Reaction products of 1,4-cyclohexanedimethanol with propylene oxide and ammonia

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:

2010-04-22 to 2010-05-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals.

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 1,4-cyclohexanedimethanol, propylene oxide and ammonia

Cas Number:

1220986-58-2

Molecular formula:

C11H24N2O

IUPAC Name:

Reaction products of 1,4-cyclohexanedimethanol, propylene oxide and ammonia

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Jeffamine RFD-270 Amine
- Substance type: Colourless liquid
- Physical state: Liquid
- Lot/batch No.: 8802-8-9
- Analytical purity: 92%
- Composition of test material, percentage of components: 0.02 wt% water
- Purity test date: 2010-07-15
- Storage condition of test material: Sample stored in cool, well-ventilated storage area prior to testing

Specific details on test material used for the study:

- Name of test material (as cited in study report): Jeffamine RFD-270 Amine
- Substance type: Colourless liquid
- Physical state: Liquid
- Lot/batch No.: 8802-8-9
- Analytical purity: 92%
- Composition of test material, percentage of components: 0.02 wt% water
- Purity test date: 2010-07-15
- Storage condition of test material: Sample stored in cool, well-ventilated storage area prior to testing

Method

Target gene:

histidine (Salmonella strains) and tryptophan (E.coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1 (initial toxicity - mutation assay): 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 (confirmatory mutagenicity assay): 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: A solubility test was conducted using water to determine the highest soluble or workable stock concentration, up to 50 mg/mL.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 ; TA98, TA1535 and TA1537 = 1.0 µg/plate ; TA100 = 2.0 µg/plate ; WP2 uvrA = 10 µg/plate

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9 ; TA98 = 1.0 µg/plate

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 ; TA100, TA1535 = 1.0 µg/plate

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 ; TA1537 = 75 µg/plate

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 ; WP2 uvrA = 1000 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation method

DURATION:
- Exposure duration: 48 to 72 hours

The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate.
Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity.

NUMBER OF REPLICATIONS:
Initial toxicity-mutation assay: vehicle control, positive controls and eight dose levels of the test article were plated, two plates per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9.
Confirmatory mutagenicity assay: All dose levels of test article (minimum five), vehicle control and positive controls were plated in triplicate.

NUMBER OF CELLS EVALUATED: Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of approximately 0.3E09 cells per milliliter.

DETERMINATION OF CYTOTOXICITY: reduction in mean number of revertants

Evaluation criteria:

For the test article 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 article.
Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.
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 will be evaluated as negative, if it is neither positive nor equivocal.

Statistics:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

(no precipitate and no toxicity were observed)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

(no precipitate and no toxicity were observed)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: During incubation of the assay plates from the initial toxicity-mutation assay, a technical error in deviation from the protocol was discovered in which the assay plates from the initial assay did not incubate for a full 48-hour period. The entire initial toxicity-mutation was not evaluated but was retested. In the retest of the initial toxicity-mutation assay, the maximum dose tested was 5000 µg/plate; this dose level was achieved using a concentration of 50 mg/mL and a 100 µL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate. No positive mutagenic responses were observed with any of the tester strains in the presence of S9 activation or with tester strains TA98, TA100, TA1535 and WP2 uvrA in the absence of S9. No precipitate was observed. Toxicity was observed at 5000 µg per plate with tester strain TA100 in the absence of S9 activation only. Due to an unacceptable vehicle control value, tester strain TA1537 in the absence of S9 activation was not evaluated for mutagenicity but was retested based on the precipitate and toxicity profiles observed. Based on the findings of the retest of the initial toxicity-mutation assay, the maximum dose plated in the retest and confirmatory mutagenicity assays was 5000 µg/plate.
- Water solubility: The test article was considered to be soluble in water.
- Sterility results: No contaminant colonies were observed on the sterility plates for the vehicle control, the test article dilutions and the S9 and Sham mixes.

COMPARISON WITH HISTORICAL CONTROL DATA: The number of revertants per plate for the negative and positive control substances were in the range of the historical control values of the laboratory.

Any other information on results incl. tables

In Experiment B2 an unacceptable vehicle control value has been observed and tester strain TA1537 in the absence of S9 activation was not evaluated for mutagenicity but was retested in Experiment B3 based on the precipitate and toxicity profiles observed. Based on the findings of the retest of the initial toxicity-mutation assay, the maximum dose plated in the second retest and confirmatory mutagenicity assays was 5000 μg per plate. In Experiment B3 (Second Retest of the Initial Toxicity-Mutation Assay), no positive mutagenic response was observed with tester strain TA1537 in the absence of S9 activation. The dose levels tested were 50, 150, 500, 1500 and 5000 μg per plate. Neither precipitate nor appreciable toxicity was observed. A reduction in revertants was observed at 5000 μg per plate with tester strain TA1535 in the presence of S9 activation.

Confirmatory mutagenicity assay:

The dose levels tested were 50, 150, 500, 1500 and 5000 µg per plate. Neither precipitate nor background lawn toxicity was observed. In this experiment no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

Applicant's summary and conclusion

Conclusions:

The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, the substance did not cause a positive mutagenic response in either the presence or absence of Aroclor-induced rat liver S9.