Reaction products of 4,4'-methylenebis(cyclohexylamine) and 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

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)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with 4,4'-methylenebis(cyclohexylamine)

EC Number:

500-103-5

EC Name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with 4,4'-methylenebis(cyclohexylamine)

Cas Number:

38294-67-6

Molecular formula:

C

IUPAC Name:

Reaction products of 4,4'-methylenebis(cyclohexylamine) and 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Test material form:

liquid: viscous

Details on test material:

Sample ID :PACM BADGE adduct
Chemical Name :4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with 4,4'-methylenebis(cyclohexylamine)
Physical State liquid
CAS # 38294-67-6
EU # 500-103-5
Manufacturer Air Products and Chemicals, Inc.
Batch # / Lot # 20160226-PDU-PACM 828

Method

Target gene:

WP2uvrA)

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver S9-mix induced Aroclor 1254)

Test concentrations with justification for top dose:

Seven concentrations of the test item, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

Vehicle / solvent:

DMSO

Controls

Controls 1

Positive controls:

yes

Positive control substance:

sodium azide

Controls 2

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Controls 3

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Controls 4

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

PACM BADGE adduct was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay both in the absence and presence of S9-mix (rat liver S9-mix induced Aroclor 1254). An additional direct plate assay was performed in the absence and presence of S9-mix.
The study procedures described in this report were based on the most recent OECD, EC and MITI guidelines.
Batch 20160226-PDU-PACM 828 of the test item was dissolved in dimethyl sulfoxide.
In the first mutation assay, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. The test item precipitated on the plates at dose levels of 1600 and 5000 μg/plate. Toxicity was observed in all tester strains in at least 5 concentrations both in the absence and presence of S9-mix. Since not enough dose levels without toxicity were tested an additional experiment was performed.
In the additional experiment, the test item was tested up to concentrations of 52 μg/plate in the absence and presence of S9-mix. The test item did not precipitate on the plates at this dose level. Toxicity was observed in all tester strains.
In the second mutation assay, the test item was again tested up to concentrations of 52 μg/plate in the pre-incubation assay. The test item did not precipitate on the plates at this dose level. Toxicity was observed in all tester strains.
The test item did not induce a significant and biologically relevant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.
In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)
Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)
The Salmonella typhimurium strains were regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment (Ref.1). The strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.

Statistics:

Colony counting
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually. Evidence of test article precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
Interpretation
Acceptability of the assay
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at WIL Research Europe.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Results and discussion

Test results

Test results 1

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

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

Untreated negative 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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

First mutation experiment: Direct plate assay
The test item was tested in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA with concentrations of 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix.
Precipitate
Precipitation of the test item on the plates was observed at the start and at the end of the incubation period at the concentrations of 1600 and 5000 μg/plate.
Toxicity
To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. Toxicity was observed in all tester strains at dose levels of 52 μg/plate and upwards in the absence and presence of S9-mix. In tester strains TA1537 (absence of S9-mix) and TA100 (absence and presence of S9-mix) toxicity was also observed at the dose level of 17 μg/plate.
Mutagenicity
No increase in the number of revertants was observed upon treatment with PACM BADGE adduct under all conditions tested.
Additional experiment: Direct plate assay
Since too many dose levels showed toxicity in the first experiment, the experiment was repeated. The test item was tested in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA with concentrations of 0.056, 0.18, 0.55, 1.7, 5.4, 17 and 52 μg/plate in the absence and presence S9-mix.
Precipitate
Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in all tester strains.
Toxicity
Toxicity was observed in all tester strains at the dose level of 52 μg/plate. In tester strain TA100, a decrease in the number of revertants was also observed at dose levels of 17 μg/plate (absence and presence of S9) and 5.4 μg/plate (absence of S9).
Mutagenicity
In tester strain TA1537, an exceedingly low (2 revertants/plate versus historical mean of 7 revertants/plate) solvent control value resulted in a mean number of revertant colonies three (3) times the concurrent control at the mid dose of 1.7 μg/plate without metabolic activation (Table 2). However, no similar 3-fold increase or any dose-related increases in revertants/plate were observed in TA1537 without metabolic activation in the independently repeated experiment. All other bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.
Second mutation assay: Pre-incubation assay
To obtain more information about the possible mutagenicity of the test item, a second mutation experiment was performed in the absence and presence of S9-mix. Based on the results of the first mutation experiment, the test item was tested up to concentrations of 52 μg/plate.
Precipitate
Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in all tester strains.
Toxicity
In the absence of S9-mix, toxicity was observed in all tester strains at dose levels of 5.4 μg/plate and upwards. Except for tester strain WP2uvrA where toxicity was observed at dose levels of 17 and 52 μg/plate. In the presence of S9-mix, toxicity was observed in all tester strains at the dose level of 52 μg/plate
Mutagenicity
No increase in the number of revertants was observed upon treatment with PACM BADGE adduct under all conditions tested.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that PACM BADGE adduct is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Based on the results of this study it is concluded that PACM BADGE adduct is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.