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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All methacrylate esters share a common and similar level of chemical reactivity through the C=C double bond and Michael addition, which appears not to be linked to mutagenicity in the case of the other esters. This analysis is consistent with a review of the mutagenic potential of lower alkyl methacrylates and closely related methacrylate esters by Albertini (2017). The review addresses mode of action, similarity of esters and all available test data at the point of publications from reactivity and genotoxicity data to full mutagenicity studies.

Therefore, it is concluded that all constituents of NUMA which all are methacrylic esters, are non-mutagenic in bacterial gene mutation assays.

Link to relevant study records

Referenceopen allclose all

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:
27-03-2008 - 08-04-2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 471, GLP. Study according to relevant guideline.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted May 26, 1983
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix fraction of Aroclor 1254-induced, male Sprague-Dawley rats and male Syrian hamster livers
Test concentrations with justification for top dose:
Main experiment:
-S9 mix; 0; 33; 100; 333; 1000; 2500; 5000 µg/plate
+S9 mix; 0; 33; 100; 333; 1000; 2500; 5000 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.


Each chemical was tested initially at half-log dose intervals up to a dose that elicited toxicity, or to a dose immediately below one which was toxic in
the preliminary toxicity test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Untreated negative controls:
yes
Remarks:
Concurrent untreated and solvent control: DMSO were performed
Negative solvent / vehicle controls:
yes
Remarks:
vehicle: THF
True negative controls:
yes
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
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: > 99%, supplier: MERCK, D-64293 Darmstadt, 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: preincubation assay as described by Haworth et al. 1983, with some differences.

- Salmonella typhimurium strains were obtained from Dr. Bruce Ames (University of California, Berkeley, U.S.A.) and were stored as recommended (Maron and Ames, 1983).
- Cultures were grown overnight with shaking at 37 °C in Oxoid No. 2 broth, and their phenotypes were analyszed prior to their use for mutagenicity assays.

Test conditions:
System of testing:
-Metabolic activation system: S9 from rat liver, induced with phenobarbital and 5,6-benzoflavone.
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:
According to OECD guideline 471, a statistical analysis of the data is not mandatory.
Key result
Species / strain:
other: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results:
negative

In conclusion, it can be stated that during the decribed mutagenicity test and under the experimental conditions reported, the test item did not
induce gene mutations by base pair changes or frameshifts in the genome of the strains tested.
Therefore, Isodecyl methacrylate has to be judged as nonmutagenic in the presence and absence of mammalian metabolic activation
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 Salmonella typhimurium were exposed to Isodecyl methacrylate at concentrations of up to 5000 µg/plate in the presence and absence of mammalian metabolic activation S9 -mix. 

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 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 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 biological relevance.

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.

Therefore, Isodecyl methacrylate is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

 

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

 

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Method and results sufficient described, similar to OECD method.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
his-
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat and hamster S9-mix
Test concentrations with justification for top dose:
33 - 4000 µg/plate
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
2-Aminoanthracene; 4-Nitro-o-phenylenediamine; Sodium azide; 9-Aminoacridine
Evaluation criteria:
Positive result indicated by a reproducible, dose related increase.
Statistics:
Margolin et al., 1981
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information):
negative

Using a scientifically valid methods, MAA was negative for mutagenicity in Salmonella tester strains.
Executive summary:

Using a scientifically valid methods, MAA was negative for mutagenicity in Salmonella tester strains.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Peer reviewed data. No more information available.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Genotoxicity:
negative
Conclusions:
Interpretation of results (migrated information):
negative

Using a valid scientific method, Dodecyl methacrylate was negative for genotoxicity in the Salmonella typhimurium reverse mutation assay.
Executive summary:

Using a valid scientific method, Dodecyl methacrylate was negative for genotoxicity in the Salmonella typhimurium reverse mutation assay.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
 S9 : Rat liver, induced with phenobarbital and 5,6-benzoflavone 
Test concentrations with justification for top dose:
-S9 mix; 39.1, 19.5, 9.77, 4.88, 2.44, 1.22, 0.610 μg/plate(TA100, TA1535, TA1537); 313, 625, 1250, 2500, 5000 μg/plate(WP2 uvrA,TA98)
+S9 mix; 625, 313, 156, 78.1, 39.1, 19.5, 9.77 μg/plate (TA100, TA1535, TA1537); 39.1, 78.1, 156, 313, 625, 1250, 2500 μg/plate(WP2 uvrA, TA98)
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9 mix; AF-2 (TA100, TA98), Sodium azide (TA1535), ENNG (WP2 uvrA) and 9-Aminoacridine (TA1537). +S9 mix; 2-Aminoanthracene (all strains) 
Details on test system and experimental conditions:
Pre-incubation method
Plates/test : 3 
Number of replicates : 2
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
9.77 µg/plate (TA100, TA1535 and TA1537) without metabolic activation, and 156 µg/plate (TA100 and TA1535) and 625 µg/plate (TA98) and 313 µg/plate (TA1537) with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
625 µg/plate with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA

This chemical did not induce gene mutations in the S.typhimurium and E. coli strains. For details on resulöts see attached tables.

Conclusions:
Interpretation of results (migrated information):
negative

In a valid guideline study, the test substance did not induce gene mutations in the S.typhimurium and E. coli strains. 
Executive summary:

In a valid guideline study, the test substance did not induce gene mutations in the S.typhimurium and E. coli strains. 

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium, other: Strains TA98, TA100, TA 102, TA 1535 and TA 1537
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from liver homogenates from rats induced with Aroclor 1254 (500 mg/kg) by intraperitoneal route
Test concentrations with justification for top dose:
Without S9 mix: 12.5, 25, 50, 100 and 200 µg/plate,
With S9 mix: 312.5, 625, 1250, 2500 and 5000 µg/plate in the first test and 31.25, 62.5, 125, 250 and 500 µg/plate in the second test
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9 mix: Sodium azide (NaN3) (TA 1535- TA 100), 9-Aminoacridine (9AA) (TA 1537), 2-Nitrofluorene (2NF) (TA 98) and Mitomycin C (MMC) (TA 102). + S9 mix: 2-Anthramine (2AM) (TA 1535 - TA 1537 - TA 98 - TA 100) and Danthron (DTH) (TA 102)
Details on test system and experimental conditions:
PRELIMINARY TOXICITY ASSAY
To assess the toxicity of the test substance to the bacteria, 6 doses (1 plate/dose) were tested in the TA 100 strain, with and without S9 mix.

MUTAGENICITY ASSAY
Five dose levels of test article along with appropriate vehicle control and positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and TA102 on selective agar in the presence and absence of Aroclor induced rat liver S9. All dose levels of test article, vehicle control and positive controls were plated in triplicate.

TEST PROCEDURE
Plate incorporation method:
One half (0.5) milliliter of S9 or Sham mix, 100 µL of tester strain and 100 µL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45°C. After vortexing, the mixture was overlaid onto the surface of minimal bottom agar.
Preincubation method:
One-half (0.5) milliliter of S9 or sham mix, 100 µL of tester strain and 100 µL of vehicle or test article dilution were added to glass culture tubes. Alter vortexing, these mixtures were incubated with shaking for 60 minutes at 37°C. Following the preincubation, 2.0 mL of selective top agar was added to each tube and the mixture was vortexed and overlaid onto the surface of minimal bottom agar.

When plating the positive controls, the test article aliquot was replaced by the of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37°C. Revertant colonies for a given tester strain and activation condition were counted by automated colony counter.

Evaluation criteria:
Acceptance criteria
This study was considered valid because the following criteria were fully met:
. the number of revertants of the controls was within the range of our historical data,
. the number of revertants of the positive controls was higher than that of the controls and was within the range of our historical data.

Evaluation criteria
The following criteria were used as an aid for determining a positive response:
. a reproducible and significant dose relationship using a linear regression analysis, considered as significant if p <= 0.05 (for n = 18 values, the correlation coefficient must be r >= 0.47).
and/or
. a reproducible and significant increase (i.e. a doubling in the number of revertants for at least one of the tested strains when compared to that of the controls) for at least one of the doses.
A test substance is considered as non-mutagenic in this test system if the above two criteria are not fully met.

Biological and statistical significances were considered during the evaluation.
Statistics:
Linear regression analysis.
Species / strain:
S. typhimurium, other: Strains TA98, TA100, TA 102, TA 1535 and TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 100 µg/plate without S9, >= 5000 µg/plate with S9 (plate incorporation assay) and >= 250 µg/plate (preincubation assay)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The negative and solvent control results were equivalent to those usually obtained in the Laboratory. The number of revertants induced by the positive controls was higher than the spontaneous one, which demonstrated the sensitivity of this test and the efficacy of the S9 mix throughout this study.

The test substance 2-ETHYLHEXYL METHACRYLATE did not induce any significant increase in the number of revertants, with or without S9 mix, in any of the 5 strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

0 : solvent control (DMSO)

T : toxicity :                                                   P : precipitate of the test substance :

- : no                                                                  - :no

+ : slight                                                             + : slight

+ + : moderale                                                      + + : moderate

+ + + : considerable to total                                    + + + : strong

Table 1: First mutagenicity test without metabolic activation

strains

concentrations

T

P

mean

standard

ratio

(µg/plate)

deviation

TA 1535

SR

-

-

12

1

-

0

-

-

10

1

-

12.5

-

-

9

3

0.9

25

-

-

7

1

0.7

50

+

-

8

1

0.8

100

+

-

6

1

0.6

200

+ +

-

7

2

0.7

NaN3(1)

-

-

266

6

26.6

TA 1537

SR

-

-

10

3

-

0

-

-

11

1

-

12.5

-

-

10

4

0.9

25

-

-

8

2

0.8

50

+

-

14

2

1.3

100

+

-

10

2

0.9

200

+ +

-

10

6

1.0

9AA(50)

-

-

96

23

9.0

TA 102

SR

-

-

281

20

-

0

-

-

236

41

-

12.5

-

-

254

17

1.1

25

-

-

255

15

1.1

50

-

-

262

13

1.1

100

-

-

245

21

1.0

200

-

-

239

6

1.0

MMC(0.5)

-

-

1976

220

8.4

TA 98

SR

-

-

21

2

-

0

-

-

25

5

-

12.5

-

-

21

8

0.8

25

-

-

16

4

0.6

50

-

-

15

8

0.6

100

-

-

15

6

0.6

200

+

-

10

2

0.4

2NF(0.5)

-

-

211

30

8.3

TA 100

SR

-

-

103

24

-

0

-

-

106

18

-

12.5

-

-

92

12

0.9

25

-

-

83

9

0.8

50

-

-

91

6

0.9

100

-

-

88

10

0.8

200

+

-

70

25

0.7

NaN3(1)

-

-

400

32

3.8


Table 2: Second mutagenicity test without metabolic activation

strains

concentrations

(µg/plate)

T

P

mean

standard

deviation

ratio

TA 1535

SR

-

-

8

1

-

0

-

-

8

2

-

12.5

-

-

8

3

1.0

25

-

..

7

1

0.8

50

+

-

8

5

0.9

100

+

-

9

3

1.0

200

++

-

3

5

0.4

NaN3(1)

-

-

217

22

26.1

TA 1537

SR

-

-

8

4

-

0

-

-

9

1

-

12.5

-

-

9

3

1.0

25

-

-

7

1

0.8

50

-

-

8

2

0.9

100

+

-

8

2

0.9

200

+

-

8

2

0.9

9AA(50)

-

-

129

34

14.3

TA 102

SR

-

-

248

17

-

0

-

-

278

30

-

12.5

-

-

263

10

0.9

25

-

-

250

6

0.9

50

-

-

229

21

0.8

100

-

-

239

15

0.9

200

-

-

220

11

0.8

MMC(0.5)

-

-

1591

82

5.7

TA 98

SR

-

-

18

1

-

0

-

-

19

6

-

12.5

-

-

14

3

0.7

25

-

-

16

3

1.2

50

-

-

12

9

0.7

100

+

-

11

3

0.6

200

+

-

9

1

0.5

2NF(0.5)

-

-

127

11

6.6

TA 100

SR

-

-

97

15

-

0

-

-

104

16

-

12.5

-

-

80

5

0.8

25

-

-

86

3

0.8

50

-

-

84

10

0.8

100

-

-

82

12

0.8

200

+

-

88

1

0.8

NaN3(1)

-

-

290

10

2.8

Conclusions:
Interpretation of results (migrated information):
negative

2-ethylhexyl methacrylate in the presence or absence of metabolic activation did not induce any significant increases in the revertant numbers
Executive summary:

A Salmonella typhimurium reverse mutation assay was performed with 2-ethylhexyl methacrylate according to OECD Guideline 471 and GLP. S. typhimurium strains, TA98, TA100, TA102, TA1535 and TA1537 were exposed to five concentrations of 2-ethylhexyl methacrylate in the presence and absence of a metabolic activation system using the plate incorporation and the preincubation methods. All doses of 2-ethylhexyl methacrylate, vehicle control and positive controls were plated in triplicate and incubated for 48-72 hours at 37 ºC. The positive controls gave expected responses. 2-ethylhexyl methacrylate was cytotoxic at >= 100 µg/plate without S9, >= 5000 µg/plate with S9 (plate incorporation assay) and >= 250 µg/plate (preincubation assay). Treatment of the S. typhimurium strains with 2-ethylhexyl methacrylate in the presence or absence of metabolic activation did not induce any significant increases in the revertant numbers.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
27-03-2008 - 08-04-2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-study OECD 471, GLP. Study according to relevant guideline.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix fraction of Phenobarbital/beta-Naphthoflavone-induced, male Wistar rat liver
Test concentrations with justification for top dose:
Pre experiment:
0; 3; 10; 33; 100; 333; 1000; 2500; 5000 µg/plate

Main experiment I and II:
-S9 mix; 0; 33; 100; 333; 1000; 2500; 5000 µg/plate
+S9 mix; 0; 33; 100; 333; 1000; 2500; 5000 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.


Each chemical was tested initially at half-log dose intervals up to a dose that elicited toxicity, or to a dose immediately below one which was toxic in the preliminary toxicity test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Untreated negative controls:
yes
Remarks:
Concurrent untreated and solvent control: DMSO were performed
Negative solvent / vehicle controls:
yes
Remarks:
vehicle: Ethanol
True negative controls:
yes
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: 4.0 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: palate incorporation test (experiment I) and preincubation assay (experiment II)

- Salmonella typhimurium strains were obtained from Trinova Biochem GmbH (D-35394 Gießen, Germany) and were stored as stock cultures in ampoules with nutrient broth + 5% DMSO in liquid nitrogen

Test conditions:
System of testing:
-Metabolic activation system: S9 from rat liver, induced with phenobarbital and beta-Naphthoflavone.
Administration:
-Number of replicates: 2
-Plate per test: 3
Evaluation criteria:
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:
According to OECD guideline 471, a statistical analysis of the data is not mandatory.
Species / strain:
other: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
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 item did not
induce gene mutations by base pair changes or frameshifts in the genome of the strains tested.
Therefore, Monomer Mix (Methacrylic acid ester of an alcohol mixture with a average C-number of 14,8) has to be judged as nonmutagenic in the presence and absence of mammalian metabolic activation 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 Salmonella typhimurium were exposed to Monomer Mix (Methacrylic acid ester of an alcohol mixture with a average C-number of 14,8) at concentrations of up to 5000 µg/plate in the presence and absence of mammalian metabolic activation S9-mix. 

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 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 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 biological relevance.

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.

Therefore, Monomer Mix (Methacrylic acid ester of an alcohol mixture with a average C-number of 14,8) is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

 

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

 

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study well documented, meets generally accepted scientific principles, acceptable for assessment.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Method: Ames test
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
his-
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Aroclor 1254 induced Sprague Dawley rat liver and male Syrian Hamster liver.
Test concentrations with justification for top dose:
Dosis: 33 - 10000 µg/plate
At least five doses tested in triplicate.
Vehicle / solvent:
DMSO (Dimethyl sulfoxid)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9: sodium azide, 9-aminoacridine and 4-nitro-o-phenylenediamine. With S9: 2-aminoanthracene
Details on test system and experimental conditions:
Salmonella typhimurium reverse mutation assay
Two preincubation assays were conducted.
Evaluation criteria:
An individual trial was judged (+) mutagenic if a dose related increase over the corresponding solvent control was seen, and it was judged weakly mutagenic (+W) if a low dose response was seen. A trial was considered questionable (?) if a dose related increase was judged insufficiently high to justify a call of "+W" if only a single dose was elevated over control or if a non-dose related increase was seen. A chemical was judged weakly mutagenic "+W' or mutagenic "+" if it produced a reproducible, dose related increase in his+ revertants over the corresponding solvent controls in replicate trials.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Testing laboratory: CWR
S9 mix from Aroclor 1254 induced Sprague Dawley rat liver and male Syrian Hamster liver (10% in test concentration)
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results: negative

Using a valid scientific method, n-Decyl methacrylate was negative for genotoxicity in the Salmonella typhimurium reverse mutation assay.
Executive summary:

Using a valid scientific method, n-Decyl methacrylate was negative for genotoxicity in the Salmonella typhimurium reverse mutation assay.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Method: Ames test
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
his-
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 97
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Aroclor 1254 induced Sprague Dawley rat liver and male Syrian Hamster liver (10 or 30 %).
Test concentrations with justification for top dose:
At least five doses tested in triplicate.
S. typhimurium TA 100: 100 - 10000 µg/plate
S. typhimurium TA 1535: 100 - 10000 µg/plate
S. typhimurium TA 1537: 100 - 1000 µg/plate
S. typhimurium TA 98: 33 - 10000 µg/plate
S. typhimurium TA 97: 1 - 10000 µg/plate
Vehicle / solvent:
95 % ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9: sodium azide, 9-aminoacridine and 4-nitro-o-phenylenediamine. With S9: 2-aminoanthracene
Details on test system and experimental conditions:
Salmonella typhimurium reverse mutation assay
Two preincubation assays were conducted.
Evaluation criteria:
An individual trial was judged (+) mutagenic if a dose related increase over the corresponding solvent control was seen, and it was judged weakly mutagenic (+W) if a low dose response was seen. A trial was considered questionable (?) if a dose related increase was judged insufficiently high to justify a call of "+W" if only a single dose was elevated over control or if a non-dose related increase was seen. A chemical was judged weakly mutagenic "+W' or mutagenic "+" if it produced a reproducible, dose related increase in his+ revertants over the corresponding solvent controls in replicate trials.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
S9 mix from Aroclor 1254 induced Sprague Dawley rat liver and male Syrian Hamster liver (10% or 30 % in test concentration)
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results: negative

Using a valid scientific method, n-Octyl methacrylate was negative in the presence or absence of metabolic activation for genotoxicity in the Salmonella typhimurium reverse mutation assay.
Executive summary:

A Salmonella typhimurium reverse mutation assay was performed with n-Octyl methacrylate similar to OECD Guideline 471 (GLP compiance not specified). Salmonella typhimurium strains, TA 97, TA98, TA100, TA1535 and TA1537 were exposed to eight concentrations (0 - 10000 µg/plate) of n-Octyl methacrylate in the presence and absence of a metabolic activation system using the plate incorporation and the preincubation methods. All doses of n-Octyl methacrylate, vehicle control and positive controls were plated in duplicate and incubated for 48-72 hours at 37 ºC. The positive controls gave expected responses. Treatment of the S. typhimuriums trains with n-Octyl methacrylate in the presence or absence of metabolic activation did not induce any significant increases in the revertant numbers.

Using a valid scientific method, n-Octyl methacrylate was negative for genotoxicity in the Salmonella typhimurium reverse mutation assay.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included as attachment. Please also see attached justification.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The constituents of the UVCB substance NUMA (Nonyl-Undecyl methacrylate) are structurally related mono alkyl methacrylate esters differing only in the respective alcoholic moieties. The main proportion of these esters are of the n-type the minor proportion of the iso type. Considering the small amount of iso-types and the negligible differences in (eco-) toxicological properties between the n- and iso types, in this assessment both types of one ester with one specific chain length are assessed together as a whole.
Further information is included as attachment. Please also see attached justification Chapter 1

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Further information is included as attachment. Please also see attached justification Chapter 1.

3. ANALOGUE APPROACH JUSTIFICATION
Please see attached justification Chapter 1 and 3.

4. DATA MATRIX
Please see attached justification Chapter 1.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Species / strain:
S. typhimurium, other: Based on read across it is concluded that all constituents of NUMA which all are methacrylic esters, are non-mutagenic in bacterial gene mutation assays
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Based on read across it is concluded that all constituents of NUMA which all are methacrylic esters, are non-mutagenic in bacterial gene mutation assays
Cytotoxicity / choice of top concentrations:
not determined
Conclusions:
Based on read across it is concluded that all constituents of NUMA which all are methacrylic esters, are non-mutagenic in bacterial gene mutation assays.
Executive summary:

All methacrylate esters share a common and similar level of chemical reactivity through the C=C double bond and Michael addition, which appears not to be linked to mutagenicity in the case of the other esters. This analysis is consistent with a review of the mutagenic potential of lower alkyl methacrylates and closely related methacrylate esters by Albertini (2017). The review addresses mode of action, similarity of esters and all available test data at the point of publications from reactivity and genotoxicity data to full mutagenicity studies.

Therefore, it is concluded that all constituents of NUMA which all are methacrylic esters, are non-mutagenic in bacterial gene mutation assays.

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

Additional information

Justification for classification or non-classification