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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994-04-15 - 1994-06-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 471, GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted May 26, 1983 and EEC Directive 92/69, L 383 A, Annex V, B 14, dated December 29, 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
additive
Test material form:
liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): N-Dimethylaminopropyl methacrylamide
- Substance type: organic
- Physical state at room temperature: liquid
- Stability under test conditions: Stability in water: > 160 hours in water; pure: stable for 3 month
- Storage condition of test material: 4 °C, light protected

Method

Species / strain
Species / strain / cell type:
other: Salmonella typhimurium TA1535, TA1537, TA98, TA100 and TA102
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix fraction of Aroclor 1254-induced, male Wistar rats livers
Test concentrations with justification for top dose:
Main experiment:
-S9 mix; 0, 33.3, 100.0, 333.3, 1000.0, 2500.0, 5000.0 µg/plate
+S9 mix; 0, 33.3, 100.0, 333.3, 1000.0, 2500.0, 5000.0 µg/plate
According to the results of the pre-experiment the concentrations applied in the main experiments were chosen.
The maximum concentration was 5000.0 µg/plate. The concentration range include two logarithmic decades. Six adequately spaced concentrations were tested. Two independent experiments were performed.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (purity: >= 99.5%)
- Justification for choice of solvent/vehicle: The solvent was chosen because of its relative nontoxicity for the bacteria.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Concurrent untreated and solvent control: DMSO were performed
Negative solvent / vehicle controls:
yes
Remarks:
vehicle: Dimethylsulfoxide (DMSO)
True negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
for TA 1535, TA 100,
Positive control substance:
sodium azide
Remarks:
sodium azide (purity: >= 99.0%, supplier: Serva, D-69042 Heidelberg, Germany) dissolved in aqua dest.; concentration: 10 µg/plate Migrated to IUCLID6: without metabolic activation
Positive controls:
yes
Remarks:
for TA 1537, TA 98
Positive control substance:
other: 4-nitro-o-phenylenediamine, without metabolic activation
Remarks:
4-nitro-o-phenylenediamine (purity: > 99.9%, supplier: Sigma, D-82041 Deisenhofen, Germany) dissolved in DMSO; concentration: 10 µg/plate
Positive controls:
yes
Remarks:
for TA 102
Positive control substance:
methylmethanesulfonate
Remarks:
methylmethanesulfonate (purity: > 99.0%, supplier: Merck-Schuchardt, D-85662 Hohenbrunn, Germany) dissolved in aqua dest.; concentration: 1.0 µl/plate Migrated to IUCLID6: without metabolic activation
Positive controls:
yes
Remarks:
for TA 1535, TA 1537, TA 98, TA 100, TA 102
Positive control substance:
other: 2-aminoanthracene, with metabolic activation
Remarks:
2-aminoanthracene (purity: 97.5%, supplier: Sigma, D-82041 Deisenhofen, Germany) dissolved in DMSO; concentration: 2.5 µg/plate (10 µg/plate in TA 102)
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation assay

- Salmonella typhimurium strains were obtained from Dr. Heinz Träger (Knoll AG, D-67008 Ludwigshafen, Germany) and were stored as stock cultures in ampoules with nutrient broth with 5% DMSO (Merck, D-64293 Darmstadt, Germany) in liquid nitrogen.

Pre-experiment:
To evaluate the toxicity of the test article a pre-experiment was performed with strains TA98 and TA 100. 8 concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described below for the experiment I.

Test conditions:
System of testing:
-Metabolic activation system: S9 from rat liver, induced with Aroclor 1254.
Administration:
-Number of replicates: 2
-Plate per test: 3
-Application: pre-incubation
Evaluation criteria:
EVALUATION OF RESULTS
The generally accepted conditions for the evaluation of the results are:

- corresponding background growth on both negative controls and test plates
- normal range of spontaneous reversion rates.

Range of spontaneous reversion frequencies*
------------------------------------------------------------------------------------
TA 135 TA 1537 TA 98 TA 100 TA 102
10 - 29 5 - 28 15 - 57 77 - 189 121 - 293

* These values refer to the negative control without metabolic activation and represent our historical control range in 1993.

According to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be
recommended for analysis of data from the bacterial assays at this time.
A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a significant and reproducible
increase for at least one test concentration is induced.
A test article producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive
response at any one of the test points is considered non-mutagenic in this system.

A significant response is described as follows:
A test article is considered mutagenic if in strain TA 100 and TA 102 the number of reversions is at least twice as high and in strains TA 1535,
TA 1537, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate. Also, a dose-dependent and reproducible
increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the
highest dose induced the above described enhancement factors or not.
Statistics:
No appropriate statistical method is available.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA98, TA100 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Toxicity was not observed up to 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: S. typhimurium TA1535, TA1537, TA98, TA100 and TA102
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Tables of the results of experiment I and experiment II please see Attached background material.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the decribed mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains tested.
Therefore, N-Dimethylaminopropyl methacrylamide has to be judged as nonmutagenic up to 5000 µg/plate in the presence and absence of
mammalian metabolic activation S9-mix according to the Ames test results.
Executive summary:

In a reverse gene mutation assay in bacteria (Ames test), strains TA1535, TA1537, TA98, TA100, and TA102 of S. typhimurium were exposed to N-Dimethylaminopropyl methacrylamide ( 98.78% stabilised with 643 ppm HQME) at concentrations of up to 5000 µg/plate in the presence and absence of mammalian metabolic activation S9 -mix

 

N-Dimethylaminopropyl methacrylamid was tested to limit concentration of 5000 µg/plate. 

No toxic effects occurred in the test groups with and without metabolic activation. The plates incubated with the test article showed normal background growth up to 5000.0 ug/plate with and without S9-mix in all strains used. No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with N-Dimethylaminopropyl methacrylamide at any concentration level, either in the presence or absence of metabolic activation (S9 -mix). There was also no tendency to higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance.

Appropriate reference mutagens were used as positive controls.

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

 

This study is classified as acceptable. This study satisfies the requirement for tests for in vitro mutagenicity (bacterial reverse gene mutation) data.

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