Fatty acids, C16-18, tetraesters with 3,3'-oxybis[1,2-propanediol]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

14 - 24 Sep 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Oleic acid, monoester with oxybis(propanediol)

EC Number:

256-367-4

EC Name:

Oleic acid, monoester with oxybis(propanediol)

Cas Number:

49553-76-6

IUPAC Name:

3-(2,3-dihydroxypropoxy)-2-hydroxypropyl octadec-9-enoate

Details on test material:

- Name of test material (as cited in study report): Diglycerol monooleate
- Physical state: off-white, yellow paste
- Analytical purity: 100%
- Composition of test material, percentage of components: 6.3% Free polyglycerol, 0.1% Free fatty acids, 2.0% Monoglycerides, 84.0% Diglycerol monoester, 3.5% Triglycerol monoester, 2.5% Diglycerol diester and 0.3% Diglycerides
- Purity test date: 2012-10-23
- Lot/batch No.: 4011768766
- Stability under test conditions: stable under the conditions of the study (no evidence of instability was observed)

Method

Target gene:

his operon (for S. typhimurium) and trp operon (for E. coli)

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:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.

Test concentrations with justification for top dose:

Preliminary cytotoxicity test (Experiment I): 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 µg/plate with and without metabolic activation
Main assay (Experiment II): 333, 667, 1000, 3333, and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was chosen as the dosing vehicle based on the solubility of the test substance and compatibility with the target cells. The test substance was soluble in DMSO at 50 mg/mL, the highest stock concentration that was prepared for use on this study.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

-S9: 2-NF (1 µg/plate, TA 98), SA (2 µg/plate, TA 100 and TA 1535), ICR-191 (2 µg/plate, TA 1537), 4-NQO (1 µg/plate, WP2 uvrA); +S9: BaP (2.5 µg/plate, TA 98); 2-AA (2.5 µg/plate, TA 100, TA 1535 and TA 1537; 25 µg/plate, WP2 uvrA)

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: acridine mutagen ICR-191 and 2-aminoanthracene

Remarks:

BaP: benzo[a]pyrene; 4-NQO: 4-nitroquinoline N-oxide; acridine mutagen ICR-191: ICR-191; SA: sodium azide; 2-aminoanthracene: 2-AA; 2-NF: 2-nitrofluorene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48-72 h

NUMBER OF REPLICATIONS: duplicate plates in the preliminary cytotoxicity test and triplicate plates in the main assay

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

Evaluation criteria:

Criteria for a positive response:
- Strains TA1535 and TA1537: data were judged positive if the increase in mean revertants at the highest numerical dose response was ≥ 3.0-fold the mean concurrent negative control value (vehicle control). This increase in the mean number of revertants per plate had to be accompanied by a dose response associated with increasing concentrations of the test substance unless observed at the top dose level only.
- Strains TA98, TA100 and WP2uvrA: data sets were judged positive if the increase in mean revertants at the highest numerical dose response was ≥ 2.0-fold the mean concurrent negative control value (vehicle control). This increase in the mean number of revertants per plate has to be accompanied by a dose response associated with increasing concentrations of the test substance unless observed at the top dose level only.

Statistics:

For each tester strain, the mean of the number of revertants and the standard deviations were calculated.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate: TA 1537 (-S9) and TA 1535 (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in the absence of S9 mix, test substance precipitation was observed starting at 3333 μg/plate for all tester strains with the exception of WP2 uvrA where precipitation was observed only at 5000 μg/plate. In the presence of S9 mix, precipitation was observed starting at 3333 μg/plate for all tester strains with the exception of TA 100 and WP2uvrA. Precipitation was not observed with WP2 uvrA in the activated condition and was only observed at 5000 μg/plate with tester strain TA 100. However, none of the precipitates prevented accurate colony counting.

RANGE-FINDING/SCREENING STUDIES: in the preliminary cytotoxicity test, a 50% reduction in revertant colonies was observed with TA 1537 in the presence of metabolic activation starting at 3333 µg/plate. No cytotoxicity was observed in any of the other tester strains after treatment with concentrations ranging from 33.3 to 5000 µg/plate in the presence and absence of metabolic activation, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA: the mean revertant number of the vehicle and positive controls was within their respective historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY: a >50% reduction in revertant colonies was observed at 5000 μg/plate for both TA1537 in the absence of S9 activation, as well as for TA1535 in the presence of S9 activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table1. Test results of first experiment – preliminary cytotoxicity test

Bacterial Reverse Mutation Assay, mean revertant colonies/plate (n=2 ± SD)
EXPERIMENT I (plate incorporation)
S9-Mix	Without
Concentration (per plate)	TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
SC (DMSO)	17 ± 1	111 ± 4	16 ± 0	5 ± 1	31 ± 1
Test item
33.3 µg	20 ± 4	98 ± 6	20 ± 2	14 ± 1	38 ± 7
66.7 µg	24 ± 6	110 ± 11	11 ± 3	10 ± 1	33 ± 8
100 µg	23 ± 6	101 ± 16	14 ± 7	3 ± 3	33 ± 4
333 µg	25 ± 5	91 ± 0	14 ± 4	5 ± 0	35 ± 4
667 µg	21 ± 4	84 ± 18	18 ± 4	4 ± 1	39 ± 6
1000 µg	28 ± 1	100 ± 6	9 ± 6	7 ± 0	38 ± 14
3333 µg	22 ± 4P	80 ± 4P	9 ± 6P	10 ± 7P	39 ± 8
5000 µg	23 ± 7P	89 ± 1P	18 ± 0P	6 ± 6P	26 ± 4P
PC
2-NF (1.0 µg)	143 ± 0	-	-	-	-
SA (2.0 µg)	-	1295 ± 50	1115 ± 115	-	-
ICR-191 (2.0 µg)	-	-	-	1440 ± 53	-
4-NQO (1.0 µg)	-	-	-	-	540 ± 13
S9-Mix	With
Concentration (per plate)	TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
SC (acetone)	33 ± 8	139 ± 1	12 ± 2	15 ± 4	56 ± 2
Test item
33.3 µg	41 ± 9	132 ± 4	17 ± 1	11 ± 1	61 ± 4
66.7 µg	39 ± 6	135 ± 8	11 ± 3	10 ± 1	49 ± 5
100 µg	27 ± 4	140 ± 11	11 ± 1	13 ± 0	54 ± 8
333 µg	33 ± 4	116 ± 7	11 ± 1	12 ± 3	55 ± 8
667 µg	32 ± 4	73 ± 7	12 ± 2	11 ± 4	45 ± 4
1000 µg	34 ± 0	87 ± 20	11 ± 1	12 ± 6	49 ± 1
3333 µg	31 ± 6P	80 ± 9	10 ± 4P	5 ± 6P	47 ± 4
5000 µg	25 ± 1P	94 ± 16P	8 ± 2P	7 ± 1P	51 ± 8
PC
BP (2.5 µg)	485 ± 87	-	-	-	-
2-AA (2.5 µg)	-	2501 ± 137	196 ± 1	181 ± 45	-
2-AA (25 µg)	-	-	-	-	257 ± 8
SC = Solvent control; PC = Positive control substances; SD = standard deviation; BaP: benzo[a]pyrene; 4-NQO: 4-nitroquinoline N-oxide; acridine mutagen ICR-191: ICR-191; SA: sodium azide; 2-aminoanthracene: 2-AA; 2-NF: 2-nitrofluorene P = precipitate; M = manual counting necessary

Table 2. Test results of second experiment – Main assay

Bacterial Reverse Mutation Assay, mean revertant colonies/plate (n=3 ± SD)
EXPERIMENT II (plate incorporation)
S9-Mix	Without
Concentration (per plate)	TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
SC (DMSO)	18 ± 2	86 ± 7	11 ± 8	8 ± 2	25 ± 3
Test item
333 µg	18 ± 3	74 ± 2	6 ± 4	5 ± 1	25 ± 5
667 µg	14 ± 3	74 ± 3	9 ± 3	4 ± 1	28 ± 6
1000 µg	16 ± 1	69 ± 2	8 ± 3	5 ± 1	24 ± 2
3333 µg	18 ± 5P	78 ± 16P	9 ± 3P	6 ± 3P	26 ± 4
5000 µg	19 ± 3P	74 ± 3P	6 ± 4P	3 ± 1P, M	26 ± 3P
PC
2-NF (1.0 µg)	150 ± 3	-	-	-	-
SA (2.0 µg)	-	1031 ± 19	956 ± 37	-	-
ICR-191 (2.0 µg)	-	-	-	1649 ± 123	-
4-NQO (1.0 µg)	-	-	-	-	456 ± 90
S9-Mix	With
Concentration (per plate)	TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
SC (acetone)	28 ± 6	99 ± 11	15 ± 2	5 ± 1	33 ± 1
Test item
333 µg	27 ± 1	94 ± 24	8 ± 2	9 ± 2	32 ± 5
667 µg	23 ± 4	72 ± 5	7 ± 3	6 ± 1	29 ± 3
1000 µg	20 ± 4P	72 ± 13	8 ± 2	5 ± 4	29 ± 4
3333 µg	19 ± 2P	63 ± 6P	8 ± 3P	5 ± 3P	35 ± 9P
5000 µg	22 ± 6P	63 ± 7P	6 ± 4P	4 ± 2P	30 ± 10P
PC
BP (2.5 µg)	284 ± 12	-	-	-	-
2-AA (2.5 µg)	-	2337 ± 174	155 ± 5	119 ± 22	-
2-AA (25 µg)	-	-	-	-	200 ± 19
SC = Solvent control; PC = Positive control substances; SD = standard deviation; BaP: benzo[a]pyrene; 4-NQO: 4-nitroquinoline N-oxide; acridine mutagen ICR-191: ICR-191; SA: sodium azide; 2-aminoanthracene: 2-AA; 2-NF: 2-nitrofluorene P = precipitate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative