEscherichia coliWP2 uvrA (pKM101): 5 µg/plate of4-Nitroquinoline 1-oxide

Table 3: SUMMARY OF COLONY COUNTS OF REVERTANTS OF TRIAL-I, PREINCUBATION METHOD

Treatment	Test Concentration  (mg/plate)		No. of Revertants (Mean of 3 Plates)
			With S9						Without S9
			Salmonella typhimurium				E.coliWP2 uvrA (pKM 101)		Salmonella typhimurium				E.coliWP2 uvrA (pKM 101)
			TA 98	TA 100	TA 1535	TA 1537			TA 98	TA 100	TA 1535	TA 1537
Vehicle Control	100 µL of Distilled Water	Mean	19.7	106.0	20.0	8.0	174.3		22.7	98.3	18.7	9.3	172.3
		±SD	6.7	8.0	2.6	2.6	5.5		6.0	3.1	2.5	3.2	5.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
Hostacerin DGMS	2	Mean	15.3	85.3	19.3	7.3	173.3		21.3	98.0	16.7	8.3	170.3
		±SD	1.2	11.1	0.6	4.0	12.5		3.2	9.8	1.5	5.1	1.2
	Fold Increase		0.6	0.9	1.0	0.8	1.0		0.8	1.0	0.8	0.9	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.63	Mean	25.7	93.3	20.0	9.3	164.0		21.7	98.0	19.0	8.0	170.3
		±SD	2.5	4.5	4.4	1.5	6.0		5.5	3.5	4.4	3.6	11.2
	Fold Increase		1.0	1.0	1.0	1.0	1.0		0.8	1.0	0.9	0.8	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.2	Mean	17.7	93.7	19.7	8.7	167.3		22.7	97.0	17.0	11.0	169.3
		±SD	3.1	8.1	5.1	4.2	6.1		4.7	9.8	5.3	2.0	6.5
	Fold Increase		0.7	1.0	1.0	0.9	1.0		0.8	1.0	0.8	1.1	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.06	Mean	18.7	101.7	19.7	10.3	171.0		20.0	86.7	20.7	9.3	168.0
		±SD	4.2	8.7	2.5	4.7	15.7		4.4	7.1	2.5	2.1	5.3
	Fold Increase		0.7	1.0	1.0	1.1	1.0		0.7	0.9	1.0	1.0	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.02	Mean	15.3	94.7	16.7	7.3	169.3		19.0	89.0	16.7	8.0	166.0
		±SD	1.2	13.3	5.5	3.1	7.4		3.6	7.2	3.1	1.7	6.2
	Fold Increase		0.6	1.0	0.8	0.8	1.0		0.7	0.9	0.8	0.8	0.9
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
Positive Control	100 µL of respective Positive Control	Mean	330.3	367.7	134.3	111.0	379.0		397.3	351.7	133.7	115.7	380.0
		±SD	14.3	8.1	11.0	13.7	15.5		35.0	33.7	13.3	13.2	17.7
	Fold Increase		12.9	3.8	6.6	11.9	2.3		14.5	3.7	6.3	12.0	2.2
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+

Values of Revertants are in Mean±SD

Positive controls:

For with S9:

ForSalmonella typhimuriumTA98, TA100, TA1535 and TA1537 = 4 µg/plate of 2-Aminoanthracene

ForEscherichia coliWP2 uvrA (pKM101)strain +S9 = 30µg/plate of 2-Aminoanthracene

For without S9:

For TA98: 2 µg/plate of 2-Nitrofluorene

For TA100 and TA1535: 1 µg/plate of Sodium azide.

For TA1537: 50 µg/plate of 9-Aminoacridine

ForEscherichia coliWP2 uvrA (pKM101): 5 µg/plate of4-Nitroquinoline 1-oxide

Applicant's summary and conclusion

Conclusions:

Based on the results of the study, it can be concluded that the registration substance is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 2 mg/plate under the test conditions.

Executive summary:

The genotoxicity of the registration substance was investigated according to the Guideline OECD 471. No mutagenic activity was observed with and without metabolic activation of the concentration of 2 mg/plate in two independent experiments.