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

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:

2017-03-20 to 2017-10-30

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, ethoxylated

IUPAC Name:

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

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 / DEG4338635
- Expiration date of the batch: 2018-10-04
- Purity test date: 2016-10-04


STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Solubility of the test substance in the vehicle: Acetone
- Reactivity of the test substance with the vehicle of the cell culture medium: Soluble

Method

Target gene:

Histidine & 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:

Sodium phenobarbitone and β-naphthoflavone induced rat liver S9 homogenate was used as the metabolic activation system

Test concentrations with justification for top dose:

In mutation assay the test item was tested at the concentrations of 0.05, 0.16, 0.50, 1.58 and 5 mg/plate. The test concentrations for mutation assay were selected based on the results of solubility, precipitation and initial cytotoxicity test.

Vehicle / solvent:

Vehicle used:acetone
Justification for choice of vehicle: The test item was soluble in acetone at a concentration of 50 mg/mL

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-amioantracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar -plate incorporation & preincubation; with and without metabolic activation

DURATION
Plate incorporation: 65 hrs
- Preincubation period:30 minutes
- Postincubation period: 63 hrs 45 minutes

NUMBER OF REPLICATIONS: triplicates

- OTHER: cytotoxicity by lawn evaluation & mutation assay by counting revertant colonies.

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.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:

other: Salmonella typhimurium TA98, TA100, TA1535, and TA1537 and Escherichia coli WP2 uvrA (pKM101).

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: No, soluble in acetone
- Precipitation: The test item resulted in precipitation at 3, 4 and 5 mg/plate and minimal precipitation was observed at 2 mg/plate and no precipitation was observed at 1, 0.9, 0.8,0.7 mg/plate.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:

Plate incorporation method
Tester Strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (With S9)
Mean 401.7 383. 140.1 119.2 383.5
SD ±23.4 ±16.9 ±8.0 ±6.6 ±9.8
Min 256 246 118 100 320
Max 440 419 1 82 149 421

Tester Strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (Without S9)
Mean 396.6 379.7 141.1 120.1 383.1
SD ±27.4 ±18.3 ±9.9 ±7.3 ±10.4
Min 290 270 110 98 371
Max 430 446 190 158 416

Pre incubation method
Tester Strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (With S9)
Mean 402.5 383.9 140.6 118.6 382.9
SD ±18.5 ±16.1 ±9.3 ±6.3 ±10.4
Min 290 293 103 99 330
Max 429 425 187 151 412

Tester Strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (Without S9)
Mean 3398.3 381.3 141.3 119.5 382.8
SD ±24.5 ±16.2 ±11.0 ±6.4 ±10.3
Min 281 312 110 97 321
Max 428 450 200 173 418

Any other information on results incl. tables

TABLE 1.           SUMMARY OF INITIAL CYTOTOXICITY TEST-SALMONELLA TYPHIMURIUMTA100                                        

Initial Cytotoxicity Test                                                                                                 

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	106	98	105	103	4.4	4+	103	93	110	102	8.5	4+
2	91	88	101	93	6.8	4+	85	96	102	94	8.6	4+
1	102	108	104	105	3.1	4+	93	101	104	99	5.7	4+
0.9	93	102	102	99	5.2	4+	105	98	91	98	7.0	4+
0.8	84	103	89	92	9.8	4 +	107	101	94	101	6.5	4+
0.7	108	92	111	104	10.2	4+	107	109	90	102	10.4	4+

 

Follow-up Initial Cytotoxicity Test                                                                                                 

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	107	96	101	101	5.5	4+	98	102	92	97	5.0	4+
5	90	87	92	90	2.5	4+	85	94	91	90	4.6	4+
4	99	84	93	92	7.5	4+	92	79	88	86	6.7	4+
3	80	92	89	87	6.2	4+	88	100	93	94	6.0	4+
2.75	86	94	90	90	4.0	4+	78	101	90	90	11.5	4+
2.5	87	94	105	95	9.1	4+	81	98	94	91	8.9	4+
2.25	104	92	96	97	6.1	4+	102	95	89	95	6.5	4+
2	91	88	85	88	3.0	4+	81	87	98	89	8.6	4+
1	90	95	80	88	7.6	4+	96	84	81	87	7.9	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 Acetone	Mean	25.0	104.0	19.3	11.0	175.0		23.3	99.7	20.3	9.7	173.7
		±SD	3.6	10.4	2.5	2.0	9.2		4.0	4.7	3.1	2.5	5.5
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
Hostacerin DGSB	5	Mean	17.3	99.0	20.3	7.3	164.0		18.7	101.7	18.7	7.3	166.3
		±SD	3.1	14.5	3.8	2.5	5.6		2.5	10.5	2.5	2.1	5.5
	Fold Increase		0.7	1.0	1.1	0.7	0.9		0.8	1.0	0.9	0.8	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	1.58	Mean	18.0	94.7	19.0	6.0	167.3		18.7	87.7	18.3	7.0	173.7
		±SD	3.6	12.5	4.6	2.0	6.4		4.0	5.1	3.8	3.6	7.1
	Fold Increase		0.7	0.9	1.0	0.5	1.0		0.8	0.9	0.9	0.7	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.5	Mean	19.3	102.3	18.0	7.3	163.3		18.0	92.0	16.0	8.3	165.3
		±SD	4.2	7.1	1.7	4.2	8.0		4.4	11.0	2.0	2.9	6.7
	Fold Increase		0.8	1.0	0.9	0.7	0.9		0.8	0.9	0.8	0.9	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.16	Mean	18.0	91.7	15.7	8.7	165.3		18.7	97.7	16.7	9.0	170.7
		±SD	1.0	9.1	1.5	1.2	14.0		4.7	9.7	3.2	2.0	9.5
	Fold Increase		0.7	0.9	0.8	0.8	0.9		0.8	1.0	0.8	0.9	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.05	Mean	19.7	102.3	16.3	6.7	167.0		19.0	99.7	18.0	9.0	169.3
		±SD	2.5	8.6	3.5	2.5	11.4		2.6	5.9	2.0	3.6	16.0
	Fold Increase		0.8	1.0	0.8	0.6	1.0		0.8	1.0	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	372.3	390.7	142.3	115.7	397.0		319.7	380.3	137.3	113.7	385.0
		±SD	20.4	19.4	12.3	8.5	15.1		22.1	22.5	7.0	5.7	28.4
	Fold Increase		14.9	3.8	7.4	10.5	2.3		13.7	3.8	6.8	11.8	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 N-oxide

TABLE 3.           SUMMARY OF COLONY COUNTS OF REVERTANTS OF TRIAL-II

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 Acetone	Mean	25.3	106.3	22.3	10.7	177.7		24.0	102.0	21.3	12.0	178.7
		±SD	3.1	12.1	4.2	1.2	7.8		3.6	4.0	4.0	2.0	3.5
	Lawn Intensity		4+	4+	23	4+	4+		4+	4+	4+	4+	4+
Hostacerin DGSB	5	Mean	20.7	106.7	18.7	9.3	175.0		20.7	100.7	20.7	7.0	168.0
		±SD	4.2	6.8	6.1	2.5	9.2		4.7	4.5	2.1	3.0	10.5
	Fold Increase		0.8	1.0	0.8	0.9	1.0		0.9	1.0	1.0	0.6	0.9
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	1.58	Mean	20.3	102.7	18.3	8.0	172.3		17.7	92.0	18.3	6.7	174.3
		±SD	2.5	3.5	3.5	2.6	8.3		3.5	4.6	4.0	1.5	5.5
	Fold Increase		0.8	1.0	0.8	0.8	1.0		0.7	0.9	0.9	0.6	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.5	Mean	21.0	106.0	17.3	8.0	172.0		17.0	94.0	16.7	8.7	172.0
		±SD	1.7	7.5	3.2	3.6	7.2		3.6	9.0	3.1	2.5	9.5
	Fold Increase		0.8	1.0	0.8	0.8	1.0		0.7	0.9	0.8	0.7	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.16	Mean	19.3	102.0	18.0	8.7	174.0		18.0	90.3	19.7	7.7	169.7
		±SD	2.1	6.0	3.6	4.7	4.6		4.6	6.5	3.1	2.1	13.2
	Fold Increase		0.8	1.0	0.8	0.8	1.0		0.8	0.9	0.9	0.6	0.9
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.05	Mean	23.0	100.7	17.7	7.7	167.0		18.3	91.3	20.3	7.3	165.3
		±SD	2.0	12.4	2.5	2.9	13.5		2.1	10.5	4.0	3.1	9.1
	Fold Increase		0.9	0.9	0.8	0.7	0.9		0.8	0.9	1.0	0.6	0.9
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
Positive Control	100 µL of respective Positive Control	Mean	380.7	398.7	147.0	115.7	394.0		340.0	382.3	133.3	121.7	393.0
		±SD	18.9	9.3	9.6	9.7	18.7		21.7	10.0	4.5	10.4	11.5
	Fold Increase		15.0	3.7	6.6	10.8	2.2		14.2	3.7	6.3	10.1	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 N-oxide

 

Applicant's summary and conclusion

Conclusions:

Based on the results of the study, it can be concluded that the test item Hostacerin DGSB, is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 5 mg/plate under the test condition.

Executive summary:

The test item, Hostacerin DGSB obtained from Clariant India Limited,was evaluated for mutagenicity in Bacterial Reverse Mutation Test as per the OECD guidelinefor testing of chemicals No. 471, “Bacterial Reverse Mutation Test”, adopted on21^stJuly 1997.

The tester strains used in the mutation assay were Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coliWP2 uvrA (pKM101).

The test concentrations tested in the mutation assay were selected based on the results of solubility, precipitation and initial cytotoxicity test. The two independent trials (trial I and II) were conducted by plate incorporation method and pre incubation method in the presence and absence of metabolic activation system. In mutation assay the test item was tested at the concentrations of0.05, 0.16, 0.50, 1.58 and 5mg/plate. Vehicle control (acetone) and appropriate positive controls (2-nitrofluorene, sodium azide and 9-Aminoacridine, 4-nitroquinoline 1-oxide for trials “without metabolic activation” and 2-Aminoanthracene for trials “with metabolic activation”) were tested simultaneously. On the basis of test item solubility and precipitation tests, the initial cytotoxicity test was performed at 0.7, 0.8, 0.9, 1, and 2 mg/plate. The follow-up initial cytotoxicity test was performed at the 1, 2, 2.25, 2.50, 2.75, 3, 4 and 5 mg/plate concentrations. Initial cytotoxicity test was performed with TA100 both in the presence and absence of metabolic activation system. For the tester strain TA100 treated with Hostacerin DGSB at the concentration of 5 mg/plate both in the presence and absence of metabolic activation no cytotoxicity and lawn reduction was observed. Hence on the basis of cytotoxicity results 5 mg/plate was considered as the highest test concentration for mutation assay.

From the experimental results obtained, the mean numbers of revertant colonies at the tested concentrations were comparable to those of the 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.The number of revertant colonies in the positive controls resulted in 2.2 to 15.0 fold increase under identical conditions.