Formaldehyde, oligomeric reaction products with 4,4'-isopropylidenediphenol and diethylenetriamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Formaldehyde, oligomeric reaction products with 4,4'-isopropylidenediphenol and diethylenetriamine

EC Number:

500-263-6

EC Name:

Formaldehyde, oligomeric reaction products with 4,4'-isopropylidenediphenol and diethylenetriamine

Cas Number:

77138-45-5

Molecular formula:

(C15H16O2.C4H13N3.CH2O)x

IUPAC Name:

2-[({2-[(2-aminoethyl)amino]ethyl}amino)methyl]-4-(2-{3-[({2-[(2-aminoethyl)amino]ethyl}amino)methyl]-4-hydroxyphenyl}propan-2-yl)phenol; 2-[({2-[(2-aminoethyl)amino]ethyl}amino)methyl]-4-[2-(4-hydroxyphenyl)propan-2-yl]phenol; bis(2-aminoethyl)amine

Test material form:

liquid

Method

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

Vehicle / solvent:

DMSO

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:

The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976).

Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).

Salmonella tester strains were derived from Dr. Bruce Ames’ cultures; E. coli tester strains

Evaluation criteria:

All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene.
All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10 - 50; TA100, 80 - 240; TA1535, 5 - 45; TA1537, 3 - 21; WP2 uvrA, 10 - 60.

To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x109 cells/mL.
The mean of each positive control must exhibit at least a 3.0-fold increase in the number of revertants over the mean value of the respective vehicle control.
A minimum of three non-toxic dose levels is required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) At least a moderate reduction in the background lawn (background code 3, 4 or 5).

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

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, EK110 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, EK110, 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. No precipitate was observed. Toxicity was observed beginning at 333, 1000 or 3333 μg per plate. Based upon these results, the maximum doses tested in the mutagenicity assay were 5000 μg per plate (for tester strains TA98 and WP2 uvrA in the presence and absence of S9 activation and TA1537 in the presence of S9 activation) and 500 μg per plate (for tester strains TA100 and TA1535 in the presence and absence of S9 activation and TA1537 in the absence of S9 activation).

In the mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate (for tester strains TA98 and WP2 uvrA in the presence and absence of S9 activation and TA1537 in the presence of S9 activation) and 1.50, 5.00, 15.0, 50.0, 150 and 500 μg per plate (for tester strains TA100 and TA1535 in the presence and absence of S9 activation and TA1537 in the absence of S9 activation). No precipitate was observed. Toxicity was observed beginning at 150, 500 or at 1500 μg per plate with all conditions except tester strain TA1537 in the absence of S9 activation. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Due to lack of toxicity with tester strain TA1537 in the absence of S9 activation, the maximum dose tested in the retest of mutagenicity assay was 5000 μg per plate for tester strain TA1537 in the absence of S9 activation.

In the retest of mutagenicity assay, the dose levels tested were 0.500, 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. No precipitate was observed. Toxicity was observed beginning at 1500 μg per plate. No positive mutagenic response was observed with tester strain TA1537 in the absence of S9 activation.

These results indicate EK110 was negative for the 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.