9-Decenamide, N,N-dimethyl-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro: Gene mutation (Bacterial reverse mutation assay / Ames test): S.typhimurium TA1537, Ta98, TA100, TA1535 and E.coli WP2uvrA. Negative with and without activation (OECD guideline 471, GLP) Chromosome aberration: negative in CHO cells (read-across to N,N-Dimethyldecan-1-amide, mixture with N,N-Dimethyloctan-1-amide) Mammalian cell gene mutation:negative in HGPRT assay performed according to OECD TG 476 (read-across to N,N-Dimethyldecan-1-amide, mixture with N,N-Dimethyloctan-1-amide). In vivo: Chromosome aberration (micronucleus assay): negative (according to OECD 474; 3 concentrations ( 75, 150, 300 mg/kg ) orally administered to male and female mice).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 March 2014 to 20 May 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD 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

Target gene:

Histidine and Tryptophan

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

The concentrations tested in the initial assay were 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/plate.
The concentrations tested in the confirmatory assay were 50, 100, 250, 500, 1000, 2500 and 5000 µg/plate using the plate incorporation method.

Vehicle / solvent:

-Solvent used: Dimethyl sulphoxide
- Justification for choice of solvent/vehicle: Specified in the study protocol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see remarks

Remarks:

ICR 191 acridine, 2-nitroflourene, sodium azide, 4-nitroquinolene-N-oxide, 2-aminoanthracene. Depending on tester strain and presence or absence of S9.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Exposure duration: 2 days
- Expression time (cells in growth medium): 2 days

NUMBER OF REPLICATIONS: Triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Growth of background lawn of bacteria.

Evaluation criteria:

Criteria for Acceptability and Interpretation of Assay
The following criteria were used as guidelines in evaluating the activity of a testsubstance in this system. These criteria are not absolute. The Study Director, based on sound scientific judgment, may take additional factors into consideration when determining the final test results.

Assay Acceptance Criteria
The vehicle and positive control substance plates for the current study were compared against revertant count ranges that are found in historical data ranges.

Criteria for Positive Response
The test substance was considered positive for mutagenicity if it induced an increase of revertants per plate with increasing concentration. The increases should be at least two times the vehicle control substance background frequency for strains with high spontaneous levels (i.e., TA100) and three times for those with low spontaneous levels (TA1537, TA98, TA1535 and WP2 uvrA). These increases should be seen in at least two or more successive concentrations or the response should be repeatable at a single concentration.

Criteria for Negative Response
The testsubstance was considered to be negative for inducing mutagenicity if it did not induce a response which fulfills the criteria for a positive response.

Criteria for Equivocal Response
Cases which did not clearly fit into the positive or negative criteria may be judged equivocal. In these cases the Study Director, based on sound scientific judgment, may take additional factors into consideration in evaluating the test results.

Statistics:

None performed

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

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:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The concentrations tested in the initial assay were 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/plate. Precipitates were not observed in any strain either with or without metabolic activation. Cytotoxicity (i.e., reduction in the background lawn and/or mean number of revertant colonies) was observed at ≥1000 µg/plate in TA1537 with and without metabolic activation, and ≥2500 µg/plate in TA98, TA100, TA1535, and WP2 uvrA with and without metabolic activation. Criteria for a negative response were met for all tester strains with and without metabolic activation.

The concentrations tested in the confirmatory assay were 50, 100, 250, 500, 1000, 2500 and 5000 µg/plate using the plate incorporation method. Precipitates were not observed in any strain either with or without metabolic activation. Cytotoxicity (i.e.,reduction in the background lawn and/or mean number of revertant colonies) was observed at ≥1000 µg/plate in TA98 with metabolic activation, and ≥2500 µg/plate in TA1537, TA100, TA1535, and WP2 uvrAwith and without metabolic activation and TA98 without metabolic activation. Criteria for a negative response were met for all tester strains with and without metabolic activation.

Conclusions:

The data from the vehicle and positive control substances demonstrated the validity and sensitivity of this test system for detecting chemical mutagens with and without metabolic activation. Mean increases in the number of revertant colonies indicative of a positive response were not observed with the substance in the Salmonella strains TA1537, TA98, TA100, and TA1535 and in the E. coli strain WP2 uvrA, with and without metabolic activation, under the conditions of this assay. Therefore, the substance is considered to be negative for inducing mutagenicity in this assay.

Executive summary:

Introduction

The substance was evaluated for mutagenic activity in the invitro Salmonella-E. coli/mammalian microsome reverse mutation assay.

Method

Four tester strains of Salmonella typhimurium(TA1537, TA98, TA100, and TA1535) and one Escherichia coli strain (WP2 uvrA) were used. The substance was prepared as a formulation in dimethylsulfoxide (DMSO) at a concentration at or below 50 mg/mL for both the initial and confirmatory assays. Mutagenicity testing was performed in triplicate at each concentration with and without an Aroclor™ 1254-induced rat liver S9 metabolic activation system using the plate incorporation method. In the initial assay, The substance was tested at 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/plate. In the confirmatory assay, the substance was tested at 50, 100, 250, 500, 1000, 2500 and 5000 µg/plate using the plate incorporation method.

Results

Precipitates were not observed in any strain either with or without metabolic activation. Cytotoxicity (i.e.,reduction in the background lawn and/or mean number of revertant colonies) was observed at ≥1000 µg/plate in strain TA1537 with and without metabolic activation (initial assay only) and TA98 with metabolic activation (confirmatory assay only), and at ≥2500 µg/plate in TA98 without metabolic activation, TA100, TA1535 and WP2 uvrA with and without metabolic activation, TA98 with metabolic activation (initial assay only), and TA1537 with and without metabolic activation (confirmatory assay only).

In both assays, criteria for a negative response were met for all tester strains with and without metabolic activation. Mean number of revertant colonies was comparable to historical control ranges at all concentrations for all tester strains with and without metabolic activation. The data from the vehicle and positive control substances demonstrated the validity and sensitivity of this test system for detecting chemical mutagens with and without metabolic activation.

Conclusion

These data support the conclusion that the substance is negative for mutagenic activity in the Salmonella strains TA1537, TA98, TA100, and TA1535 and in the E. coli strain WP2 uvrA, with and without metabolic activation, under the conditions of this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In vitro Ames test

The substance was evaluated for mutagenic activity in the in vitro Salmonella-E. coli/mammalian microsome reverse mutation assay using a method based on OECD TG 471 [Wells M, 2014]. Four tester strains of Salmonella typhimurium (TA1537, TA98, TA100, and TA1535) and one Escherichia coli strain (WP2 uvrA) were used. The substance was prepared as a formulation in dimethylsulfoxide (DMSO) at a concentration at or below 50 mg/mL for both the initial and confirmatory assays. Mutagenicity testing was performed in triplicate at each concentration with and without an Aroclor™ 1254-induced rat liver S9 metabolic activation system using the plate incorporation method. In the initial assay, the substance was tested at 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/plate. In the confirmatory assay, the substance was tested at 50, 100, 250, 500, 1000, 2500 and 5000 µg/plate using the plate incorporation method. Precipitates were not observed in any strain either with or without metabolic activation. Cytotoxicity (i.e. reduction in the background lawn and/or mean number of revertant colonies) was observed at ≥1000 µg/plate in strain TA1537 with and without metabolic activation (initial assay only) and TA98 with metabolic activation (confirmatory assay only), and at ≥2500 µg/plate in TA98 without metabolic activation, TA100, TA1535 and WP2 uvrA with and without metabolic activation, TA98 with metabolic activation (initial assay only), and TA1537 with and without metabolic activation (confirmatory assay only).

In both assays, criteria for a negative response were met for all tester strains with and without metabolic activation. Mean number of revertant colonies was comparable to historical control ranges at all concentrations for all tester strains with and without metabolic activation. The data from the vehicle and positive control substances demonstrated the validity and sensitivity of this test system for detecting chemical mutagens with and without metabolic activation.

These data support the conclusion that the substance is negative for mutagenic activity in the Salmonella strains TA1537, TA98, TA100, and TA1535 and in the E. coli strain WP2 uvrA, with and without metabolic activation, under the conditions of this assay.

This study is supported by a study available for the read-across substance N,N-dimethyldecanamide (CAS No. 14433-76-2) in which the substance was found to be negative, with or without metabolic activation when tested according to OECD TG 471 against Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 [Wollny H-E, 1999].

In vitro Chromosome aberration test

An in vitro study is not available for the substance, however a study is available for the read-across substance N,N-Dimethyldecan-1-amide, mixture with N,N-Dimethyloctan-1-amide (CAS No. 67359-57-3).

In this study, Chinese hamster ovary cells were treated with N,N-Dimethyldecan-1 -amide, mixture with N,N-Dimethyloctan-1-amide at the concentrations of 10, 40 and 160 µg/ml medium without S9 mix and at the concentrations of 7.2, 36 and 180 µg/ml with S9 mix [Gahlmann R, 1995]. The test substance induced cytotoxic effects in cells exposed to the highest doses with and without metabolic activation at the early harvest time of 8 hours after the beginning of the treatment. The mitotic indices were markedly reduced to 66.7 and 43.2 % relative to control cells.

With one exception, no statistically significant or biologically relevant increases of numbers of metaphases with aberrations were detected 8, 24 or 30 hours after the beginning of the four hour treatment with the test substance with and without S9 mix (Tables 2-5). A statistically significant increase of the numbers of cells with aberrations was calculated for cells exposed to the dose of 180 µg/ml with metabolic activation and the harvest time of 8 hours. However, the absolute number of cells with aberrations (3.5 %) was within the normal range as compared to the values for other solvent controls within this study and for other studies performed in this laboratory. This statistically significant increase was therefore considered to be caused by the unusually low number of cells with aberrations in the corresponding solvent control. This statistically significant result is therefore considered not to be biologically relevant.

The positive controls mitomycin C and cyclophosphamide induced clear clastogenic effects and demonstrated the sensitivity of the test system.

In vitro mammalian cell gene mutation assay

An in vitro study is not available for the substance, however a study is available for the read-across substance N,N-Dimethyldecan-1-amide, mixture with N,N-Dimethyloctan-1-amide (CAS No. 67359-57-3).

The test material, N,N-Dimethyldecan-1 -amide, mixture with N,N-Dimethyloctan-1-amide, was assayed for mutagenic activity at the HGPRT locus in V79 cells from 25 to 250 µg/ml both, with and without metabolic activation according to OECD guideline 476 [Brendler-Schwaab S, 1994]. Under both treatment conditions, cytotoxic effects were induced. The vehicle control mutant frequencies were all in the normal range of background frequencies for the assay. In contrast, the positive controls EMS and DMBA induced a distinct mutagenic effect in mutant frequency, which was significantly increased over the negative controls demonstrating the sensitivity of the test system and the ability to detect known mutagens.

In vivo Micronucleus test

The potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCEs) in mouse bone marrow was assessed in a study performed according to OECD TG 474 [Weinburg, 2014].

In the range finding study the test substance was administered orally by gavage once daily for 3 consecutive days to 3 groups (Groups 2-4) of Crl:CD1(ICR) mice. Dosage levels were 300, 400, and 500 mg/kg/day for Groups 2, 3, and 4, respectively. A concurrent control group (Group 1) received deionized water on a comparable regimen. The dose volumes based upon specific gravity were 0.57, 0.34, 0.46, and 0.57 mL/kg for all Groups 1-4, respectively. Each group (Groups 1-4) consisted of 3 animals/sex. All surviving animals were euthanized on study day 3 and discarded.

All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed at the time of dose administration and approximately 1 to 2 hours following dosing. Detailed physical examinations were performed and individual bodyweights were collected at least once weekly during acclimation, 1 week (± 2 days) prior to randomization, at the time of animal selection for randomization, on study day 0, and prior to the scheduled euthanasia. Individual food weights were recorded for 1 week (± 2 days) prior to randomization and from study day 0 until just prior to the scheduled euthanasia.

A single 300 mg/kg/day group female (no. 15542) was euthanized in extremis and a single 500 mg/kg/day group male (no. 15472) was in moribund condition and was scheduled to be euthanized in extremis on study day 2; however the animal died prior to euthanasia. Clinical observations noted for these animals on the day of death consisted of prostration, dermal atonia, pale or cool extremities, cool body, laboured respiration, gasping, decreased respiration rate, and/or yellow material on selected body surfaces (around mouth, urogenital area, anogenital area, and/or ventral trunk). Although no macroscopic findings were noted for either of the 2 early death animals and a microscopic evaluation was not conducted, the early death of the 500 mg/kg/day group male was considered potentially test substance-related. Test substance-related clinical observations in animals surviving to the scheduled euthanasia were limited to single incidences of yellow material around the urogenital area and/or dermal atonia in the 400 and 500 mg/kg/day group males Test substance-related mean bodyweight losses were noted in all test substance-treated male and female groups and correlated with lower mean food consumption in all male groups and in the 300 and 500 mg/kg/day group females.

In the main study the test substance was administered orally by gavage once daily for 3 consecutive days to 3 groups (Groups 2-4) of Crl:CD1(ICR) mice. Dosage levels were 75, 150, and 300 mg/kg/day for Groups 2, 3, and 4, respectively. A concurrent negative control group (Group 1) received deionized water on a comparable regimen. A positive control group (Group 5) received a single oral dose of 60 mg/kg cyclophosphamide monohydrate (CPS) at a dose volume of 10 mL/kg on study day 2, the day prior to the scheduled euthanasia. The dose volumes based upon specific gravity were 0.34, 0.09, 0.17, and 0.34 mL/kg for Groups 1-4, respectively. Each group (Groups 1-5) consisted of 6 animals/sex. All animals were euthanized on studyday3 and discarded following bone marrow collection.

All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed at the time of dose administration and approximately 1 to 2 hours following dosing. Detailed physical examinations were performed and individual bodyweights were collected at least once weekly during acclimation, 1 week (± 2 days) prior to randomization, at the time of animal selection for randomization, on study day 0, and prior to the scheduled euthanasia. Individual food weights were recorded for 1 week (± 2 days) prior to randomization and from study day 0 until just prior to the scheduled euthanasia. Bone marrow collection for micronucleus evaluation was performed for 5 of 6 animals/sex/group at the scheduled euthanasia (study day 3). All animals were discarded without necropsy. Bone marrow smears were prepared and the coded slides were counted for polychromatic, normochromatic, and micronucleated polychromatic erythrocytes following the final bone marrow sample collection on study day 3.

There were no test substance-related effects on survival. Test substance-related clinical observations of rales and yellow material on selected body surfaces (around the mouth and/or urogenital area) were noted in all test substance-treated male and female groups as early as study day 1, at time of or 1-2 hours post-dosing. Additional test substance-related clinical observations on study days 2 and 3 included dermal atonia, decreased defecation, small faeces, laboured respiration, gasping, pale extremities, swollen abdominal area, and/or yellow material on the ventral trunk. Test substance-related mean bodyweight losses were noted in all test substance-treated male and female which resulted in 10.4% to 16.3% lower mean bodyweights in male groups and 10.2% to 12.1% lower mean bodyweights in the female groups at the end of the study when compared to the control group. The bodyweight losses correlated with lower food consumption in all test substance-treated male and female groups. The test substance did not produce an increase in the mean number of micronucleated polychromatic erythrocytes (%MN-PCEs) compared to the negative control group. No bone marrow cytotoxicity(decreases in the ratio of polychromatic to total erythrocytes, PCE:TE ratio) was noted in any test substance-treated group. Therefore, the test substance met the criteria for a negative response for bone marrow cytotoxicity and clastogenicity and/or disruption of the mitotic apparatus under the conditions of this assay.

Based on the results of the definitive phase of this study, oral administration of the test substance once daily to Crl:CD-1 mice for 3 consecutive days resulted in a negative response for induction of bone marrow micronuclei at dosage levels up to 300 mg/kg/day.

Justification for selection of genetic toxicity endpoint
GLP study conducted according to OECD 471 test guideline, Klimisch grade 1.

Justification for classification or non-classification

No Classification is needed for mutagenicity as negative results were obtained in in vitro and in vivo studies performed using the substance itself and the read-across substances.