Reaction mass of 4,4'-Isopropylidenediphenol, 1-chloro-2,3-epoxypropane and ethylenediamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 September 2016 to 26 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD Guideline 471, updated and adopted 21 July 1997 ISO/IEC 17025:2005

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)

GLP compliance:

yes

Remarks:

Refer to main study report

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

EDA-BADGE-EDA

Cas Number:

854009-15-7

Molecular formula:

C25H40N4O4

IUPAC Name:

EDA-BADGE-EDA

Constituent 2

Reference substance name:

EDA-BADGE-EDA-BADGE-EDA

Molecular formula:

C48H72N6O8

IUPAC Name:

EDA-BADGE-EDA-BADGE-EDA

Constituent 3

Reference substance name:

EDA-BADGE α-glycol

Molecular formula:

C23H34N2O5

IUPAC Name:

EDA-BADGE α-glycol

Constituent 4

Reference substance name:

reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine

IUPAC Name:

reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine

Constituent 5

Reference substance name:

Unidentified reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine

IUPAC Name:

Unidentified reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine

Test material form:

liquid: viscous

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material: BBF01102V1 (provided by Sponsor)- Expiration date of the lot/batch: 01 January 2021 (per Sample Label)STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Room temperature, protected from light- Solubility and stability of the test substance in the solvent/vehicle: Water initially was the vehicle of choice based on solubility of the test substance and compatibility with the target cells. After sonication at 31.2ºC for five minutes, the test substance formed a clear solution in water at a concentration of approximately 50 mg/mL in the solubility test conducted at BioReliance. A second solubility test also was conducted in DMSO, and the test substance formed a clear solution in DMSO at a concentration of approximately 500 mg/mL. Subsequently, DMSO was used as the vehicle in the retest of the preliminary toxicity and mutagenicity assays.TREATMENT OF TEST MATERIAL PRIOR TO TESTING- Final preparation of a solid: To achieve a solution, the most concentrated dilution was vortexed for seven minutes and sonicated at 35.3ºC for approximately three minutes in the preliminary toxicity assay. Initially, test substance dilutions were prepared for the mutagenicity assay in water. The most concentrated dilution (15.0 mg/mL) was vortexed for eight minutes, and a solution was achieved with a few undissolved fiber-like particles. However, subsequent dilutions from 0.0500 to 5.00 mg/mL were workable suspensions. Due to solubility issues, the test substance dilution was aborted, and the entire preliminary toxicity assay was retested in DMSO at the Sponsor’s request. In the retest of the preliminary toxicity assay and mutagenicity assay conducted in DMSO, the most concentrated dilution was vortexed for four to eight minutes to achieve a solution.FORM AS APPLIED IN THE TEST (if different from that of starting material): Undissolved fiber-like particles and workable suspension in Water. Solution in DMSO

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. These strains 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 and in the retest of 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.In the mutagenicity assay, the dose levels tested were 0.150, 0.500, 1.50, 5.00, 15.0, 50.0 and 150 µg per plate with tester strains TA98, TA100 and TA1535 in the absence of S9 activation and TA100, TA1535 and TA1537 in the presence of S9 activation; 1.50, 5.00, 15.0, 50.0, 75.0, 100, 150, 200 and 500 μg per plate with tester strain TA98 in the presence of S9 activation; and 1.50, 5.00, 15.0, 50.0, 150, 500 and 1500 μg per plate with tester strains TA1537 and WP2 uvrA in the absence of S9 activation and WP2 uvrA in the presence of S9 activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water and DMSO for the test substance; 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: Water initially was the vehicle of choice based on solubility of the test substance and compatibility with the target cells. DMSO was used for the retest of preliminary toxicity assay and mutagenicity assay based on solubility determined in the initial trial of preliminary toxicity assay.

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 hoursNUMBER OF REPLICATIONS: 2 in the initial-toxicity mutation assay; 3 in the confirmatory mutagenicity assayNUMBER OF CELLS EVALUATED: >/= 0.3 x 10^8 cells/plateDETERMINATION 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 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 TA1537Data 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 uvrAData 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 a biologically relevant 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. A response was evaluated as negative if it was neither positive nor equivocal.

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

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the initial and retest of preliminary toxicity assay, toxicity was observed beginning at concentrations from 10.0 to 667 µg per plate. In the mutagenicity assay, toxicity was observed beginning at 15.0, 50.0 or 500 μg per plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Precipitation: In the initial and retest of preliminary toxicity assay, precipitate was observed at 5000 µg per plate. No precipitate was observed in the mutagenicity assay.HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)See main study report appendix

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

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, 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediamine did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor induced rat liver S9.

Executive summary:

The test substance, 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediamine, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonellatyphimuriumand at the tryptophan locus of Escherichia colistrain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Water initially 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 at 5000 µg per plate. Toxicity was observed beginning at concentrations from 66.7 to 667 µg per plate. Based on these results, the maximum doses tested in the mutagenicity assay were intended to be 1.50 µg per plate with all Salmonella tester strains in the presence and absence of S9 activation and 15.0 µg per plate with tester strain WP2uvrA in the presence and absence of S9 activation. However, due to solubility issues in water during dilution of the test substance for the mutagenicity assay, the dilution was aborted, and the entire preliminary toxicity assay was retested using the same dose levels and DMSO as the vehicle.

In the retest of the preliminary toxicity assay, precipitate was observed at 5000 µg per plate. Toxicity was observed beginning at concentrations from 10.0 to 667 µg per plate. A 1.7-fold increase in revertant counts was observed with tester strain TA98 in the presence of S9 activation. The increase was not clearly dose-responsive; however, the revertant count at the peak of the response was outside of the 95% historical control limit for this test condition, and the dose level above the response exhibited toxicity. Based upon these results, the maximum doses tested in the mutagenicity assay were 150 µg per plate with tester strains TA98, TA100 and TA1535 in the absence of S9 activation and TA100, TA1535 and TA1537 in the presence of S9 activation; 500 μg per plate with tester strain TA98 in the presence of S9 activation; and 1500 μg per plate with tester strains TA1537 and WP2 uvrA in the absence of S9 activation and WP2uvrA in the presence of S9 activation, using DMSO as the vehicle.

In the mutagenicity assay, the dose levels tested were 0.150, 0.500, 1.50, 5.00, 15.0, 50.0 and 150 µg per plate with tester strains TA98, TA100 and TA1535 in the absence of S9 activation and TA100, TA1535 and TA1537 in the presence of S9 activation; 1.50, 5.00, 15.0, 50.0, 75.0, 100, 150, 200 and 500 μg per plate with tester strain TA98 in the presence of S9 activation; and 1.50, 5.00, 15.0, 50.0, 150, 500 and 1500 μg per plate with tester strains TA1537 and WP2uvrA in the absence of S9 activation and WP2uvrA in the presence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 15.0, 50.0 or 500 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

These results indicate 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediamine was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonellatyphimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

All criteria for a valid study were met as described in the protocol.