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Genetic toxicity in vitro

Description of key information

2015a: Bacteria reverse mutation assay (Ames) OECD 471, GLP, S. typhimurium, 31.6 - 5000 µg/plate, +/- S), Result negative

2015b: In vitro Mammalian Cell Gene Mutation Test (HPRT) OECD 476, GLP, V79 cells of the Chinese Hamster, 0.1 - 10 mM , +/-S9. Result negative.

2015c: In vitro Mammalian ChromosomeAberrationTest, OECD 473, GLP, human lyphocytes, 6 -10 mM, +/-S9. Results negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: 41334368E0

Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 mix: liver from male Wistar rats (induced with phenobarbital and ß-naphthoflavone)
Test concentrations with justification for top dose:
Experiment I + II: 31.6; 100; 316; 1000; 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: aqua dest.

Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine; 2-aminoanthracene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments

TREATMENT AND HARVEST SCHEDULE:
Standard plate test (Experiment II):
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control)
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation)
100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain)
2000 µL Overlay agar

Preincubatintest (Experiment II):
For the pre-incubation method 100 µL of the test item preparation was pre-incubated with the tester strains (100 µL and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37°C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate. For each strain and dose level, including the controls, three plates were used. After solidification the plates were inverted and incubated at 37°C for at least 48 h in the dark.

Evaluation criteria:
The mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control.
A test item is considered as mutagenic if:
- a clear and dose-related increase in the numer of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows.
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higer than the reversion rate of the solvent control.
Species / strain:
other: TA 98, TA 100, TA 1535, TA 1537, TA 102
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
Positive controls validity:
valid
Additional information on results:
No precipitation of the test item was observed in any tester strain usen in experiment I and II (with and without metabolic activation). No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II.
No biologically relevant increases in revertant colony numers of any of the five tester strains were observed follwoing treatment with N,N-Dimethylacetamide at any concentratin level, neither in the presence nor absence of metabolic activation in experiment I and II. The references mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, N,N-Dimethylacetamide did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, N,N-Dimethylacetamide is considered to be non-mutagenic in theis bacterial reverse mutation assay.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: 41334368E0
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
For cell lines:
- Absence of Mycoplasma contamination: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: MEM
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 mix : liver from male Wistar rats (induced with Phenobarbital and ß-Naphtoflavone)
Test concentrations with justification for top dose:
Experimant I: 0.1, 0.2, 0.5, 1, 2, 5, 7, 9 and 10 mM (4 h; with and without metabolic activation)
Experiment II: 0.2, 0.5, 1, 2, 5, 7, 9 and 10 mM (20 h; without metabolic activation); 0.30, 0.75, 1.5, 3, 4, 6, 8 and 10 mM (4 h; with metabolic activation)

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 24 h
- Exposure duration/duration of treatment: 4 and 20 h

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 6-9 days
- Selection time (if incubation with a selective agent): 7 days (11 µg/mL thioguanine (TG)
- Fixation time (start of exposure up to fixation or harvest of cells): day 13 - 18

Evaluation criteria:
A test is considered to be negative if there is no biological relevant increase in the number of mutants.
There are several criteria for determining a positve result:
- a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations
- a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of the mutant frequency is not observed
- if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed
Statistics:
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No precipitation of the test item was noted in the experiments.
No biologically relevant groth inhibition was observed in experiment I and II with and without metabolic activation.
In experiment I without metabolic activation the relative growth was 93.9% for the highest concentration (10mM)evaluated. The higest biologically relevant concentration evaluated with metabolic activatin was 10 mM with arelative growth of 99.1%.
In experiment II without metabolic activation the relative growth was 98.6% for the highest concentratin (10mM) evaluated. Teh highest concentration evaluated with metabolic activation was 10 mM with a relative of 103.0%.
Conclusions:
In conclusion, in the described in vitro cell gene mutagenicity test under the experimental conditions reported, the test item N,N-Dimethylacetamide is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 41334368E0
Species / strain / cell type:
other: human lymphocytes
Details on mammalian cell type (if applicable):
For lymphocytes:
- blood was collected only from a single doner (healthy and non-smoking)
- whole blood samples treated with an anti-coagulant (e. g. heparin) were pre-cultured in the presence of mitogen (phytohaematogglutinin, PHA).
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : male Wistar rats (induced with phenobarbital and \-naphthoflavone)
Test concentrations with justification for top dose:
Experiment I: 6, 8 and 10 mM (with and without methabolic activation); 4 h treatment, 24 h preparation interval
Experiment II: 6, 8 and 10 mM (without metabolic activation); 24 h treatment, 24 h preparation interval
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: RPMI cell culture medium ((RPMI + 0% FBS and RPMl+15% FBS)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Experiment I: 4 h (with and without S9 mix); Experiment II: 24 h (without S9 mix)

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): colcomid 0.2 µg/ml was added 2 h before harvesting
- STAIN (for cytogenetic assays): Giemsa
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 150 metaphases per culture

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: mitotic index (MI), proliferation index
Evaluation criteria:
There are several criteria for determining a positive result: a clear and dose-related increase in the number of cells with aberrations; a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (0.0% - 3.0% aberrant cells (without and with metabolic activation)).
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results.
Statistics:
A statistical evaluation was used as an aid for interpretation of the results. Statistical significance at the 5% level (p < 0.05) was evaluated by the Fischer's exact test. The p value was used as a limit in judging for significance levels in comparison with the corresponding negative control.
Species / strain:
other: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No precipitation and no toxic effects of the test item were noted without and with metabolic activation in all dose groups evaluated in experiment I and II.

In experiment I and II no biologically relevant decreases of proliferation index were observed.

In experiment I and II no biologically relevant increase of the aberration rates was noted after treatment with the test item without and with metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.

In experiment I with metabolic activation the negative control showed slightly increased values with 3.7% compared to the historical data range (0.0-3.0%). However, as the blood donor is well established and regularly used for our experiments, did at the time of blood collection not suffer from an infection and felt well, and additionally no technical mistake was noted in the performance of the assay, the experiment will be considered as valid. As the dose groups are compared with the negative control, reliable results are obtained. In accordance with the increased value for the negative control, we did observe in this experiment higher aberration rates for the positive control as well, supporting the reliability of the experiment.
Conclusions:
In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item N,N-Dimethylacetamide did not induce structural chromosomal aberrations in human lymphocyte cells. Therefore, N,N-Dimethylacetamide is considered to be non-clastogenic in this chromosome aberration test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

There is no evidence for mutagenic activity in vivo. None of the studies used for assessment is of unrestricted reliability but the available data as a whole are sufficient for evaluation of this endpoint.

NIOSH (1980): Dominant lethal assay according to OECD 478, non-GLP. Tested in male rats, 20 and 700 ppm, 5-d inhalation. Result negative.

Two additional dominant lethal assays equivalent to OECD 478, non-GLP. Tested in male mice, 680 µl/kg bw, i.p. (1976) or 1500 and 3000 mg/kg bw, dermal (1972), respectively. Result negative.

NIOSH (1980): Chromosome aberration test according to OECD 475, unknown GLP compliance. Tested in rats, 20 and 700 ppm. Result negative.

NIOSH (1980): Drosophilia SLRL assay according to OECD 477, unknown GLP. Drosophilia melanogaster, 200 ppm. Result negative.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
(partly limited documentation, e.g. no details about test substance or no tabulated details on results; no data on cytotoxicity in bone marrow; only 50 cells per rat analysed [100 cells recommended]; MTD presumably not reached)
Qualifier:
according to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
yes
Remarks:
(only 50 metaphases scored)
GLP compliance:
not specified
Type of assay:
chromosome aberration assay
Specific details on test material used for the study:
- Name of test material: dimethylacetamide (DMAC)
No details available.
Species:
rat
Strain:
other: CD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Diet and water ad libitum but not during exposure.
- Some evidence for sialodacryoadenitis (SDA Virus; natural occurring pathogen in rats) in all rats.
- Housing: males caged individually

no further details available
Route of administration:
inhalation
Vehicle:
see details of exposure
Details on exposure:
Whole body exposure; generation by bubbling nitrogen through DMAC contained in a glass gas washing bottle (50 °C); vapour mixture diluted with filtered, compressed air; atmospheres in the exposure chambers were dynamic (continuously generated for a single pass through the animal holding zone); concentrations within the exposure chambers maintained by regulating the flow of nitrogen and diluting air into the mixing vessels.

Continuously monitoring in the breathing zone by infra-red gas analysers; calibrations performed.

Chamber relative humidity and temperature was measured every hour.

After exposure rats observed for clinical signs, ear numbers checked, body weights recorded and returned to their cages.
Duration of treatment / exposure:
7 h
Frequency of treatment:
once or for five days
Post exposure period:
Three different sampling times: 6, 24 or 48 h after the last exposure rats were sacrificed; 2 h prior to sacrifice were i.p. injected with 3 mg/kg bw colchicine.
Dose / conc.:
20 ppm (nominal)
Remarks:
70 mg/m³
analytical data revealed deviations from the target concentrations of more than +-10% were limited to 20 min high (20 ppm target concentration) and 40 min low, 30 min high (700 ppm target concentration); no further details
Dose / conc.:
700 ppm (nominal)
Remarks:
2500 mg/m³
analytical data revealed deviations from the target concentrations of more than +-10% were limited to 20 min high (20 ppm target concentration) and 40 min low, 30 min high (700 ppm target concentration); no further details
No. of animals per sex per dose:
no details available
Control animals:
yes, concurrent vehicle
Positive control(s):
Yes, single gavage with 250 mg/kg bw ethyl methanesulfonate.
Tissues and cell types examined:
Bone marrow cells; investigated abnormalities: gaps, breaks, fragments, dicentrics, translocations (within the limitations of the staining methods), and pulverisation
Details of tissue and slide preparation:
Bone marrow cell suspension prepared, fixed and dried on slides and examined by light microscopical methods after staining with Giemsa R66.
50 cells with well-spread chromosomes per animal examined on a blind basis.
Evaluation criteria:
Statistical significance (a) including all chromosomal abnormalities and (b) excluding cells only exhibiting gaps.
Statistics:
The data were transformed using the Freeman-Tukey transformation.
A one-sided Student's t test was used on the transformed values.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Significant effects in positive controls (breaks with fragments, exchanges and multiple aberrations), especially after the post exposure period of 48 h. No increase in the incidences of chromosomal aberrations in the treatment groups.

Data on acute inhalation toxicity in Section 7.2.3 suggested no toxic effects at a dose level of 700 ppm.

Conclusions:
No clastogenic activity was detected in the bone marrow of rats after inhalation exposure to 700 ppm (2500 mg/m³).
Executive summary:

The Tier II mutagenic screening of the test substance was conducted according to OECD 475 in 1980. GLP compliance was not specified. A cytogenetic analysis of rat bone marrow cells was performed after in vivo exposure to 20 or 700 ppm of the test substance for seven hours per day for 1 or 5 days in male and female rats. A positive (single gavage with 250 mg/kg bw ethyl methanesulfonate) control and a negative control was included. Sampling times were 6 h, 24 h, and 48 h post-exposure. The frequencies of chromosomal aberrations were not increased significantly in the rat bone marrow cells.

Endpoint:
in vivo mammalian germ cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
(partly limited documentation, e.g. no details about test substance or no tabulated details on results)
Qualifier:
according to
Guideline:
OECD Guideline 477 (Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila melanogaster)
GLP compliance:
not specified
Type of assay:
Drosophila SLRL assay
Specific details on test material used for the study:
- Name of test material: dimethylacetamide (DMAC)
No details available.
Species:
Drosophila melanogaster
Strain:
other: wild-type Oregon-K
Sex:
male
Details on test animals and environmental conditions:
Stocks maintained in bottles with 100 mL medium.
no further data
Route of administration:
inhalation
Vehicle:
see details of exposure
Details on exposure:
Whole body exposure; generation by bubbling nitrogen through DMAC contained in a glass gas washing bottle (50 °C); vapour mixture diluted with filtered, compressed air; atmospheres in the exposure chambers were dynamic (continuously generated for a single pass through the animal holding zone); concentrations within the exposure chambers maintained by regulating the flow of nitrogen and diluting air into the mixing vessels.

Continuously monitoring by infrared spectroscopy before the vapour entered the chamber; calibrations performed.

After exposure rats observed for clinical signs, ear numbers checked, body weights recorded and returned to their cages.
Duration of treatment / exposure:
95 minutes; single exposure (exposure duration studied in preliminary toxicity tests via hatchability index)
Frequency of treatment:
once
Post exposure period:
treated males mated with virgin Müller-5 females (1 males with 2 females) the morning after exposure and remating with further females 3 and 8 days after the 1st mating (all stages of spermatogenesis tested).
Dose / conc.:
200 ppm (nominal)
Remarks:
720 mg/m³
No. of animals per sex per dose:
no details available
Control animals:
yes, concurrent no treatment
yes, historical
Positive control(s):
no data
Tissues and cell types examined:
Emergence for F1 generation flies from the pupae began about 10 days after mating; matings for the F2 generation set up 1-4 days later by mating brother with sisters.
Details of tissue and slide preparation:
F2 generation checked for wild-type males
Evaluation criteria:
(a) a compound giving frequencies below 0.5 % in duplicate experiments considered to show no evidence of mutagenic activity
(b) a compound giving frequencies greater than 1.0 % in the same brood in duplicate experiments considered to show mutagenic potential
(c) a compound giving frequencies between 0.5 % and 1.0 % shows evidence of possibly being mutagenic; although this evidence is not conclusive, the
compound clearly would-deserve further study
Statistics:
see evaluation criteria, no further data
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
reduced activity
Negative controls validity:
valid
Positive controls validity:
not examined
Additional information on results:
Only 2 lethals in stock A. In stock B 3 lethals were found (frequeny of 0.57 %), however, all 3 lethals came from the same male.
Conclusions:
No gene mutation was found in the SLRL test in Drosophila melanogaster after exposure for 95 minutes to a vapor concentration of 200 ppm (720 mg/m³).
Executive summary:

The Tier II mutagenic screening of the test substance was conducted according to OECD 477 in 1980. GLP compliance was not specified. The sex linked recessive lethal test was performed using Drosophila-melanogaster after a 95 minute exposure to 200 ppm. Three wild-type male Oregon K flies were exposed in a glass vessel at the required concentration at a rate of ca. 5 l/min. Exposed flies were kept overnight in their feeding vials in a 26°C water bath. Survivors were recorded and four males were picked and mated with four virgin females. The number of laid eggs was recorded. Recessive lethal test was conducted using treated males that were mated in a ratio 1:2 to virgin Müller-5 females. Emergence for F1 generation flied began about ten days after mating. Mating for the F2 generation were set up 1 -4 days later by mating brother with sisters.

Only 2 lethals in stock A. In stock B 3 lethals were found (frequeny of 0.57 %), however, all 3 lethals came from the same male. Results revealed no signs for a genotoxic effect of the test substance. Sex-linked recessive lethal (SLRL) frequency was not increased in Drosophilia.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
(partly limited documentation, e.g. no details about test substance or no tabulated details on results; no data on systemic toxicity in males; only 20 females per mating used
Qualifier:
according to
Guideline:
OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
Deviations:
no
GLP compliance:
no
Type of assay:
rodent dominant lethal assay
Specific details on test material used for the study:
- Name of test material: dimethylacetamide (DMAC)
No details available.
Species:
rat
Strain:
other: CD
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Diet and water ad libitum but not during exposure.
- Some evidence for sialodacryoadenitis (SDA Virus; natural occurring pathogen in rats) in all rats.
- Housing: males caged individually

no further details available
Route of administration:
inhalation
Vehicle:
see details of exposure
Details on exposure:
Whole body exposure; generation by bubbling nitrogen through DMAC contained in a glass gas washing bottle (50° C); vapour mixture diluted with filtered, compressed air; atmospheres in the exposure chambers were dynamic (continuously generated for a single pass through the animal holding zone); concentrations within the exposure chambers maintained by regulating the flow of nitrogen and diluting air into the mixing vessels.

Continuously monitoring in the breathing zone by infra-red gas analysers; calibrations daily.

Chamber relative humidity and temperature was measured every hour.

After exposure rats observed for clinical signs, ear numbers checked, body weights recorded and returned to their cages.
Duration of treatment / exposure:
7 h/day for 5 consecutive days
Frequency of treatment:
once daily
Post exposure period:
10 weeks for treated males
Dose / conc.:
20 ppm (nominal)
Remarks:
70 mg/m³
analytical data revealed deviations from the target concentrations of more than +-10 % were limited to 45 min low, 45 min high (20 ppm target concentration) and 20 min low (700 ppm target concentration); no further details
Dose / conc.:
700 ppm (nominal)
Remarks:
2500 mg/m³
analytical data revealed deviations from the target concentrations of more than +-10 % were limited to 45 min low, 45 min high (20 ppm target concentration) and 20 min low (700 ppm target concentration); no further details
No. of animals per sex per dose:
10 males in each group
Control animals:
yes, concurrent vehicle
Positive control(s):
Yes, 100 mg/kg bw/day ethyl methanesulfonate via gavage.
Tissues and cell types examined:
After the last exposure each male mated with 2 virgin females for 1 week; thereafter males transferred to fresh cages and again mated with 2 females; females sacrficed 17 days after start of matings; repeat of this procedure for totally 10 matings.
Details of tissue and slide preparation:
Uteri of sacrificed females examined for corpora lutea, early and late loss of implantations and viable fetuses.
Evaluation criteria:
Statistical significance concerning fertility index and probability of an early death analysed.
Statistics:
Some parameters were transformed using the Freeman-Tukey transformation.
Chi-square test
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
(no clinical signs but slight reduction in body weight [700 ppm; no further details])
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Significant effects in positive controls (Corpora lutea graviditatis counts significantly reduced at week 1, 2, and 3; implantations per pregnancy significantly reduced at week 1, 2, 3, and 4; early death frequency significantly increased at week 2-5).

Conclusion: No dominant lethal events in treatment groups: no significant effects on fertility index, numbers ot corpora lutea or implantations or the frequency of early deaths after repeated inhalation exposure to 700 ppm (2500 mg/m³) indicating no dominant lethal effects in the treatment groups.

Data on repeated inhalation toxicity in Section 7.5.3 suggested toxic effects at a dose level of 700 ppm.

Conclusions:
No dominant lethal events in treatment groups: no significant effects on fertility index, numbers ot corpora lutea or implantations or the frequency of early deaths after repeated inhalation exposure to 700 ppm (2500 mg/m³) indicating no dominant lethal effects in the treatment groups.
Executive summary:

The Tier II mutagenic screening of the test substance was conducted according to OECD 478 and not GLP compliant. Dominant lethal tests in male rats were performed after in vivo exposure to 20 or 700 ppm of the test substance for 7 hours per day for five consecutive days via inhalation route. Ten animals per exposure group were treated once daily (first day of exposure assessed as day 1) and observed for ten weeks post-exposure. A positive control was included, namely 100 mg/kg bw/day of ethyl methanesulfonate applied via gavage and a negative control was used. On day 5, two female rats were introduced to each of the 40 cages where male rats were housed individually. On day 22, female rats were killed and examined for pregnancy and dominant lethal effects. The procedure was repeated for ten consecutive weeks. No clinical or genotoxic effects in rats treated with the test substance were observed. The positive control revealed a positive effect.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: This study has to be carefully evaluated as it was performed at Industrial Bio-test Labs which is known to have conducted scientific fraud. Therefore, the reliability is not assignable.
Remarks:
Comparable to Guideline study with acceptable restrictions (partly limited documentation, e.g. no details about the test substance; MTD presumably not reached).
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
GLP compliance:
no
Type of assay:
rodent dominant lethal assay
Specific details on test material used for the study:
- Name of test material: dimethylacetamide (DMAC)
No details available.
Species:
mouse
Strain:
other: "Charles River strain albino"
Sex:
male
Details on test animals and environmental conditions:
no details
Route of administration:
dermal
Vehicle:
undiluted
Details on exposure:
Undiluted test substance applied to the shaved backs (presumably no coverage); controls received the same volume (high dose) sesame oil.
In preliminary toxicity studies the max. tolerated dose was determined: severe erythema was found at 10000 mg/kg bw and 1/4 mice died at 3000 mg/kg bw (also erythema); local effects (erythema) were found at all tested doses.
Duration of treatment / exposure:
single dermal exposure
Frequency of treatment:
once
Post exposure period:
7 weeks
Dose / conc.:
1 500 other: mg/kg bw
Dose / conc.:
3 000 other: mg/kg bw
No. of animals per sex per dose:
12
Control animals:
yes, sham-exposed
Positive control(s):
Yes, i.p. 100 mg/kg bw methylmethanesulfonate
Tissues and cell types examined:
After exposure each treated male mouse mated with 3 virgin females for 1 week; this procedure was repeated with further virgin females for 5 weeks (totally 6 matings in 6 weeks); males were sacrificed after 6 weeks and each female 1 week after removal from the breeding cage.
The numbers of implantation sites, resorption sites (late and early deaths), and embryos recorded; females judged to be pregnant if corpora lutea were present in the ovaries.
Evaluation criteria:
Increase in pre- and post-implantation loss and/or increase in dead embryos as a measure of dominant lethal events
Statistics:
no data
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
(only local erythema for several hours)
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Two positive controls died.
Mating index: at the low dose the mating index was reduced at week 6 but not at other weeks; no effects at the high dose level; considered not to be treatment related. In the positive control the mating index was slightly reduced at week 1 and significantly reduced at week 2.

Dominant lethal events:
The numbers of implantations, resorptions, and embryos for both treatment groups compared favorably to those for controls; positive controls showed a reduction in implantations and embryos and an increase in early resorptions at weeks 1 and 2 (normal at all other weeks).

In the dominant lethal assay 12 male mice per dose level received a single dermal application of 1500 or 3000 mg/kg bw (no coverage). The negative control was sham treated with sesame oil and the positive control received i.p. 100 mg/kg bw methylmethanesulfonate. After exposure each treated male mouse was mated with 3 virgin females for 1 week; this procedure was repeated with further virgin females for 5 weeks (totally 6 matings in 6 weeks); males were sacrificed after 6 weeks and each female 1 week after removal from the breeding cage. Negative and positive controls were valid concerning dominant lethal parameters. The numbers of implantations, resorptions, and embryos for both treatment groups compared favorably to those for controls indicating no dominant lethal effects.

Conclusions:
The test substance did not cause a dominant lethal response in male mice after single dermal exposure to 1500 or 3000 mg/kg bw.
Executive summary:

A dominant lethal assay equivalent or similar to the OECD guideline 478 was performed in male Charles River albino mice (non-GLP compliant). Twelve animals received a single dermal application of 1500 or 3000 mg/kg bw of the undiluted test substance. Undiluted test substance was applied to the shaved backs (presumably no coverage) of the animals. The negative control was sham treated with sesame oil. A positive control was included, namely 100 mg/kg bw methylmethanesulfonate administered intraperitoneal. The post exposure period was 7 weeks. After exposure, each treated male mouse was mated with three virgin females for one week; this procedure was repeated with further virgin females for five weeks (in total: 6 matings in 6 weeks). Males were sacrificed after 6 weeks and each female one week after removal from the breeding cage. Increase in pre- and post-implantation loss and/or increase in dead embryos served as a measure of dominant lethal events. Negative and positive controls were valid concerning dominant lethal parameters. The numbers of implantations, resorptions, and embryos for both treatment groups compared favorably to those for controls indicating no dominant lethal effects.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
(partly limited documentation, e.g. no details about the test substance; untreated controls; one dose and MTD not reached; no data on historical negative control; no details of results in the re-evaluation of the study by Wellcome Lab (1977))
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
Principles of method if other than guideline:
According to Roehrborn & Vogel, Deutsche Medizinische Wochenzeitschrift 92: 2315-2321 (1967) and World Health Organisation, Technical Report Series, Nr.482 (1971)
GLP compliance:
no
Type of assay:
rodent dominant lethal assay
Specific details on test material used for the study:
- Name of test material: dimethylacetamide (DMAC)
- Substance No.: XX/12
no further details available
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Wiga, Sulzfeld, Germany
- Mean body weight at initiation: 28.5 g in males (27 g in females)
- Age at initiation: 12-14 weeks
- Housing: single cages except during mating (1 male with 3 virgin females)
- Certified diet and tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Rel. air humidity: 50-60 %
- Photoperiod: 12 hrs dark/12h hrs light

Route of administration:
intraperitoneal
Vehicle:
water
Details on exposure:
Dose: 1/5 of the LD50 in male mice
Application volume: 0.1 mL per mouse
Duration of treatment / exposure:
once
Frequency of treatment:
single application
Post exposure period:
8 weeks for treated males

Dose / conc.:
680 other: µl/kg bw
Remarks:
640 mg/kg bw
No. of animals per sex per dose:
20 males
Control animals:
yes, concurrent no treatment
Positive control(s):
0.056 mg/kg bw Trenimon®
Tissues and cell types examined:
Each treated male mice mated with 3 virgin females for 1 week; this procedure was repeated with further virgin females for 7 weeks (totally 8 matings in 8 weeks); each female was sacrificed at day 18 after initiation of mating.

Determined parameters:
number of pregnant dams (conception rate)
total number of implantations
living fetuses
dead fetuses
early resorptions
late resorptions
mutation index: (dead fetuses + early resorptions + late resoptions)/total implantations
Evaluation criteria:
Statistically significant increase of mutation rate/index in the treated group.

Statistics:
Level of significance: p<=0.05
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No clinical signs in the treatment group or positive control group as well as no effects on body weight.
No effects at necropsy in any group.
No effects on conception rate in any group.
Pre-implantation loss: no effects in the treatment group but the positive control resulted in significant loss at week 1.
Post-implantation loss: significant loss in the positive control at week 1, 2, and 3. No significant effects in the treatment group throughout the study.
Conclusions:
No germ cell mutation was detected in the mouse dominant lethal test at a single i.p. dose of 640 mg/kg bw.
Executive summary:

A dominant lethal test in male NMRI mice was performed according to OECD guideline 478 in 1976 (non-GLP compliant). The in vivo exposure was conducted to a concentration of 680 µl/kg bw of the test substance once via intraperitoneal route. Twenty animals were either treated with 0.1 ml of the test substance, received the vehicle (water), or the positive control ( 0.056 mg/kg bw Trenimon®). Each treated male mice was mated with three virgin females. The post-exposure observation time was eight weeks. No clinical signs in the treatment group or positive control group as well as no effects on body weight were observed. No effects at necropsy in any group and no effects on conception rate in any group were reported. No effects on preimplantation loss in the treatment group were observed but the positive control resulted in significant loss at week 1. Significant loss in the positive control was observed at week 1, 2, and 3. No significant effects in the treatment group throughout the study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

no further information

Additional information

In the OECD SIDS document (2001; SIAM 13) on N,N-dimethylacetamide (DMAC) data were presented on this endpoint which were also documented in this report but more details were presented and an evaluation of the validity of these report. The evaluation of results is comparable to the OECD SIDS document. In the OECD SIDS document it was concluded that DMAC does not show mutagenic effects in several in vitro and in vivo tests.

 

In vitro genetic toxicity

Addtionally to the studies of the OECD SIDS document, more recent in vitro mutagenicity studies were performed:

The test substance was tested for its mutagenic potential in a reverse mutation assay (OECD 471). Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 were exposed in the plate incorporation test and preincubation test, in concentrations of 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with and without metabolic activation. The experiments were conducted in triplicate. The test substance is considered to be non-mutagenic in the bacterial reverse mutation assay (2015a, RL1).

The test substance was assessend for its potential to induce mutations at the HPRT locus (OECD 476) using V79 cells of the Chinese Hamster. Following test concentrations were used, 0.1, 0.2, 0.5, 1, 2, 5, 7, 9 and 10 mM (4 h; with and without metabolic activation) and 0.2, 0.5, 1, 2, 5, 7, 9 and 10 mM (20 h; without metabolic activation) and 0.30, 0.75, 1.5, 3, 4, 6, 8 and 10 mM (4 h; with metabolic activation). The test item is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster (2015b, RL1).

A chromosome aberration assay (OECD 473) was carried out in order to investigate a possible potential of the test substance to induce structural chromosome aberrations in human lymphocytes. The metaphases were prepared 24 h after start of treatment. The treatment interval was 4 h without and with metabolic activation (experiment I) and 24 h without metabolic activation (experiment II). Duplicate cultures were set up. The following concentrations were evaluated 6, 8 and 10 mM. The test substance is considered to be non-clasogenic in the chromosome aberration test (2015c, RL1).

Supportingly, in further studies with N,N-Dimethylacetamide in bacteria (S. typhimurium and E.coli WP2uvrA), no increase in revertants was recorded (Zeiger et al., 1988; 1976; 1977). Human embryonic intestinal cells (passage 12-35) were used to conduct an unscheduled DNA synthesis assay (NIOSH, 1980, RL2). Cells were exposed with and without metabolic activation for 3 h to concentrations up to 9.4 mg/mL (8 dose levels tested) in the presence of 3H-thymidine. Two different methods were used: exposure period: 3 h in medium containing 3H-thymidine for labelling; autoradiography. Fifty nuclei were examined for each culture (method 1). Exposure period: 4 h; Liquid scintilation measurement of radioactivity; tritiated deoxyguanosine incorporation measured. Flow 2,002 cells used (method 2). Results showed a negative genotoxic potential.

 

In vivo genetic toxicity

A cytogenetic assay according to OECD 475 was performed. Male and female CD rats were repeatedly exposed to 0, 20 or 700 ppm (0, 70, 2500 mg/m³) of the test substance for 7 h per day at 5 consecutive days (NIOSH, 1980; RL2). Sampling times were 6 h, 24 h, and 48 h post-exposure and bone marrow was prepared for light microscopical evaluation of chromosomal aberrations. The positive control received via gavage 100 mg/kg bw/day ethylmethanesulfonate; negative controls were sham exposed with vehicle. The positive control resulted in a significant increase in chromosome aberrations compared to control, but no increase in the incidences of chromosomal aberrations were found in the treatment groups.

In the dominant lethal assay male CD rats (NIOSH, 1980; RL2) were repeatedly exposed via the inhalation route to 0, 20 or 700 ppm (0, 70, 2500 mg/m³) for 7 h per day at 5 consecutive days. The positive control received 100 mg/kg bw/day ethylmethanesulfonate. After the last exposure each treated male was mated with 2 virgin females for 1 week; this procedure was repeated with further virgin females for 9 weeks (totally 10 matings in 10 weeks); females were sacrificed 17 days after start of mating. Negative and positive controls were valid concerning dominant lethal parameters. No significant effects on fertility index, numbers of corpora lutea or implantations or the frequency of early deaths were detected indicating no dominant lethal effects in the treatment groups.

In the mouse dominant lethal assay the parenteral route was used (1976, RL2). 20 male NMRI mice received a single i.p. injection of 640 mg/kg bw. Treated males, concurrent untreated and positive controls were mated with virgin females (1 male/3 females) the week after treatment; mating for one week with further females was repeated for 7 weeks (totally 8 matings for each male). Females were sacrificed on day 18 after mating initiation; conception rate and mutation index (dead fetuses and all resorptions per total number of implantations) were determined. No clinical signs and no effects on body weight were seen in males of any group; necropsy revealed no treatment related effects. Conception rate was not altered in any group. No effects on pre-implantation loss were seen in the treatment group but the positive control resulted in significant loss at weeks 1, 2, and 3. Concerning post-implantation loss a significant loss in the positive control was detected at weeks 1, 2, 3, and 4. A slight increase in post-implantation loss of the treatment group (19.6 %) was recorded but this effect was not significant (p = 0.056).

In the second mouse dominant lethal assay (1972, RL4: performed at IBT) 12 male mice per dose level received a single dermal application of 1500 or 3000 mg/kg bw (no coverage). The negative control was sham treated with sesame oil and the positive control received i.p. 100 mg/kg bw methylmethanesulfonate. After exposure each treated male mouse was mated with 3 virgin females for 1 week; this procedure was repeated with further virgin females for 5 weeks (totally 6 matings in 6 weeks); males were sacrificed after 6 weeks and each female 1 week after removal from the breeding cage. Negative and positive controls were valid concerning dominant lethal parameters. The numbers of implantations, resorptions, and embryos for both treatment groups compared favourably to those of controls indicating no dominant lethal effects.

In the SLRL test in Drosophila melanogaster (NIOSH, 1980; RL2) wild-type Oregon-K males (stock A and B) were exposed for 95 minutes to 200 ppm (720 mg/m³), the max. tolerated exposure duration at this concentration (tested in preliminary experiments). Males were mated with Müller-5 virgin females followed by mating of F1 brothers and sisters. F2 flies were checked for wild-type males. Only 2 lethals were produced in stock A. In stock B 3 lethals were found (frequeny of 0.57 %), however, all 3 lethals came from the same male. In summary, no gene mutation was found in the SLRL test in Drosophila melanogaster after exposure for 95 minutes to a vapour concentration of 200 ppm (720 mg/m³).

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.