N,N-Diethylacrylamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: Negative

Chromosome aberration in mammallian cells: Negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 1993-07-06 to 1993-07-27

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:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batach No.: not specifed
Purity: not specifed

Target gene:

Histidine locus in several strains of Salmonella typhimurium and tryptophan locus of Escherichia coli (E. coli) strain

Species / strain / cell type:

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

Species / strain / cell type:

other: S. typhimurium TA 1538

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver metabolic activation system (S-9 mix)

Test concentrations with justification for top dose:

-Dose-range Finding Test: TA 98, TA 100, TA 1538, TA 1535, TA1537 and the WP2uvrA, both with and without S9-mix: 5, 50, 500, 5000 μg/ plate.

Mutatuion Test 1: TA 98, TA 100, TA 1538, TA 1535, TA1537 and the WP2uvrA, both with and without S9-mix: 312.5, 625, 1250, 2500 and 5000 μg/ plate.

Mutatution Test 2: TA 98, TA 100, TA 1538, TA 1535, TA1537 and the WP2uvrA, both with and without S9-mix: 312.5, 625, 1250, 2500 and 5000 μg/ plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Prior to commencing testing the solubiity of the material was assessed at 50 mg/mL in water in which it was found to be completely miscible. Water was therefore used as the solvent for this study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S-9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S-9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
- Cell density at seeding: 2E+9 cells per mL

DURATION
- Exposure duration: all plates were incubated 3 days

Evaluation criteria:

The mean number of revertant colonies for all treatment groups is compared with those obtained for solvent control groups. The mutagenic activity of a test material is assessed by applying the following criteria:
a) If treatment with a test material produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of positive dose-relationship, in two separate experiments, with any bacterial strain either in presence or absence of S-9 mix it’s considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
b) If treatment with a test material does not produce reproducible increases at least 1.5 times the concurrent controls, at any does level with any bacterial strain, it’s considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
c) If the result obtained fail to satisfy the criteria for a clear “positive” or “negative” response given in paragraphs (a) and (b), the following approach is taken in order to resolve the issue of the material’s mutagenic activity in this test system.
(1) Repeat test may be performed using modifications of the experimental method. These modifications include (but are not restricted to), the use of narrower dose range that already tested; the use of different levels of liver homogenate S-9 fraction in the S-9 mix. Should an increase in revertant colony numbers be observed which satisfies paragraph (a) the material is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(2) If no clear “positive” response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et al. (1989).

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538

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

Key result

Species / strain:

E. coli WP2 uvr A

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 substantial increases in revertant colony numbers of any the tester strains were observed following treatment with the test item at any dose level, in the presence or absence of S-9 mix in either mutation test.
Tester strain TA 98 was contaminated in the second mutation test but this contamination did not affect the counting of the plates.

Conclusions:

When tested in water, the test item shows no evidence of mutagenic activity in this bacterial system.

Executive summary:

In this in vitro assessment of the mutagenic potential of test item, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and a tryptophan dependent mutant of Escherichia coli (WP2 uvrA) were exposed to the test material, diluted in water which was used as a negative control.

 

Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induce rats.

 

In the preliminary dose range finding study with dose levels of up to 5000μg/plate no toxicity was observed. A top dose level of 5000μg/plate was chosen for subsequent mutation study. other dose levels used in mutation assays were: 2500, 1250, 625, 312.5μg/plate.

 

No evidence of mutagenic activity was seen at any dose level of test item in either mutation test.

 

The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolizing activity of the liver preparations.

 

It's concluded that, when tested in water, the test item shows no evidence of mutagenic activity in this bacterial system.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2013-08-26 to 2013-10-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

GLP compliance:

yes

Type of assay:

other: in vitro chromosome aberration

Specific details on test material used for the study:

Bacth No.: not specified
Purity: 99.95%

Species / strain / cell type:

other: Chinese hamster lung cell line

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Chinese hamster lung cells. The cell is obtained from American Type Culture Collection (2010-04, USA).
- Suitability of cells: it has been widely used and there are abundant basic information for chromosome aberration test.
-Store: in liquid nitrogen was thawed and cultured for 2~3 generation times.

Culture media and condition
- Media: minimum Essential Medium (Gibco) 500 mL supplemented with Fetal Bovine Serum (Gibco) 50 mL was used.
Condition: cultures were incubated at (37 ± 1) °C in a humidified atmosphere of (5 ± 1)% CO2. The CHL cells were subsequently subcultured every 3~4 days.

Metabolic activation:

with and without

Metabolic activation system:

Sprague-Dawley male rat liver induced by Aroclor-1254 (S9)

Test concentrations with justification for top dose:

Main test with and without S9: 1300, 650 and 325 μg/mL
Confirmation test with and without S9: 1300, 650 and 325 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: the tets item was soluble in sterile distilled water, so sterile distilled water was chosen as a vehicle and used in diluting the test substance.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

other: Cyclophosphamide monohydrate (CPA)

Details on test system and experimental conditions:

- Cell density at seeding: 4000 cells/mL

DURATION
- Preincubation period: 3 days
- Exposure duration: 6 (24) hours without S9, 6 (6) hours with S9

Evaluation criteria:

Chromosome aberration was determined by following criteria.
Negative (-): less than 5%
Equivocal (+): from 5% to less than 10%
Positive (+): 10% or more

Key result

Species / strain:

mammalian cell line, other: CHL cell

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:

-Decision of concentration
In the concentration range-finding test, cytotoxicity was less than 50% at all test concentration in absence of (S9) and presence (S9) of metabolic activation system. The highest concentration was selected at 1300 μg/mL for main test and confirmation test. Precipitation of test was not observed.

-Without metabolic activation I (S9- 6h) –Main test
In the absence (S9-) of metabolic activation I, the frequency of structural aberrant cells were 0.5%, 0.0%, 1.0% and 0.5% at the concentrations of 0, 325, 650 and 1300 μg/mL, respectively. All the results were less than 5% which was evaluated negative from criteria.

-With metabolic activation I (S9+ 6h) –Main test
In the presence (S9+) of metabolic activation I, the frequency of structural aberrant cells were 0.5%, 0.5%, 1.0% and 1.0% at the concentrations of 0, 325, 650 and 1300 μg/mL, respectively. All the results were less than 5% which was evaluated negative from criteria.

-Without metabolic activation II (S9- 24h) –Confirmation test
In the absence (S9-) of metabolic activation II, the frequency of structural aberrant cells were 0.5%, 0.5%, 0.0% and 0.0% at the concentrations of 0, 325, 650 and 1300 μg/mL, respectively. All the results were less than 5% which was evaluated negative from criteria.

-With metabolic activation II (S9+ 6h) –Confirmation test
In the presence (S9+) of metabolic activation II, the frequency of structural aberrant cells were 1.0%, 0.0%, 0.5% and 1.0% at the concentrations of 0, 325, 650 and 1300 μg/mL, respectively. All the results were less than 5% which was evaluated negative from criteria.

Conclusions:

The test item was determined not induce chromosome aberration in CHL cell under the presence experimental condition.

Executive summary:

The test substance was evaluated for its potential to induce chromosome aberration performing the chromosome aberration test with cultured Chinese hamster lung cell line (CHL) in the absence (S9-) and presence (S9+) of metabolic activation system under OECD Guideline 473. The test substance was prepared by dissolving in sterile distilled water.

 

In the main and confirmation test, the highest concentration was determined at 1300 μg/mL (about 10 mM) in either the absence (S9-) and presence (S9+) of metabolic activation system, because no growth inhibition more than 50% was observed in the concentration rang-finding test.

 

Five groups were included in both main and confirmation test, 3 dose level (325, 650 and 1300 μg/mL), negative and positive groups.

As a result of main and confirmation test, the frequency of structural aberrant cells was less than 5% at all test concentrations, and there was no increase of chromosome aberrations according to the concentration dependent manner compared with negative control.

 

Therefore, the test substance was determined not induce chromosome aberration in CHL cell under the presence experimental condition. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Negative result both in Ames study and In vitro chromosome aberration study.

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.5.1, this substance should not be classified as mutagen.