[No public or meaningful name is available]

Genetic toxicity: in vitro

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:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was performed similarly to the OECD 471 Guideline with acceptable restrictions.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

: no certificate of analysis, no historical control data, no true assessment of the cytotoxicity, only 2 plates/concentration, only the pre-incubation method is used, no indication on the precipitation.

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid

IUPAC Name:

Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid

Test material form:

other: amber liquid at room temperature

Details on test material:

- Name of test material (as cited in study report): Mixture of N-[2-(2-oxo-1-imidazolidinyl)ethyl]methacrylamide and methacrylic acid, other name: WAM II
- Stability under test conditions: stable for 28 days in the dark, at ambient temperature

Method

Target gene:

Histidine gene for S. typhimurium strain, Trptophan for E. coli strain

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: additional mutations in rfa and uvrB genes. For the strains TA98, TA100 and WP2 uvrA presence of an additional plasmid pKM101 in order to enhance their sensitivity of detection of some mutagens. See Table 7.6.1/1

Metabolic activation:

with and without

Metabolic activation system:

Self-produced S9 fraction by induction of male SD rats' liver with administration of PB and 5,6-BF directly to stomach

Test concentrations with justification for top dose:

The amount used for the test was calculated as concentration of N-[2-(2-oxo-1-imidazolidinyl)ethyl]methacrylamide.
preliminary study: 0; 50; 100; 200; 500; 1000; 2000 and 5000 µg/plate
Main test: 313; 625; 1250; 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no data

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-(2-furyl)-3(5-nitro-2-furyl)acrylamide (AF-2); 2-methoxy-6-chloro-9-[3-(2-chloroehyl)-aminopropylamino]acridine.2HCl (ICR-191) and 2-aminoanthracene (2AA)

Remarks:

see details in table 7.6.1/2

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period:20 min
- Exposure duration: 48h
- Expression time (cells in growth medium): 48h
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: 2 plates(triplicate for the negative control), 2 independant experiments

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: no data

Evaluation criteria:

no data

Statistics:

no data

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See details in Table 7.6.1/3 to 7.6.1/6

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

As there was no decrease in the number of revertant, the test item can be considered as non cytotoxic.

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See details in Table 7.6.1/3 to 7.6.1/6

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

As there was no decrease in the number of revertant, the test item can be considered as non cytotoxic.

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See details in Table 7.6.1/3 to 7.6.1/6

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

As there was no decrease in the number of revertant, the test item can be considered as non cytotoxic.

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See details in Table 7.6.1/3 to 7.6.1/6

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

As there was no decrease in the number of revertant, the test item can be considered as non cytotoxic.

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See details in Table 7.6.1/3 to 7.6.1/6

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

As there was no decrease in the number of revertant, the test item can be considered as non cytotoxic.

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: > 50%
no other data

RANGE-FINDING/SCREENING STUDIES:the range-finding study was performed in the same conditions as the main study, the number of revertants was calculated by the mean of the 3 replicate plates perfomed for each concentration of test item. The range of concentration of the preliminary study is broader than in the main study. Thus, this preliminary can be considered as a mutagenic experiment as such.

COMPARISON WITH HISTORICAL CONTROL DATA: no historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/3:Number of revertants per plate in the absence of metabolic activation in the first test

Test item Concentration (µg/plate)	TA 1535	TA1537	TA 98	TA 100	WP2 uvrA
	Mean$	Mean$	Mean$	Mean$	Mean$
0	14	12	25	139	37
50	15	15	19	142	47
100	15	9	28	155	40
200	10	7	21	127	38
500	15	9	21	151	52
1000	8	9	24	151	47
2000	12	11	25	144	38
5000	11	8	20	138	53
Positive control**	259	1685	513	916	458

$: Mean of triplicate

**Mutagens positive controls:

- NaN₃(0.5 µg/plate) in TA1535 strain

- ICR-191 (1 µg/plate) in TA1537 strain

- AF-2 (0.01 µg/plate) in TA 100 and WP2 uvrA strains

- AF-2 (0.1 µg/plate) in TA 98 strain

 

 

Table 7.6.1/4: Number of revertants per plate in the presence of metabolic activation (S9 mix) in the first test 

Test item Concentration (µg/plate)	TA 1535	TA1537	TA 98	TA 100	WP2 uvrA
	Mean$	Mean$	Mean$	Mean$	Mean$
0	13	17	36	144	50
50	9	19	37	144	52
100	14	18	34	161	54
200	9	20	36	129	49
500	13	15	40	145	54
1000	11	15	43	143	61
2000	15	21	30	147	61
5000	14	26	47	203	58
Positive control**	202	172	200	948	547

$: Mean of triplicate

**Mutagens positive controls:

- 2AA (2 µg/plate) in TA1537 and TA1535 strains

- 2AA (1 µg/plate) in TA100 strain

- 2AA (0.5 µg/plate) in TA98 strain

- 2AA (10 µg/plate) in WP2 uvrA strain

 

Table 7.6.1/5: Number of revertants per plate in the absence of metabolic activation in the second test

Test item Concentration (µg/plate)	TA 1535	TA1537	TA 98	TA 100	WP2 uvrA
	Mean$	Mean$	Mean$	Mean$	Mean$
0	13	7	23	110	36
313	9	8	28	124	43
625	12	8	31	131	34
1250	10	11	20	123	39
2500	16	13	16	122	42
5000	12	11	27	130	42
Positive control**	257	1414	488	511	510

$: Mean of triplicate

**Mutagens positive controls:

- NaN₃(0.5 µg/plate) in TA1535 strain

- ICR-191 (1 µg/plate) in TA1537 strain

- AF-2 (0.01 µg/plate) in TA 100 and WP2 uvrA strains

- AF-2 (0.1 µg/plate) in TA 98 strain

 

Table 7.6.1/6: Number of revertants per plate in the presence of metabolic activation (S9 mix) in the second test

 

Test item Concentration (µg/plate)	TA 1535	TA1537	TA 98	TA 100	WP2 uvrA
	Mean$	Mean$	Mean$	Mean$	Mean$
0	11	20	38	122	43
313	12	17	26	131	47
625	14	18	29	142	53
1250	12	12	29	133	40
2500	12	16	44	143	39
5000	16	19	46	147	51
Positive control**	121	89	232	587	352

$: Mean of triplicate

**Mutagens positive controls:

- 2AA (2 µg/plate) in TA1537 and TA1535 strains

- 2AA (1 µg/plate) in TA100 strain

- 2AA (0.5 µg/plate) in TA98 strain

- 2AA (10 µg/plate) in WP2 uvrA strain

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the test conditions, Reaction mass of methacrylic acid and N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide did not show any mutagenic activity in the bacterial reverse mutation test using Salmonella typhimurium and E. coli.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD No.471 guideline, Reaction mass of methacrylic acid and N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide diluted in water was tested in S. typhimurium TA1535, TA1537, TA100, TA98 and E. coli WP2 uvrA in the presence and the absence of mammalian metabolic activation (S9) using the preincubation method. The amount used for the test was calculated as concentration of N-[2-(2-oxo-1-imidazolidinyl)ethyl]methacrylamide (48.61%). Six known mutagens (Sodium azide; 2-(2-furyl)-3(5-nitro-2-furyl)acrylamide (AF-2); 2-methoxy-6-chloro-9-[3-(2-chloroehyl)-aminopropylamino]acridine.2HCl (ICR-191) and 2-aminoanthracene (2AA)) were used to check the sensitivity of the test system. They gave appropriate response, therefore the test was considered as valid.

In the first assay the test item was used at the following concentration: 0; 50; 100; 200; 500; 1000; 2000 and 5000 µg/plate (duplicate). There was a slight increase in the number of revertants in the TA98, TA100 and TA1537 strains with S9 mix at the highest dose, but this increase did not exceed twice the negative control (<1.5). Therefore it was not considered as relevant regarding the mutagenicity. For the other conditions, there was no increase in the number of revertants compared to the negative control.

In the second assay the test item was used at the following concentration: 313; 625; 1250; 2500 and 5000 µg/plate (duplicate). There was a slight increase in the number of revertants in the TA1537 without S9 mix at 2500 µg/plate, but the mean revertants did not exceed twice the mean obtained for the negative control and there was no dose-response relationship. Therefore it was not considered as relevant regarding the mutagenicity. For the other conditions, there was no increase in the number of revertants compared to the negative control.

As there was no decrease in the number of revertants compared to the negative control, the test item can be considered as non cytotoxic up to the recommended limit concentration of 5000 µg/plate.

Under the test conditions, Reaction mass of methacrylic acid and N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide did not show any mutagenic activity in the bacterial reverse mutation test using Salmonella typhimurium and E. coli. This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 471.