Isooctadecanoic acid, mixed esters with oxybis[propanediol]

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:

14 July - 05 August 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Isooctadecanoic acid, mixed esters with oxybis[propanediol]

Molecular formula:

not applicable, substance is UVCB

IUPAC Name:

Isooctadecanoic acid, mixed esters with oxybis[propanediol]

Method

Target gene:

his operon (S. typhimurium strains), trp operon (E. coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, induced with phenobarbital and beta-naphtha flavone

Test concentrations with justification for top dose:

Experiment 1 - Plate Incorporation Method (all tester strains): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
The maximum concentration was chosen as recommended in the guideline followed.

Experiment 2 – Pre-Incubation Method (all tester strains): 15, 50, 150, 500, 1500, 5000 µg/plate
Six test item dose levels per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Lot/Batch: 1679355 (experiment 1); 1719831 (experiment 2)
- Purity: 99.98% (experiment 1); > 99% (experiment 2)
- Expiry date: 11/2021 (experiment 1); 07/2022 (experiment 2)

- Justification for choice of solvent/vehicle: The test item was immiscible in dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at 100 mg/mL in solubility checks performed. Acetone was therefore selected as the vehicle. Distilled water was not evaluated as a vehicle in this test system as information provided by the sponsor suggested it was unstable.

Controls

Untreated negative controls:

yes

Remarks:

untreated plates

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation for experiment 1) and preincubation (experiment 2)

DURATION
- Preincubation period: 20 min (at 37 ± 3 °C)
- Exposure duration: approx. 48 h (at 37 ± 3 °C)

NUMBER OF REPLICATIONS: triplicate in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of bacterial background lawn and number of revertant colonies

Evaluation criteria:

Acceptance criteria:
- All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks
- All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls)
- All tester strain cultures should be in the range of 0.9 to 9 x 10E9 bacteria per mL
- Positive controls must be included to demonstrate both the intrinsic sensitivity of the tester strains and the integrity of the S9-mix
- All of the positive controls should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation
- There should be a minimum of four non-toxic test item dose levels
- There should be no evidence of excessive contamination

Evaluation criteria:
Any, one, or all of the following are used to determine the overall result of the study.
- A dose-related increase in mutant frequency over the dose range tested
- A reproducible increase at one or more concentrations
- Fold increase greater than two times the concurrent solvent control for any tester strain

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Individual plates were counted for revertant colonies. The average and standard deviation of the number of revertant colonies were calculated. Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. No further statistical analysis was not performed.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at 1500 and 5000 µg/plate

HISTORICAL CONTROL DATA: data are attached as separated pdf document (History Profile of Vehicle and Positive Control Values.pdf)

Any other information on results incl. tables

Table 1: Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1 (Plate Incorporation)

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
67		14		21		18		13
90	(82)	20	(21)	20	(20)	24	(21)	15	(12)
90		28		20		22		9

   Experiment 2 (Pre-Incubation) 

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
80		30		18		25		9
73	(78)	19	(23)	23	(26)	25	(24)	12	(11)
82		21		27		21		13

Table 2: Test Results Experiment 1 – Without Metabolic Activation (Plate Incorporation)

Test Period		From: 24 July 2017					To: 27 July 2017
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (Acetone)	78 83 89	(83) 5.5	28 27 31	(29) 2.1	20 22 26	(23) 3.1	23 14 21	(19) 4.7	18 9 12	(13) 4.6
	1.5 µg	79 88 97	(88) 9.0	23 26 22	(24) 2.1	21 13 21	(18) 4.6	19 25 23	(22) 3.1	9 14 13	(12) 2.6
	5 µg	82 74 77	(78) 4.0	29 22 31	(27) 4.7	19 19 29	(22) 5.8	11 11 12	(11) 0.6	10 8 8	(9) 1.2
	15 µg	101 86 62	(83) 19.7	21 24 24	(23) 1.7	25 15 30	(23) 7.6	25 24 13	(21) 6.7	16 16 22	(18) 3.5
	50 µg	75 90 81	(82) 7.5	18 23 32	(24) 7.1	12 23 20	(18) 5.7	22 23 27	(24) 2.6	7 20 19	(15) 7.2
	150 µg	69 90 87	(82) 11.4	32 14 21	(22) 9.1	25 29 27	(27) 2.0	16 16 20	(17) 2.3	20 25 11	(19) 7.1
	500 µg	86 93 88	(89) 3.6	25 22 28	(25) 3.0	12 21 23	(19) 5.9	19 20 16	(18) 2.1	14 13 14	(14) 0.6
	1500 µg	84 78 68	(77) 8.1	21 24 25	(23) 2.1	26 22 28	(25) 3.1	11 16 19	(15) 4.0	15 18 12	(15) 3.0
	5000 µg	70 P 97 P 63 P	(77) 18.0	18 P 22 P 23 P	(21) 2.6	23 P 29 P 25 P	(26) 3.1	27 P 13 P 18 P	(19) 7.1	17 P 21 P 15 P	(18) 3.1
Positive controls S9-Mix (-)	Name DoseLevel No. of Revertants	ENNG		ENNG		ENNG		4NQO		9AA
		3 µg		5 µg		2 µg		0.2 µg		80 µg
		598 522 475	(532) 62.1	318 290 352	(320) 31.0	804 596 630	(677) 111.6	200 254 261	(238) 33.4	127 157 305	(196) 95.3

Table 3: Test Results Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period		From: 24 July 2017					To: 27 July 2017
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (Acetone)	80 79 102	(87) 13.0#	24 24 21	(23) 1.7	25 38 27	(30) 7.0	16 15 22	(18) 3.8	14 19 13	(15) 3.2
	1.5 µg	79 102 67	(83) 17.8	26 20 24	(23) 3.1	31 26 26	(28) 2.9	18 23 22	(21) 2.6	8 17 13	(13) 4.5
	5 µg	98 83 100	(94) 9.3	20 27 24	(24) 3.5	18 30 26	(25) 6.1	22 19 21	(21) 1.5	10 11 14	(12) 2.1
	15 µg	85 100 87	(91) 8.1	18 24 26	(23) 4.2	23 34 25	(27) 5.9	25 26 34	(28) 4.9	13 10 9	(11) 2.1
	50 µg	78 114 80	(91) 20.2	24 20 28	(24) 4.0	20 26 17	(21) 4.6	17 21 19	(19) 2.0	17 16 12	(15) 2.6
	150 µg	84 86 68	(79) 9.9	25 29 15	(23) 7.2	30 36 30	(32) 3.5	16 30 16	(21) 8.1	16 22 13	(17) 4.6
	500 µg	102 94 103	(100) 4.9	20 22 20	(21) 1.2	30 28 30	(29) 1.2	24 25 17	(22) 4.4	13 19 21	(18) 4.2
	1500 µg	65 89 106	(87) 20.6	26 20 29	(25) 4.6	22 23 17	(21) 3.2	14 26 30	(23) 8.3	10 21 12	(14) 5.9
	5000 µg	106 P 104 P 92 P	(101) 7.6	22 P 22 P 34 P	(26) 6.9	28 P 36 P 30 P	(31) 4.2	12 P 21 P 18 P	(17) 4.6	16 P 16 P 12 P	(15) 2.3
Positive controls S9-Mix (+)	Name DoseLevel No. of Revertants	2AA		2AA		2AA		BP		2AA
		1 µg		2 µg		10 µg		5 µg		2 µg
		614 554 574	(581) 30.6	317 336 356	(336) 19.5	143 145 143	(144) 1.2	182 203 226	(204) 22.0	409 440 514	(454) 53.9

Table 4: Test Results Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period		From: 02 August 2017					To: 05 August 2017
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (Acetone)	72 86 81	(80) 7.1#	15 18 23	(19) 4.0	31 24 32	(29) 4.4	27 23 25	(25) 2.0	16 9 11	(12) 3.6
	15 µg	84 80 75	(80) 4.5	17 27 18	(21) 5.5	31 27 29	(29) 2.0	18 23 21	(21) 2.5	9 11 15	(12) 3.1
	50 µg	81 74 70	(75) 5.6	12 17 21	(17) 4.5	37 20 26	(28) 8.6	29 20 19	(23) 5.5	12 8 11	(10) 2.1
	150 µg	73 81 90	(81) 8.5	23 23 15	(20) 4.6	30 30 28	(29) 1.2	21 24 23	(23) 1.5	19 11 8	(13) 5.7
	500 µg	86 74 81	(80) 6.0	17 14 17	(16) 1.7	26 34 31	(30) 4.0	26 28 19	(24) 4.7	18 11 9	(13) 4.7
	1500 µg	73 70 85	(76) 7.9	14 14 18	(15) 2.3	28 26 31	(28) 2.5	19 21 27	(22) 4.2	14 11 9	(11) 2.5
	5000 µg	83 P 86 P 87 P	(85) 2.1	23 P 16 P 19 P	(19) 3.5	29 P 29 P 29 P	(29) 0.0	24 P 19 P 24 P	(22) 2.9	7 P 15P 8 P	(10) 4.4
Positive controls S9-Mix (-)	Name DoseLevel No. of Revertants	ENNG		ENNG		ENNG		4NQO		9AA
		3 µg		5 µg		2 µg		0.2 µg		80 µg
		1290 1191 1001	(1161) 146.9	1617 1787 1834	(1746) 114.2	1241 1249 1147	(1212) 56.7	342 298 379	(340) 40.6	386 327 427	(380) 50.3

Table 5: Test Results Experiment 2 – With Metabolic Activation (Pre-Incubation) 

Test Period		From: 02 August 2017					To: 05 August 2017
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (Acetone)	76 82 88	(82) 6.0#	28 26 20	(25) 4.2	30 35 38	(34) 4.0	23 25 34	(27) 5.9	15 17 18	(17) 1.5
	15 µg	74 77 83	(78) 4.6	22 27 21	(23) 3.2	35 35 30	(33) 2.9	24 33 28	(28) 4.5	16 13 11	(13) 2.5
	50 µg	72 64 78	(71) 7.0	20 21 20	(20) 0.6	27 34 32	(31) 3.6	30 32 32	(31) 1.2	18 11 17	(15) 3.8
	150 µg	87 81 87	(85) 3.5	27 14 27	(23) 7.5	28 22 36	(29) 7.0	24 31 27	(27) 3.5	16 14 16	(15) 1.2
	500 µg	85 83 73	(80) 6.4	17 27 24	(23) 5.1	39 32 25	(32) 7.0	34 31 32	(32) 1.5	19 21 17	(19) 2.0
	1500 µg	80 70 78	(76) 5.3	25 20 21	(22) 2.6	26 25 29	(27) 2.1	26 23 28	(26) 2.5	17 14 14	(15) 1.7
	5000 µg	69 P 78 P 86 P	(78) 8.5	13 P 19 P 20 P	(17) 3.8	31 P 35 P 29 P	(32) 3.1	32 P 28 P 35 P	(32) 3.5	11 P 19 P 18 P	(16) 4.4
Positive controls S9-Mix (+)	Name DoseLevel No. of Revertants	2AA		2AA		2AA		BP		2AA
		1 µg		2 µg		10 µg		5 µg		2 µg
		1095 979 1053	(1042) 58.7	291 266 256	(271) 18.0	120 138 147	(135) 13.7	196 180 174	(183) 11.4	455 444 437	(445) 9.1

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

2AA: 2-Aminoanthracene

BP: Benzo(a)pyrene

P: Test itemprecipita

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative