Reaction mass of undec-8-enal and undec-9-enal and undec-10-enal

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:

02-12-2016 to 19-01-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

May 30, 2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of undec-8-enal and undec-9-enal and undec-10-enal

EC Number:

907-237-7

Molecular formula:

C11H20O

IUPAC Name:

Reaction mass of undec-8-enal and undec-9-enal and undec-10-enal

Method

Target gene:

Histidine and tryptophan genes

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:

Rat liver S9-mix

Test concentrations with justification for top dose:

Experiment 1 (plate incorporation method)

Without S9: Initial: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate ; Amended: TA100, TA1535 and TA1537 (without S9): 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.
With S9: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate for TA100, TA98, TA1537 and E.coli. For TA1535: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
Results from the first mutation test showed significant toxicity of the test item to the bacterial tester strains and consequently an insufficient number of non-toxic dose levels.

Experiment 2 (pre-incubation method)

Without S9: 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate (based on results of experiment 1).
With S9: 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate (based on results of experiment 1).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: according to guidelines

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other:

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment 1: Plate incorporation methodology
Experiment 2: Pre-incubation methodology

DURATION
- Preincubation period: with and without S9-mix 20 minutes (prior to exposure in experiment 2 only)
- Exposure duration: 48 hours (experiment 1 and 2)

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn measurement and reduction in revertant colonies compared to the controls

Evaluation criteria:

There are several criteria for determining a positive result. Any one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: TA1535, TA1537, TA98, TA100 and WP2 uvrA

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 150 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

other: TA1535, TA1537, TA98, TA100 and WP2uvrA

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 5 μg/plate (TA 1535), 15 μg/plate (TA 1537 & TA 100) and 50 μg/plate (TA98 and WP2uvrA).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
-Precipitation: No precipitation was observed.

HISTORICAL CONTROL DATA:
-A history profile of vehicle, untreated and positive control values (reference items) can be found in the attached illustration.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
-In exp 1 (plate incorporation method): the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 50 μg/plate in the absence of S9-mix and 150 μg/plate in the presence of S9-mix. Consequently the toxic limit was employed as the maximum dose in the second mutation test.
-In exp 2 (pre-incubation method): The test item induced a stronger toxic response in the second mutation test (pre-incubation method), with weakened bacterial background lawns noted in the absence of S9-mix from 5 μg/plate (TA1535), 15 μg/plate (TA100 and TA1537) and 50 μg/plate (TA98 and WP2uvrA). In the presence of S9-mix, weakened bacterial background lawns were noted to all of the tester strains from 150 μg/plate.

ADDITIONAL INFORMATION ON TEST RESULTS
-In exp 1 (plate incorporation method): There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).
-In exp 2 (pre-incubation method): no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix). Statistically significant increases in TA1535 revertant colony frequency were observed in the second mutation test at 150 μg/plate in the presence of S9-mix. These increases were considered to have no biological relevance because weakened bacterial background lawns were also noted at the same dose level. Therefore these responses would be due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, Intreleven aldehyde was determined to be not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The mutagenic activity of Intreleven aldehyde was evaluated in accordance with OECDG 471 and GLP principles. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to nine dose levels, in triplicate, both with and without the addition of S9-mix. Based on results of experiment 1 (plate incorporation), the dose range used for experiment 2 (pre-incubation method) was between 0.05 and 150 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants and background lawn, was observed. In the first mutation test (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 50 μg/plate in the absence of S9-mix and 150 μg/plate in the presence of S9-mix. Consequently the toxic limit was employed as the maximum dose in the second mutation test. Weakened bacterial background lawns were noted in the absence of S9-mix from 5 μg/plate (TA1535), 15 μg/plate (TA100 and TA1537) and 50 μg/plate (TA98 and WP2uvrA) in the second experiment. In the presence of S9-mix, weakened bacterial background lawns were noted to all of the tester strains from 150 μg/plate. These increases were considered to have no biological relevance. No precipitation was observed at any of the concentrations. Adequate negative and positive controls were included. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) for both experiments. Intreleven aldehyde was therefore considered to be non-mutagenic under the conditions of this test.