Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
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

Constituent 1
Reference substance name:
Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid
EC Number:
934-058-1
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
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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.