reaction mass of 2-[[1-(chloromethyl)-2-[[4-(oxiran-2-ylmethoxymethyl)cyclohexyl]methoxy]ethoxy]methyl]oxirane and cis-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane and trans-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane

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:

2018

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Remarks:

No deviations occurred that impacted the integrity of the study.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[[1-(chloromethyl)-2-[[4-(oxiran-2-ylmethoxymethyl)cyclohexyl]methoxy]ethoxy]methyl]oxirane

Molecular formula:

C17H29ClO5

IUPAC Name:

2-[[1-(chloromethyl)-2-[[4-(oxiran-2-ylmethoxymethyl)cyclohexyl]methoxy]ethoxy]methyl]oxirane

Constituent 2

Reference substance name:

cis-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane

Cas Number:

1197197-64-0

Molecular formula:

C14H26O4

IUPAC Name:

cis-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane

Constituent 3

Reference substance name:

trans-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane

Cas Number:

158307-92-7

Molecular formula:

C14H26O4

IUPAC Name:

trans-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane

impurity 1

Reference substance name:

[4-(oxiran-2-ylmethoxymethyl)cyclohexyl]methanol

Molecular formula:

C11H20O3

IUPAC Name:

[4-(oxiran-2-ylmethoxymethyl)cyclohexyl]methanol

impurity 2

Reference substance name:

[4-[[3-chloro-2-(oxiran-2-ylmethoxy)propoxy]methyl]cyclohexyl]methanol

Molecular formula:

C14H25ClO4

IUPAC Name:

[4-[[3-chloro-2-(oxiran-2-ylmethoxy)propoxy]methyl]cyclohexyl]methanol

impurity 3

Reference substance name:

2-[[3-chloro-2-[[4-[[3-chloro-2-(oxiran-2-ylmethoxy)propoxy]methyl]cyclohexyl]methoxy]propoxy]methyl]oxirane

Molecular formula:

C20H34Cl2O6

IUPAC Name:

2-[[3-chloro-2-[[4-[[3-chloro-2-(oxiran-2-ylmethoxy)propoxy]methyl]cyclohexyl]methoxy]propoxy]methyl]oxirane

impurity 4

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3M Company
- Expiration date of the lot/batch: 26 March, 2022
- Purity test date: 05 December, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article was dissolved in dimethyl sulfoxide (DMSO)

Method

Target gene:

Histidine operon, tryptophan operon

Species / strain

Species / strain / cell type:

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

Remarks:

E. coli WP2uvrA

Metabolic activation:

with and without

Metabolic activation system:

Rat Liver S9 Mix

Test concentrations with justification for top dose:

54, 164, 512, 1600 and 5000 ug/plate. The top dose was chosen based on OECD test guideline recommendations and slight precipitation at 5000 ug/plate in several plates without inducing cytotoxicity.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: Sodium azide, ICR-191, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:in agar (plate incorporation)
- Cell density at seeding (if applicable): 10^9 cells/mL

DURATION
- Preincubation period: None
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

SELECTION AGENT (mutation assays): Tryptophan and histidine-minimal agar.

DETERMINATION OF CYTOTOXICITY
- Method: bacterial background lawn

Rationale for test conditions:

Per OECD Test Guideline 471

Evaluation criteria:

No formal hypothesis testing was done.
A test item is considered negative (not mutagenic) in the test if:
- The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two
(2) times the concurrent control, and unless the total number of revertants in tester strain
TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
- The negative response should be reproducible in at least one follow up experiment.
A test item is considered positive (mutagenic) in the test if:
- The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2)
times the concurrent control, or the total number of revertants in tester strain TA1535,
TA1537 or TA98 is greater than three (3) times the concurrent control.
- In case a repeat experiment is performed when a positive response is observed in one of
the tester strains, the positive response should be reproducible in at least one follow up
experiment.

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

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

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

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

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Based on the results of the study, the test article is positive in the Ames assay in the presence and absence of metabolic activation.

Executive summary:

The mutagenic potential of the test article was evaluated in the bacterial reverse mutation assay (Ames assay) with S. typhimurium strains TA98, TA100, TA1535, and TA1537 and E.coli strain WP2 uvrA in the presence and absence of metabolic activation. The study was conducted according to OECD 471 in compliance with OECD GLP. Based on the results of a dose-range finding test, the test article was tested in the mutation assay at a concentration range of 52 to 5000 μg/plate in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the absence of S9-mix, the test item induced dose-related increases in the number of revertant colonies compared to the solvent control in three tester strains (TA100, WP2uvrA and TA1535). The increases observed were above the laboratory historical control data range, and were up to 7.9-, 3.0-, and 16-fold the concurrent vehicle controls in the tester strains TA100, WP2uvrA and TA1535, respectively.

In the presence of S9-mix, the test item induced dose-related increases in the number of revertant colonies compared to the solvent control in three tester strains (TA100, WP2uvrA and TA1535). The increases observed were above the laboratory historical control data range, and were up to 11-, 2.6-, and 112-fold the concurrent vehicle controls in the tester strains TA100, WP2uvrA and TA1535, respectively.

The two other bacterial strains showed negative responses over the entire dose range, i.e. no dose related increase in the number of revertants. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Since 2.6- to 112-fold, dose related increases were observed in three tester strains, both in the absence and presence of S9-mix, these increases are considered biologically relevant and the test article is mutagenic in the absence and presence of S9-mix.

Based on the results of the study, the test article is positive in the Ames assay in the presence and absence of metabolic activation.