Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- genetic toxicity in vitro: non-mutagenic, study conducted according to OECD TG 471, GLP

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-04-21 to 2017-06-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21st July, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 30, 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August, 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
his
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 liver mix
Test concentrations with justification for top dose:
0.0316, 0.100, 0.316, 1.00, 3.16, 10.0, 31.6 and 100 µg/plate based on the results of a preliminary test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The vehicle was diluted prior to the experiment and the solvent was compatible with the survival of the bacteria and the S9 activity.
Untreated negative controls:
yes
Remarks:
A.dest.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (without metabolic activation), 2-aminoanthracene (with metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation method


DURATION
- Preincubation period: 60 min at 37°C
- Exposure duration: 48 h at 37°C


NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: other: Cytotoxicity can be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ± 0.5 in relation to the solvent control.

Rationale for test conditions:
The test was performed as recommended by the OECD guideline 471, adopted in 1997.
Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.
Key result
Species / strain:
other: TA 98, 100, 102, 1535, 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
detected in TA 98, 102, 1535 at 31.6 µg and higher (w/o met. ac.) and at 100 µg and higher (w. met.act.)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was found in all tester strains at concentrations of 31.6 µg/plate and higher (with and without metabolic activation) in experiment 1.

RANGE-FINDING/SCREENING STUDIES: Preliminary study performed with 3.16, 10.0, 31.6, 100,316, 1000, 2500 and 5000 µg/plate test substance


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Historical Laboratory Control Data of the Positive Control (in 2014 - 2016) without S9 ( - S9)
TA 98 TA 100 TA 1535 TA 1537 TA 102
Substance
Canc./plate 4-NOPD 10µg NaN3 10 µg NaN3 10 µg 4-NOPD 40µg MMS 1 µL
Mean 430.7 612.1 792.0 94.5 1729.2
SD 155.5 220.0 299.5 22.7 518.8
Min 141 132 38 35 272
Max 1830 1423 1854 273 3321
RSD [%] 36.1 35.9 37.8 24.0 30.0
n 971 1188 931 929 682

Historical Laboratory Control Data of the Positive Control (in 2014 - 2016) with S9 ( - S9)
TA 98 TA 100 TA 1535 TA 1537 TA 102
Substance
Canc./plate 2-AA 2.5 µg 2-AA 2.5 µg 2-AA 2.5 µg 2-AA 2.5 µg 2-AA 10 µg
Mean 1880.5 1727.7 133.9 234.1 801.2
SD 708.5 522.0 134.9 101.4 223.7
Min 70. 169 22 26 137
Max 3606 3132 1954 682 3588
RSD [%] 37.7 30.2 100.8 43.3 27.9
n 966 1184 927 925 678



- Negative (solvent/vehicle) historical control data:
Historical Laboratory Control Data of the Negative Control (in 2014 - 2016) without S9 ( - S9)
TA 98 TA 100 TA 1535 TA 1537 TA 102
Mean 24.2 90.7 13.8 8.2 270.4
SD 6.7 15.6 6.7 2.9 55.0
Min 11 49 4 3 141
Max 58 155 41 35 472
RSD [%] 27.7 17.2 48.6 35.3 20.3
n 972 1191 929 931 682

Historical Laboratory Control Data of the Negative Control (in 2014 - 2016) with S9 ( - S9)
TA 98 TA 100 TA 1535 TA 1537 TA 102
Mean 29.0 96.4 10.5 8.3 339.7
SD 6.8 14.1 4.5 3.1 71.3
Min 15 62 3 3 157
Max 59 160 38 36 586
RSD [%] 23.4 14.6 42.7 37.4 21.0
n 967 1189 925 926 676

S9: metabolic activation
Conc.: concentration
Mean: mean of revertants/plate
Min.: minimum of revertants/plate
Max.: maximum of revertants/plate
SD: Standard Deviation
RSD: Relative Standard Deviation
n: Number of control values



Experiment I:

Toxic effects were observed for TA 98 at 3.16 µg/plate and higher (without metabolic activation) and at 100 µg/plate (with metabolic activation),

in TA 100 at 10 µg/plate and higher (without metabolic activation) and at 100 µg/plate (with metabolic activation),

in TA 1535 at 3.16 µg/plate and higher (without metabolic activation) and at 31.6 µg/plate and higher (with metabolic activation),

in TA 1537 at 1.00 µg/plate and higher (without metabolic activation) and at 31.6 µg/plate and higher (with metabolic activation),

in TA 102 at 31.6 µg/plate and higher (with and without metabolic activation).

Reduction of the number of revertants to ≤ 0.5 was found in TA 1535 at 3.16 µg/plate (with metabolic activation) and in TA 1537 at 0.0316 µg/plate (without metabolic activation), these reductions were considered not biologically relevant due to lack of a dose-response relationship.

Experiment II:

Toxic effects were observed for TA98, TA 1535, TA 102 at 31.6 µg/plate and higher (without metabolic activation) and at 100 µg/plate (with metabolic activation),

in TA 100 at 10.0 µg/plate and higher (without metabolic activation) and at 31.6 µg/plate and higher (with metabolic activation),

in TA 1537 at 31.6 µg/plate and higher (with and without metabolic activation).

Reduction of the number of revertants to ≤0.5 occurred in TA 1537 at a concentration of 31.6 µg/plate (without metabolic activation) and was regarded as biologically not relevant due to lack of a dose-response relationship.

Table 1:Number of revertants per plate (mean of 3 plates) Experiment 1

 

[TA98]

[TA100]

[TA1535]

[TA1537]

[TA102]

Conc.
[unit]

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

0

 27

 37

 no

 101

 90

 no

 20

 14

 no

13

10

 no

205

343

 no

DMSO

23

26

no

69

86

no

22

17

no

9

6

no

184

244

no

0.0316

 27

 30

 no

 58

 71

 no

 16

 11

 no

3

11

 no

195

275

 no

0.100

 21

 22

 no

 58

 83

 no

 24

 14

 no

7

15

 no

195

245

 no

0.316

 23

 23

 no

 74

82

 no

 17

 15

 no

7

13

 no

153

174

 no

1.00

 22

 21

 no

 57

 88

 no

 13

 10

 no

3

14

 no

148

199

 no

3.16

 18

 24

 yes

 60

88

 no

 17

 9

 yes

2

8

 yes

110

166

 no

10.0

20

33

yes

7

81

yes

8

10

yes

3

9

yes

113

192

no

31.6

8

24

yes

0

 61

yes

6

12

yes

0

8

yes

65

54

yes

100

0

17

yes

0

41

yes

0

0

yes

0

0

yes

0

0

yes

Positive control

 479

 732

 

 1106

 416

 

 754

 110

 

133

118

 

1619

790

 

Table 4: Number of revertants per plate (mean of 3 plates) Experiment 2

 

[TA98]

[TA100]

[TA1535]

[TA1537]

[TA102]

Conc.
[unit]

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

0

 22

27

 no

 92

90

 no

 19

 27

 no

8

12

 no

203

289

 no

DMSO

26

25

 

93

78

 

20

17

 

14

12

 

156

240

 

0.0316

 17

21

 no

 82

 75

 no

 23

 17

 no

9

19

 no

293

240

 no

0.100

 25

 17

 no

 96

 74

 no

 17

 21

 no

12

12

 no

268

285

 no

0.316

 34

 17

 no

 87

 78

 no

 19

 14

 no

12

9

 no

287

327

 no

1.00

 21

 22

 no

 91

 70

 no

 15

 30

 no

11

9

 no

267

327

 no

3.16

 20

 20

 no

 85

 78

 no

 17

 18

 no

7

9

 no

231

231

 no

10.0

15

22

no

31

87

yes

11

16

no

9

7

no

261

224

no

31.6

12

21

no

23

70

yes

11

20

yes

4

8

yes

150

245

yes

100

12

12

yes

0

0

yes

2

5

yes

1

2

yes

101

122

yes

Positive control

 415

 1602

 

 816

 1917

 

 982

 235

 

125

110

 

2528

909

 

Conclusions:
In the present study conducted according to OECD guideline 471 the S. typhimurium tester strains 98, 100, 102, 1535 and 1537 were incubated with 0.0316, 0.100, 0.316, 1.00, 3.16, 10.0, 31.6 and 100 µg/plate m-Xylylenebismaleimide with and without metabolic activation for 48h. Cytotoxicity was detected in all tester strains at different concentrations, a reduction of the number of revertants was detected in TA 1535 and TA 1537 but due to lack of a dose-response relationship this effect was regarded biologically not relevant. No increases in the number of revertants was detected, thus, the test item is considered non-mutagenic under the present test conditions.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997), Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 were exposed to m-Xylylenebismaleimide in DMSO in concentrations of 0 (control),0.0316, 0.100, 0.316, 1.00, 3.16, 10.0, 31.6 and 100 µg/platein all strains in the absence and presence of mammalian metabolic activation (rat liver S9 mix) for 48 h. The assay was performed using the plate incorporation and the preincubation method.

The test substance was tested up to cytotoxic concentrations. Cytotoxic effects were noted in strains TA98, TA 1535 and TA1537 starting at 3.16 µg/plate without metabolic activation, and at 100 and 31.6 µg/plate in with metabolic activation in experiment 1, respectively. In experiment 2 cytotoxic effects were noted in strains TA 102, TA 1535 and TA1537 at 31.6 µg/plate, in TA98 at 100 µg/plate and in TA 100 at 10 µg/plate without metabolic activation. In strains TA100 and TA1537 cytoxicity occured at 31.6 µg/plate and in TA98 and TA1537 at 100 µg/plate with metabolic acitvation.Precipitation was observed at 31.6 and 100 µg/plate only in experiment 1.The positive controls induced the appropriate responses in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.

There was no evidence of an increase in the number of revertant colonies that exceeded twice background in any of the five tester strains (TA98, TA 100, TA102, TA1535 or TA1537) examined at dose levels up to 100 µg/plate in the absence and presence of a metabolic activation source (S9) . Therefore, test substance was considered to be non-genotoxic (non-mutagenic) in Salmonella tester strains TA98, TA100, TA102, TA1535 and TA 1537 under the conditions employed (plate incorporation assay and preincubation method).

There was no evidence of induced mutant colonies over background.

Under the conditions of the study, the test substance was negative for mutagenic potential.

Justification for classification or non-classification

Based on available, reliable and relevant data m-Xylylenebismaleimide does not need to be classified as mutagenic according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemcials (GHS).