N,N'-methylenebis[methacrylamide]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-03-14 to 2017-04-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-methylenebis[methacrylamide]

EC Number:

219-102-3

EC Name:

N,N'-methylenebis[methacrylamide]

Cas Number:

2359-15-1

Molecular formula:

C9H14N2O2

IUPAC Name:

N,N'-methylenebis(2-methylacrylamide)

Test material form:

solid: particulate/powder

Details on test material:

- Designation: N,N’-Methylene-bis-methacrylamide
- Chemical name: 2-Propenamide, N,N’-methylene-bis (2-methyl)
- Characteristics: white powder
- Storage conditions: at room temperature in the dark

Method

Target gene:

histidine operon

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: uvrB-, rfa-, pKM 101, resistance to Ampicillin

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: uvrB-, rfa-, pKM 101, resistance to Ampicillin

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: wild-type, rfa-, pKM 101/pAQ1, resistance to Ampicillin

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: uvrB-, rfa-, non-resistance to Ampicillin

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

other: uvrB-, rfa-, non-resistance to Ampicillin

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, Aroclor 1254-induced, from male Sprague-Dawley rat liver (Trinova Biochem GmbH, Gießen, Germany)

Test concentrations with justification for top dose:

In two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100, no signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. Hence, 5000 µg/plate were chosen as top concentration for the main study.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: test substance could be completely dissolved in DMSO.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding: 0.1 mL of approximately 1.00E+08 viable cells in the late exponential or early stationary phase
DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 37°C for 48 h
NUMBER OF REPLICATIONS:
- Plates: 3 per concentration and experiment
- Experiments: 2 independent experiments, each with and without metabolic activation
DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is evidenced by a reduction in the number of spontaneous revertants by at least 50%, a clearing or diminution of the background lawn or by the degree of survival of the treated cultures. Insolubility could have been assessed as precipitation in the final mixture under the actual test conditions and evident to the unaided eye.

Evaluation criteria:

Acceptance Criteria:
The results of the negative and positive control cultures should be within the range of the historical data. The range of spontaneous reversion frequencies per plate is based on Kirkland (1990):
TA98: 20-60
TA100: 100-200
TA102: 240-320
TA1535: 10-35
TA1537: 3-20
Where concurrent negative or positive control data fall outside the range, they may be acceptable and considered for the inclusion into the historical control distribution as long as these data are not extreme outliers. Historical background data of revertant colony numbers of the negative and positive controls without and with metabolic activation for the experiments of the years 2015 to 2017 are given in Table 1 in section "any other details on results incl. tables".

Statistics:

Interpretation of results:
A test item is considered to show a positive response if
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
or
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation coefficient may be applied.
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
Biological relevance of the results should be considered first. A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not described
- Effects of osmolarity: not described
- Evaporation from medium: not described
- Water solubility: test item could be completely dissolved in DMSO
- Precipitation: none reported
RANGE-FINDING/SCREENING STUDIES:
Two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100 were conducted. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate. Hence, 5000 µg/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.
HISTORICAL CONTROL DATA: please see section “any other information on results incl. tables”.

Any other information on results incl. tables

Preliminary test

The test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate (see tables 2 and 3). Hence, 5000 µg/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations ranging from 31.6 to 5000 µg/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

Cytotoxicity 

No signs of cytotoxicity were noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, up to the top concentration of 5000 µg/plate in any test strain.

Mutagenicity

No increase in revertant colony numbers as compared with control counts was observed tested up to a concentration of 5000 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation test and preincubation test). The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system. The results of the negative and positive control cultures are within the range of the historical data (see table 1). The slightly higher value of revertant colony numbers of test strain TA1535 in all experiments (266.0 to 400.0) compared to the historical positive control data (49 to 270) is considered to be within the normal variation, as the mutation frequency is compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control. 

A summary of the results is given below in tables 4 to 7.

HISTORICAL CONTROL DATA

Table 1: history profile of negative and positive control values of the years 2015 to 2017 (n= 80 studies). Data obtained from plate incorporation and preincubation tests

Negative reference item
Strain	TA 98		TA 100		TA 102		TA 1535		TA 1537
S9-mix	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9
Mean	30.3	32.1	145.1	145.2	277.3	279	19.7	19.9	6.7	6.7
SD	5.6	5.9	18.4	19.2	16.5	17.2	4.4	4.6	1.7	1.8
Min	20	20	107	101	245	203	10	10	2	3
Max	49	49	195	198	323	324	36	36	10	10
Positive reference item
Strain	TA 98		TA 100		TA 102		TA 1535		TA 1537
S9-mix	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9
	2-nitro-fluorene	Benzo[a]-pyrene	Sodium azide	2-amino-anthracene	Mitomycin C	Benzo[a]-pyrene	Sodium azide	2-amino-anthracene	9-amino-acridine	Benzo[a]-pyrene
Mean	151.2	150.4	952.9	948.8	1029.7	1024.3	135.6	135.4	76.7	77.6
SD	27.9	28.8	99.7	103.7	102.3	97.1	28.8	28.4	26.5	26.4
Min	91	96	677	703	756	781	51	49	28	31
Max	293	291	1213	1195	1637	1366	266	270	185	184

Table 2: Preliminary cytotoxicity test without metabolic activation. Plate incorporation test with TA 100.

Test item (µg/plate)	Background lawn	Revertants plate 1	Revertants plate 2
0.316	normal	107	133
1	normal	130	130
3.16	normal	152	171
10	normal	171	163
31.6	normal	167	170
100	normal	170	168
316	normal	175	130
1000	normal	162	160
3160	normal	176	188
5000	normal	185	184
Solvent control 100 µL/plate	normal	118	145

 

Table 3: Preliminary cytotoxicity test with metabolic activation. Plate incorporation test with TA 100.

Test item (µg/plate)	Background lawn	Revertants plate 1	Revertants plate 2
0.316	normal	156	157
1	normal	175	176
3.16	normal	152	169
10	normal	174	141
31.6	normal	113	174
100	normal	171	172
316	normal	133	134
1000	normal	165	163
3160	normal	187	188
5000	normal	137	187
Solvent control 100 µL/plate	normal		132

 

Table 4: Plate incorporation test without metabolic activation

Test item (µg/plate)	Number of reverted colonies (mean values, n=3)
	TA98	TA100	TA102	TA1535	TA1537
31.6	26	134	276.3	16.3	6.7
100	29.7	129.7	270.7	15	6.7
316	34.7	147	283	16	5.7
1000	28.7	137	287	15	5.7
3160	28.3	137	269.7	18	8.7
5000	31	152.7	301.3	13	9
Negative reference item 100 µL/plate	28.7	158.3	297.3	22.3	8
Positive reference item	2-nitro-fluorene	Sodium azide	Mitomycin C	Sodium azide	9-amino-acridine
Concentration µg/plate	10	10	10	10	100
	154.3	943.3	962.7	323	107.7

 

Table 5: Plate incorporation test with metabolic activation

Test item (µg/plate)	Number of reverted colonies (mean values, n=3)
	TA98	TA100	TA102	TA1535	TA1537
31.6	26.3	137.7	280.3	22	7.3
100	30.3	134.7	276	19.3	5
316	33.7	145.3	287.7	12.7	5.7
1000	33	137.7	280.3	15.3	5
3160	30.7	145	273	14.3	5.3
5000	32.3	137.7	303.7	18.3	5
Negative reference item 100 µL/plate	29	159	304	15	4.7
Positive reference item	Benzo[a]pyrene	2-amino-anthracene	Benzo[a]pyrene	2-amino-anthracene	Benzo[a]pyrene
Concentration µg/plate	10	2	10	2	10
	159.7	946	926.3	400	105

 

Table 6: Preincubation test without metabolic activation

Test item (µg/plate)	Number of reverted colonies (mean values, n=3)
	TA98	TA100	TA102	TA1535	TA1537
31.6	26	158.3	268	18.3	5.3
100	28	140	271.3	18	7.3
316	37.3	130.3	291.7	15	5
1000	28.3	144.7	243	13.7	6.3
3160	24.7	156	263.3	19	8.3
5000	34	162.3	296.3	15.3	7.7
Negative reference item 100 µL/plate	32.7	165.7	280.7	26.3	7.3
Positive reference item	2-nitro-fluorene	Sodium azide	Mitomycin C	Sodium azide	9-amino-acridine
Concentration µg/plate	10	10	10	10	100
	154.3	989.7	1032.7	302.7	92.3

 

Table 7: Preincubation test with metabolic activation

Test item (µg/plate)	Number of reverted colonies (mean values, n=3)
	TA98	TA100	TA102	TA1535	TA1537
31.6	33.3	124.7	278.3	21.3	6.3
100	31.3	131.3	282.3	20	5
316	33.3	119.7	286	20	7
1000	35	128	294.3	14.3	5.7
3160	30.7	131	282.7	16	7
5000	29.3	139	287	13.7	8.7
Negative reference item 100 µL/plate	27.7	144	262.7	26.7	7.7
Positive reference item	Benzo[a]pyrene	2-amino-anthracene	Benzo[a]pyrene	2-amino-anthracene	Benzo[a]pyrene
Concentration µg/plate	10	2	10	2	10
	151.7	1052.7	1095	266	103.3

 

 

Applicant's summary and conclusion

Conclusions:

Under the present conditions, the test substance tested up to a concentration of 5000 µg/plate caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation. Thus, the test item is considered to be non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

The potential of N,N'-methylenebis[methacrylamide] to induce gene mutations was examined in 5 Salmonella typhimurium strains (TA98, TA100, TA102, TA1535 and TA1537) without and with metabolic activation (S9-fraction derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test. DMSO was employed as the negative control.

In a preliminary test, no signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate. In the main study, six concentrations ranging from 31.6 to 5000 µg/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation. No signs of cytotoxicity were noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, up to the top concentration in any test strain.

No increase in revertant colony numbers as compared with control counts was observed for the test item, tested up to a concentration of 5000 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation test and preincubation test).

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

ln conclusion, under the present test conditions, the test substance tested up to a concentration of 5000 µg/plate caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.