Reaction products of Soya and Linseed Oil Fatty Acids with Bisphenol A diglycidylether

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 August 2018 to 20 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-study according to OECD Test Guideline 471

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guideline 471, updated and adopted 21 July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

EC Number:

216-823-5

EC Name:

2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Cas Number:

1675-54-3

Molecular formula:

C21H24O4

IUPAC Name:

2,2'-[propane-2,2-diylbis(4,1-phenyleneoxymethylene)]dioxirane

Constituent 2

Reference substance name:

Fatty acids, linseed-oil, polymers with bisphenol A, epichlorohydrin and soya fatty acids

EC Number:

613-885-9

Cas Number:

66070-79-9

Molecular formula:

(C15 H16 O2 . C3 H5 Cl O . Unspecified . Unspecified)x

IUPAC Name:

Fatty acids, linseed-oil, polymers with bisphenol A, epichlorohydrin and soya fatty acids

Test material form:

liquid: viscous

Details on test material:

Clear colourless viscous liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: F288I1K351
- Expiration date of the lot/batch: 20 Jan 2020
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:Room temperature, protected from light
- Stability under test conditions: While the Certificate of Analysis indicates an expiry date (20 Jan 2020), it does not indicate the acceptable storage parameters for the test substance. Thus, the stability of the test
substance has not been determined to cover the period of shipment and storage at BioReliance
- Solubility and stability of the test substance in the solvent/vehicle: The test substance formed a clear solution in DMSO at a concentration of approximately 500 mg/mL with sonication at 31.7ºC for 5 minutes
in the solubility test conducted at BioReliance. Soya/Linseed Oil Fatty Acid-BADGE reaction product in DMSO, at concentrations of 106.4 and 0.320 mg/mL, was stable at room temperature for at least 3.6 hours
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: To achieve a solution, the most concentrated dilution was vortexed for 4 to 5 minutes in each assay.

Method

Target gene:

The Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. These strains contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain from the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrB gene, which codes for a protein of the DNA nucleotide excision repair system, resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chi) and biotin (bio) genes (bacteria
require biotin for growth). Tester strains TA98 and TA100 contain the R-factor plasmid, pKM101. Thesestrains are reverted by a number of mutagens that are detected weakly or not at all with the non-R-factor parent strains. pKM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system, which is normally present in these organisms. The tester strain Escherichia coli WP2 uvrA carries the defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagens, which substitute one base for another. Additionally, the strain is deficient in the DNA nucleotide excision
repair system.

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:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg per plate. The top dose was per the guideline Based upon preliminary toxicity assay results, the maximum dose tested in the mutagenicity assay was 5000 μg per plate. In the mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:DMSO for the test substance and all positive controls were diluted in dimethyl sulfoxide (DMSO) except for sodium azide, which was diluted in sterile water
- Justification for choice of solvent/vehicle: DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION: - Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: 1 in the preliminary toxicity assay; 3 in the mutagenicity assay
NUMBER OF CELLS EVALUATED: >/= 0.3 x 10^8 cells/plate
DETERMINATION OF CYTOTOXICITY
- Method: other: Counting of revertant colony numbers and evaluation of the condition of the bacterial background lawn.

Evaluation criteria:

The revertant colony numbers were determined for each plate (counted either manually or by automatic colony counter). The mean and standard deviation of the number of revertants per plate were calculated
and reported.
For each replicate plating, the mean and standard deviation of the number of revertants per plate were ca lculated and are reported.
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:
Strains TA1535 and TA1537
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
Strains TA98, TA100 and WP2 uvrA
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
An equivocal response is an increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited.

Statistics:

According to the test guidelines, the biological relevance of the results is the criterion for the interpretation of the results, and a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

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

Positive controls validity:

valid

Test results 5

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

Positive controls validity:

valid

Any other information on results incl. tables

Mutagenicity Assay without S9 activation, Exposure Method: Plate incorporation assay

 

Strain	Substance	Dose level μg per plate	Mean revertants per plate	Standard Deviation	Ratio treated / solvent
TA98	Soya/Linseed Oil Fatty Acid-BADGE reaction product         DMSO	5000 1500 500 150 50 15   ---	15 13 16 11 9 11   12	2 5 3 2 2 5   4	1.3 1.1 1.3 0.9 0.8 0.9   ---
TA100	Soya/Linseed Oil Fatty Acid-BADGE reaction product         DMSO	5000 1500 500 150 50 15   ---	927 761 657 548 296 167   87	51 12 63 25 6 17   7	10.7 8.7 7.6 6.3 3.4 1.9
TA1535	Soya/Linseed Oil Fatty Acid-BADGE reaction product         DMSO	5000 1500 500 150 50 15   ---	46 29 34 26 19 16   12	1 5 7 4 4 3   5	3.8 2.4 2.8 2.2 1.6 1.3
TA1537	Soya/Linseed Oil Fatty Acid-BADGE reaction product         DMSO	5000 1500 500 150 50 15   ---	12 8 7 8 8 6   8	4 0 3 2 2 1   6	1.5 1.0 0.9 1.0 1.0 0.8
WP2uvrA	Soya/Linseed Oil Fatty Acid-BADGE reaction product         DMSO	5000 1500 500 150 50 15   ---	69 55 60 43 33 23   25	15 11 10 2 6 1   10	2.8 2.2 2.4 1.7 1.3 0.9
TA98	2NF	1.00	72	19	6.0
TA100	SA	1.00	765	49	8.8
TA1535	SA	1.00	721	59	60.1
TA1537	9AAD	75	546	133	68.3
WP2uvrA	MMS	1000	606	139	24.2

 

 

 

Mutagenicity Assay with S9 activation

Plate incorporation assay

 

Strain	Substance	Dose level μg per plate	Mean revertants per plate	Standard Deviation	Ratio treated / solvent
TA98	Soya/Linseed Oil Fatty Acid-BADGE reaction product         DMSO	5000 1500 500 150 50 15   ---	27 27 23 23 26 24   21	3 5 4 4 5 6   8	1.3 1.3 1.1 1.1 1.2 1.1
TA100	Soya/Linseed Oil Fatty Acid-BADGE reaction product         DMSO	5000 1500 500 150 50 15   ---	978 580 357 152 126 104   95	226 68 34 17 12 6   3	10.3 6.1 3.8 1.6 1.3 1.1
TA1535	Soya/Linseed Oil Fatty Acid-BADGE reaction product         DMSO	5000 1500 500 150 50 15   ---	416 458 222 50 16 18   16	77 30 11 5 4 5   5	26.0 28.6 13.9 3.1 1.0 1.1
TA1537	Soya/Linseed Oil Fatty Acid-BADGE reaction product         DMSO	5000 1500 500 150 50 15   ---	11 13 10 9 10 7   11	4 0 0 2 1 2   3	1.0 1.2 0.9 0.8 0.9 0.6
WP2uvrA	Soya/Linseed Oil Fatty Acid-BADGE reaction product         DMSO	5000 1500 500 150 50 15   ---	80 41 46 39 33 38   27	28 1 13 10 12 8   9	3.0 1.5 1.7 1.4 1.2 1.4
TA98	2AA	1	219	13	10.4
TA100	2AA	2	721	111	7.6
TA1535	2AA	1	88	4	5.5
TA1537	2AA	2	43	6	3.9
WP2uvrA	2AA	15	302	46	11.2

 

Applicant's summary and conclusion

Conclusions:

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, Soya/Linseed Oil Fatty Acid-BADGE reaction
product did cause a positive mutagenic response with tester strains TA100, TA1535 and WP2 uvrA in the presence and absence of S9 activation. The study was concluded to be positive without conducting
a confirmatory (independent repeat) assay because the results were clearly positive; hence, no further testing was warranted

Executive summary:

The test substance, Soya/Linseed Oil Fatty Acid-BADGE reaction product, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of

Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg per plate. Precipitate was observed beginning at 1000 μg per plate with all conditions. Toxicity as reduction in revertant count was observed at 5000 μg per plate with tester strain TA1537 in the absence of S9 activation. Mutagenic responses were observed with tester strains TA100, TA1535 (in the presence and absence of S9 activation) and WP2 uvrA (in the absence of S9 activation). Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.

In the mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. No toxicity was observed. Precipitate was observed beginning at 1500 μg per plate with all conditions. Positive mutagenic responses were observed (2.2- to 28.6-fold, maximum increases, outside the historical control limits) with tester strains TA100, TA1535 and WP2 uvrA in the presence and absence of S9 activation.

These results indicate Soya/Linseed Oil Fatty Acid-BADGE reaction product was positive for the ability to induce reverse mutations at selected loci of selected strains of Salmonella typhimurium (TA100 and

TA1535) and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.