Reaction mass of N,N-dimethyldodecanamide and N,N-dimethyltetradecanamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Salmonella typhimurium reverse mutation assay (Reaction mass of N,N-dimethyldodecanamide and N,N-dimethyltetradecanamide; S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2), OECD 471; non-mutagenic (with and without metabolic activation) (Stepan 2017; Orovecz)

- Salmonella typhimurium reverse mutation assay (N,N-Dimethyldecanamide; TA1535,TA1537,TA98,TA100 and TA102), OECD 471:

non-mutagenic (with and without metabolic activation) (Cognis 1999; Hans-Eric Wollny)

- V79-HGPRT (mixture of N,N-Dimethyldecanamide and N,N-Dimethyloctanamide; V79), OECD 476: non mutagenic (with and without metabolic activation) (Bayer, 1994, Brendler-Schwaab S.)

- Chromosome abberation test (mixture of N,N-Dimethyldecanamide and N,N-Dimethyloctanamide; CHO cells), OECD 473:

not clastogen (with and without metabolic activation) (Bayer 1995; R. Gahlmann)

It may be predicted from this that (analogue read-across) the registered substance, Reaction mass of N,N-dimethyldodecanamide and N,N-dimethyltetradecanamide, would share a similar toxicity profile and would not be genotoxic in similar assays.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study according OECD guideline, GLP, well documented

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 (Phenobarbital, ß-Napthoflavone)

Test concentrations with justification for top dose:

Experiment I (TA 98, TA 100): 3; 10; 33; 100 and 1000 µg/plate
Experiment I (TA 1535, TA 1537, TA102): 33; 66; 100; 333; 666 and 1000 µg/plate
Experiment II (all strains): 33; 66; 100; 333; 666 and 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: chosen because of the test item solubility

Untreated negative controls:

yes

Remarks:

untreated controls

Negative solvent / vehicle controls:

yes

Remarks:

solvent controls

Positive controls:

yes

Positive control substance:

other: sodium azide, 4-nitro-o-phenylene-diamine, methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: pre mixed suspension (test solution, S9mix or buffer, bacteria suspension, overlay agar) were poured onto agar plates

Evaluation criteria:

according to guideline

Statistics:

no

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in higher concentrations, tested up to cytotoxic concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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:

cytotoxicity

Remarks:

in higher concentrations, tested up to cytotoxic concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity was checked for higher concentration in pre-experiment. The backround growth was reduced at 2500 µg/plate with and without S9 mix

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not induce gene mutations under the experimental conditions with the tested strains
SAT 990560 is considered to be non-mutagenic in the salmonella typhimurium reverse mutation assay

Executive summary:

This study was performed to investigate the potential of SAT 990560 (N,N-Dimethyldecan-1 -amide) to induce gene muta­tions according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strainsTA1535,TA1537,TA98,TA100,and TA102.

The assay was performed with and without liver microsomal activation. Due to technical reasons the results of experiment II had to be dismissed and an additional experiment was performed under identical conditions. The results of this additional experiment are included in this report as experiment II. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Experiment I(TA98,TA100): 3; 10; 33; 100; 333; and 1000 ug/plate

Experiment I(TA1535,TA1537,TA102): 33; 66; 100; 333; 666; and 1000 ug/plate Experiment II (all strains): 33; 66; 100; 333; 666; and 1000 ug/plate

Toxic effects, evident as a reduction in the number of revertants, occurred at the following concentrations:

Strain	Experiment I(µg/plate)		Experiment II (µg/plate)
	withoutS9mix	with S9 mix	withoutS9mix	withS9mix
TA1535	333 - 1000	666 - 1000	333 - 1000	666 - 1000
TA1537	333 - 1000	666 - 1000	666 - 1000	666 - 1000
TA98	333 - 1000	/	666- 1000	1000
TA100	333 - 1000	1000	666- 1000	666 - 1000
TA102	666 - 1000	/	666 - 1000	/

/ no relevant toxic effects observed

In experiment I, the background growth was reduced at the higher concentrations with and without S9 mix in all strains.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with SAT 990560 (N,N-Dimethyldecan-1 -amide) at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct in­crease of induced revertant colonies.

Conclusion

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, SAT 990560 (N,N-Dimethyldecan-1 -amide) is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across hypothesis proposed is that the organism is not exposed to common compounds but rather, as a result of structural similarity, that different compounds have similar toxicological and fate properties. In this case the ECHA Read-Across Assessment Framework (RAAF) Scenario 2 is used.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: N,N-Dimethyldecanamide [ EC 238-405-1; CAS 14433-76-2]
Target: Reaction Mass of N,N-Dimethyldodecanamide and N,N-Dimethyltetradecanamide [EC not assigned; CAS not assigned]

3. ANALOGUE APPROACH JUSTIFICATION
It may be concluded that both the registered substance (target) and the source molecules can be regarded as close structural analogues having both similar physical chemical and toxicological properties. It follows that where endpoints for the registered substance may not have experimental evidence, particularly those involving animal studies, that these can be addressed by read across grouping using an analogue approach. It is therefore proposed that, after careful assessment, experimental data from the source molecules may be used as surrogate evidence for read across to the registered substance. Please refer to attached document for information on the data available to support the read-across.

4. DATA MATRIX
Please refer to attached document

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in higher concentrations, tested up to cytotoxic concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in higher concentrations, tested up to cytotoxic concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: all strains/cell types tested / Migrated from field Test system'.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study according to guideline, well conducted (GLP) and documented

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

in vitro cytogenetic test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix from Aroclor 1254 treated wistar rats

Test concentrations with justification for top dose:

0, 10, 40, 160 µg/ml (without S9)
0, 7.2, 36, 180 µg/ml (with S9)
with harvest time 24h
highest concentration again additional with harvest time 8h and 30 h tested
Pretest with concentration up to 1000 µg/ml showed less survived cells in concentration of 160 µg/ml and above

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol (stock solution containing 500mg/ml in ethanol was diluted in culture medium up to a concentration of 1mg/ml
- Justification for choice of solvent/vehicle: solubility

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

untreated control

Positive controls:

yes

Positive control substance:

other: mitomycin C and cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h at 37°C
- Fixation time (start of exposure up to fixation or harvest of cells): 8, 24h (main study), 30h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): 5% Giemsa solution

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 1 x 10e6 cells were seeded in 20ml medium (containing 0.2ml testitem in solvent, and 1mL S9 mix if metabolic activation)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cell survival

Evaluation criteria:

The mitotic index was determined by counting 1000 cells per culture.
Chromosomes of approximately 200 metaphases per concentration, 100 metaphases from each of two parallel cultures, were
examined for structural changes.
classes of aberrations are characterized as follows:
Gaps
Break
Fragment
Deletion
Exchange
Multiple abberation

Statistics:

The statistical analysis of the results was performed by pair-wise comparison of the numbers of
metaphases with aberrations (including and excluding gaps) and of metaphases with exchanges among 200 cells for treatment
and solvent control groups.
The statistical analysis followed the recommendations outlined by Richardson et al. (1989)
Fisher exact test was used for the statistical evaluation.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

tested up to first cytotoxic concentration 160µg/ml 180µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: 2 pre studies - first pre study ranging from 10 to 1000µg/ml
- secound pre study ranging from 100 to 250 µg/ml

COMPARISON WITH HISTORICAL CONTROL DATA: historical control data were added to the study report.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Survival index determined, highest tested concentration reduces the index by a minimum of 40% (43.2 ; 160µg/ml without S9 and 66.7%; 180µg/ml with S9) harvest time of 8 h, only moderate to negligible effects (with no effects) were seen for harvest times of 24h and 30h

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Not considered to be clastogenic for mammalian cells with and without metabolic activation

Executive summary:

Chinese hamster ovary cells were treated with confidential substance name 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. Confidential substance name 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.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across hypothesis proposed is that the organism is not exposed to common compounds but rather, as a result of structural similarity, that different compounds have similar toxicological and fate properties. In this case the ECHA Read-Across Assessment Framework (RAAF) Scenario 2 is used.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: N,N-Dimethyldecan-1-amide, mixture with N,N-Dimethyloctanc-1-amide [CAS 67359-57-3]
Target: Reaction Mass of N,N-Dimethyldodecanamide and N,N-Dimethyltetradecanamide [EC not assigned; CAS not assigned]

3. ANALOGUE APPROACH JUSTIFICATION
It may be concluded that both the registered substance (target) and the source molecules can be regarded as close structural analogues having both similar physical chemical and toxicological properties. It follows that where endpoints for the registered substance may not have experimental evidence, particularly those involving animal studies, that these can be addressed by read across grouping using an analogue approach. It is therefore proposed that, after careful assessment, experimental data from the source molecules may be used as surrogate evidence for read across to the registered substance. Please refer to attached document for information on the data available to support the read-across.

4. DATA MATRIX
Please refer to attached document

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

tested up to cytotoxic conc.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

migrated from field 'test system'

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study according to guideline, well conducted (GLP) and documented

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79 cell line was originally derived from the lung of a
male Chinese hamster (Chu and Mailing, 1968) kind gift from Prof. G. Speit, University of Ulm, Germany

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix Aroclor 1254 induced from Wistar rats

Test concentrations with justification for top dose:

Cytotoxicity test: 7.9, 15.7, 31.3, 62.5, 125.0, 250.0, 500.0, 750.0, 1000.0 µg/ml
Main test: 25.0, 50.0, 100.0, 125.0, 150.0, 200.0, 250.0µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: Ethylmethanesulfonate (EMS) and Dimethylbenzanthracene (DMBA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium (containing 2% FCS)

according to Guideline

DURATION
- Preincubation period: 16-24h
- Exposure duration: 5h
- expression period: 6 days
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): 6-Thioguanine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: triplicates for cytotoxicity
Main test (repeated completly two times ): 8 dishes (from 1 flask) were examined, for each dosage 2 flask were used.

NUMBER OF CELLS EVALUATED:
for each dose level one flask with 4*10e6 cells were avaiable.

DETERMINATION OF CYTOTOXICITY
- Method: cell count, average count, cytotoxicity was expressed by comparison of
colonies in treated cultures versus vehicle control cultures (relative cloning efficiency)

Evaluation criteria:

The number of 6-TG resistant colonies in the mutation assay dishes and the number of colonies in the
cloning efficiency dishes were determined.Colonies with 50 cells or less were excluded

Statistics:

Weighted analysis of variance as well as to a weighted recursive regression, both with Poisson derived weights. Weighted analysis of
variance followed by pairwise comparisons using Dunnetts test.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

up to cytotoxic concentration

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation of the test article after addition of the test article-Ethanol solution to
the medium starting to be evident at 1500 µg/ml. Therefore, a preliminary cytotoxicity test was conducted with a series of
9 concentrations ranging from 7.9 µg/ml to 1000 µg/ml

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

the test article is considered to be nonmutagenic in the V79-HGPRT Forward Mutation Assay both with and without metabolic activation.

Executive summary:

The test material, confidential substance name, 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. 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.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across hypothesis proposed is that the organism is not exposed to common compounds but rather, as a result of structural similarity, that different compounds have similar toxicological and fate properties. In this case the ECHA Read-Across Assessment Framework (RAAF) Scenario 2 is used.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: N,N-Dimethyldecan-1-amide, mixture with N,N-Dimethyloctanc-1-amide [CAS 67359-57-3]
Target: Reaction Mass of N,N-Dimethyldodecanamide and N,N-Dimethyltetradecanamide [EC not assigned; CAS not assigned]

3. ANALOGUE APPROACH JUSTIFICATION
It may be concluded that both the registered substance (target) and the source molecules can be regarded as close structural analogues having both similar physical chemical and toxicological properties. It follows that where endpoints for the registered substance may not have experimental evidence, particularly those involving animal studies, that these can be addressed by read across grouping using an analogue approach. It is therefore proposed that, after careful assessment, experimental data from the source molecules may be used as surrogate evidence for read across to the registered substance. Please refer to attached document for information on the data available to support the read-across.

4. DATA MATRIX
Please refer to attached document

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

up to cytotoxic conc.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested / Migrated from field Test system'.

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-05-09 - 2017-05-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: S61254001
- Expiration date of the lot/batch: 2018-07-10

Target gene:

Histidine or tryptophan

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital- and beta-naphthoflavone-induced rat liver S9

Test concentrations with justification for top dose:

Initial Mutation Test: 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate at S. typhimurium strains without metabolic
activation and 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate at S. typhimurium strains with metabolic activation and 5000, 1581, 500, 158.1, 50 15.81, 5,
1.581 and 0.5 μg/plate at E. coli WP2 uvrA strain with and without metabolic activation.

Confirmatory Mutation Test: 158.1, 50, 15.81, 5, 1.581, 0.5, 0.1581 and 0.05 μg/plate at S. typhimurium strains without
metabolic activation and 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate at S. typhimurium strains with metabolic activation, and 5000, 1581, 500, 158.1,
50 15.81, 5, 1.581 and 0.5 μg/plate at E. coli WP2 uvrA strain with and without metabolic activation.

Dose selection based on results of a preliminary study

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Based on preliminary solubility test.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

other:

Remarks:

see table below

Details on test system and experimental conditions:

METHOD OF APPLICATION: Initial mutation test - plate incorporation; Confirmatory mutation test - pre-incubation procedure.

DURATION
Initial mutation test - plates incubated at 37°C for 48±1 hours.
Confirmatory mutation test - Incubated for 20 min at 37ºC in a shaking incubator, then plated and incubated at 37°C for 48±1 hours.

NUMBER OF REPLICATIONS: 3

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

Positive controls validity:

valid

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

Positive controls validity:

valid

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

Positive controls validity:

valid

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

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

-S9 Confirmatory test

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES:
In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using S. typhimurium TA98 and TA100 tester strains ±S9 Mix with appropriate untreated, negative (solvent) and positive controls.
Each sample (including the controls) was tested in triplicate.
Following concentrations were examined: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.
In the preliminary experiment, the numbers of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system).
No precipitate was observed in the Preliminary Concentration Range Finding Test in both bacterial strains with and without metabolic activation.
Absent/reduced/slightly reduced background lawn was detected in the Preliminary Range Finding Test in all Salmonella typhimurium strains on the plates at 5000, 2500, 1000 and 316 μg/plate with metabolic activation, and at 5000, 2500, 1000, 316 and 100 μg/plate concentrations without metabolic activation.

Conclusions:

The test item has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Executive summary:

The mutagenic potential of the test item was examined in a bacterial reverse mutation assay. Under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used and is therefore considered to have no mutagenic activity in this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro tests with Reaction mass of N,N-dimethyldodecanamide and N,N-dimethyltetradecanamide

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and

a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of the Compatibility Test, the test item was dissolved in Dimethyl sulfoxide (DMSO) at a concentration of 100 mg/mL. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate at Salmonella typhimurium strains without metabolic activation and 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate at Salmonella typhimurium strains with metabolic activation, in the Confirmatory Mutation Test were 158.1, 50, 15.81, 5, 1.581, 0.5, 0.1581 and 0.05 μg/plate at Salmonella typhimurium strains without metabolic activation and 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate at Salmonella typhimurium strains with metabolic activation, and in the Initial Mutation Test and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50 15.81, 5, 1.581 and 0.5 μg/plate at Escherichia coli WP2 uvrA strain with and without metabolic activation.

No precipitate was observed in the main tests in all examined strains with and without metabolic activation.

Inhibitory, cytotoxic effect of the test item (absent / reduced / slightly reduced background lawn development) was observed in the main tests in all examined strains with and without metabolic activation at several concentrations (expect in the Initial Mutation Test at Escherichia coli WP2 uvrA strain).

In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item has no mutagenic activity on the growth of the bacterial strains under the conditions of this test.

In vitro tests with pure N,N-Dimethyldecanamide:

The test substance N,N-Dimethyldecanamide was tested in a salmonella typhimurium reverse mutation assay (TA1535,TA1537,TA98,TA100and TA102) according to OECD 471 guideline (Cognis 1999; Hans-Eric Wollny) with and without metabolic activation. The test item was tested at the following concentrations:(TA98,TA100): 3; 10; 33; 100; 333; and 1000 µg/plate and(TA1535,TA1537,TA102): 33; 66; 100; 333; 666; and 1000 µg/plate.Toxic effects, evident as a reduction in the number of revertants, occurred between 333 and 1000µg/plate depending on strain and activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with N,N-Dimethyldecan-1 -amide at any dose level, neither in the presence nor absence of metabolic activation (S9 mix).In conclusion the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In vitro tests with mixtures of N,N-Dimethylamides:

A mixture of N,N-dimethyldecanamide and N,N-Dimethyloctanamide (with traces of N,N-dimethyl-dodecanamide and ~5% N,N-dimethyl-hexanamide) was tested in an in vitro mammalian chromosome abberation test (CHO cells) and in an V79-HGPRT Forward Mutation Assay (V79 cells). It is concluded that, caused by a high amount of N,N-Dimethyl-decanamide in the mixture and the fact that the residual contains homologues with a lower and higher molecular weight (mainly N,N-Dimethyloctanamide) which can be assumed to have an similar toxicological behaviour as the mixture, the mixture has an similar behaviour in the different cell tests like pure N,N-Dimethyldecanamide. Therefore the following study results will be used for assessing the genetoxic behaviour of N,N-Dimethyldecanamide.

The genotoxic potential of a C8/C10 dimethylamide mixture was tested in an in vitro mammalian chromosome abberation test according to OECD 473 (Bayer 1995; R. Gahlmann). Therefore Chinese hamster ovary cells were treated with the substance 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. The test substance induced cyto toxic 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 (which was considered as uncritical, see study summary), 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. Therefore the C8/C10 mixture not considered to be clastogenic for mammalian cells with and without metabolic activation

A further study was accomplished to observe the genotoxic potential of C8/C10 FADMA (Bayer, 1994, Brendler-Schwaab S.). Therefore the test material 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. Under both treatment conditions, cyto toxic 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 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. The test substance was considered to be non mutagenic in the V79-HGPRT Forward Mutation Assay both with and without metabolic activation.

Based on the results of the reverse mutation assay performed on the substance itself and on the C10 analogue and the in vitro mammalian chromosome abberation test (CHO cells) and in vitro V79-HGPRT Forward Mutation Assay (V79 cells) performed using N,N-dimethyldecanamide, mixture with N,N-dimethyloctanamide it can be concluded that the N,N-dimethylalkylamides, including the registered substance, Reaction mass of N,N-dimethyldodecanamide and N,N-dimethyltetradecanamide, are not mutagenic.

Justification for classification or non-classification

In vitro genotoxicity test and in vitro mutagenicity test does not reveal a positive result.

Due to criteria of GHS (Regulation (EU) 1272/2008) for germ cell mutagens ("The classification in Category 2 is based on: positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from: somatic cell mutagenicity tests in vivo, in mammals; or other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays....") the substance is not to classify as germ cell mutagen.