Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test material was examined for mutagenic activity in a reverse mutation test to Salmonella typhimurium and Escherichia coli according to OECD guideline 471 and in compliance with good laboratory practices (GLP).

In the study SOLVENT N1 did not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism.

Link to relevant study records
Reference
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:
16-MAR-2012 to 05-JUN-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Manufacturer
- Lot/batch No: SETTEMBRE 2011
- Expiration date of the lot/batch: 31 Dec 2016
- Purity test date: 27 March 2012
- Purity: 74.152 %
Target gene:
Histidine and uvrB (for S. typhimurium) or tryptophan and uvrA (for E. coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
one batch of S9 tissue fraction (Trinova Biochem GmbH) from liver of male Sprague-Dawley rat treated with phenobarbital - 5,6-benzoflavone
Test concentrations with justification for top dose:
- Preliminary experiments - plate incorporation: range for dose levels set at half-log intervals in the absence and presence of S9 metabolism (no correction factor applied for the purity of the test material)
- Main experiments - plate incorporation and pre-incubation methods: 156, 313, 625, 1250, 2500, and 5000 µg/plate in the absence and presence of S9 metabolism (no correction factor applied for the purity of the test material)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: the test item was found to be soluble in acetone at 500 mg/mL. This result permitted a maximum concentration of 5000 µg/plate to be used in the toxicity test.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
(see in Table 1 below)
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Remarks:
Sodium azide and methylmethanesulphonate (MMS) were prepared in distilled water, while the other positive control substances were prepared in dimethylsulfoxide (DMSO).
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and pre-incubation
- Type and identity of media:
* Nutrient broth: Oxoid Nutrient Broth No 2 was prepared at a concentration of 2.5% in distilled water and autoclaved prior to use. This was used for the preparation of liquid cultures of the tester strains.
* Nutrient agar: Oxoid Nutrient Broth No 2 (25 g) and Difco Bacto-agar (15 g) was added to 1 L of distilled water and autoclaved. The solution was then poured into 9 cm plastic Petri dishes and allowed to solidify and dry before use. These plates were used for the non-selective growth of the tester strains.
* Minimal Agar: minimal medium agar was prepared as 1.5% Difco Bacto-agar in Vogel-Bonner Medium E, with 2% Glucose, autoclaved and poured into 9 cm plastic Petri dishes.
* Top Agar: "Top Agar" (overlay agar) was prepared as 0.6% Difco Bacto-agar + 0.5% NaCl in distilled water and autoclaved. Prior to use 10 mL of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine (or 0.5 mM tryptophan) was added to 100 mL of the top agar.

DURATION
- Pre-incubation period: 30 min at 37°C
- Exposure duration: 72 hrs at 37°C
- Expression time (cells in growth medium): 72 hrs

SELECTION AGENT: histidine or tryptophan

NUMBER OF REPLICATIONS: in the preliminary experiments, a single plate was used at each test point and positive controls were not included. In the main experiments, three replicate plates were used at each test point, and two independent experiments were performed.

DETERMINATION OF CYTOTOXICITY
- Method: in the preliminary experiments, toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation. In the main experiments, the test item was assayed using the plate incorporation method, at a maximum dose-level of 5000 µg/plate and at 4 lower dose-levels spaced at approximately half-log intervals: 50, 158, 500, and 1580 µg/plate.
Evaluation criteria:
For the test item to be considered mutagenic, 2-fold (or more) increases in mean revertant numbers must be observed at 2 consecutive dose-levels or at the highest practicable dose-level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose-levels.
Statistics:
Regression analysis, correlation coefficient (r) calculation and Student's "t" statistical analysis.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: slight toxicity with TA100 tester strain at the highest dose level
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions.

MAIN STUDY:
In the main assay, using the plate incorporation method, the test item was assayed at the maximum dose level of 5000 µg/plate and at four lower dose levels spaced by two-fold dilutions: 313, 625, 1250, and 2500 µg/plate. An additional dose level of 156 µg/plate was employed with TA100 tester strain in the presence of S9 metabolism. No increases in revertant numbers were observed at any concentration tested.
Thus, a pre-incubation step was included for all treatments of the second experiment of the main assay. The test item was assayed at the dose levels of 156, 313, 625, 1250, 2500, and 5000 µg/plate. No increases in the number of revertant colonies were observed in the pre-incubation assay, at any dose level, with any tester strain, in the absence or presence of S9 metabolism.
Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Plate incorporation and pre-incubation methods: No precipitation of the test item was observed at the end of the incubation period at any concentration, regardless of the experiment. Slight thinning of the background lawn and reduction in revertant numbers was observed with TA100 tester strain at the highest dose level, in the absence and presence of S9 metabolism.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: plate incorporation method

Table 2: Toxicity test with and without metabolic activation

 

Without metabolic activation

With metabolic activation

Dose level

(µg/plate)

TA 1535

Rev/pl.

TA 1537

Rev/pl.

TA 98

Rev/pl.

TA 100

Rev/pl.

Wp2 uvrA

Rev/pl.

TA 1535

Rev/pl.

TA 1537

Rev/pl.

TA 98

Rev/pl.

TA 100

Rev/pl.

Wp2 uvrA

Rev/pl.

Untreated

13

20

32

130

26

22

22

36

112

24

Acetone

100 µL

25

16

34

117

23

17

15

36

114

23

50

16

13

40

123

23

23

16

34

113

28

158

16

12

32

130

22

19

22

41

146

26

500

22

20

34

105

20

23

14

31

121

20

1580

17

13

36

109

19

21

17

35

123

29

5000

24

15

34

110

32

22

25

40

91

28

 

Table 3: Genotoxicity test with and without metabolic activation - plate incorporation method (mean ± standard error)

 

Without metabolic activation

With metabolic activation

Dose level

(µg/plate)

TA 1535

Rev/pl.

TA 1537

Rev/pl.

TA 98

Rev/pl.

TA 100

Rev/pl.

Wp2 uvrA

Rev/pl.

TA 1535

Rev/pl.

TA 1537

Rev/pl.

TA 98

Rev/pl.

TA 100

Rev/pl.

Wp2 uvrA

Rev/pl.

Untreated

18 ± 2.4

20 ± 0.9

33 ± 2.2

143 ± 3.5

29 ± 1.7

18 ± 2.3

19 ± 1.8

38 ± 1.2

133 ± 3.2

29 ± 1.0

Acetone

15 ± 1.5

21 ± 2.3

36 ± 3.5

112 ± 0.6

23 ± 4.6

18 ± 1.8

24 ± 2.6

36 ± 0.7

112 ± 0.6

32 ± 2.8

156

-

-

-

-

-

-

-

-

149 ± 10.4

-

313

19 ± 1.8

15 ± 1.5

26 ± 3.4

121 ± 4.0

32 ± 1.5

18 ± 2.0

15 ± 0.9

40 ± 5.7

128 ± 5.3

32 ± 2.8

625

20 ± 2.7

15 ± 1.3

29 ± 1.7

120 ± 4.8

26 ± 4.6

19 ± 1.5

14 ± 1.5

36 ± 2.0

127 ± 6.7

29 ± 3.9

1250

18 ± 0.9

16 ± 1.2

25 ± 3.4

112 ± 6.0

26 ± 0.9

19 ± 1.2

17 ± 1.2

41 ± 7.2

134 ± 3.7

27 ± 2.5

2500

18 ± 0.9

16 ± 2.8

26 ± 5.4

98 ± 8.3

25 ± 1.2

19 ± 1.7

17 ± 2.0

43 ± 2.7

124 ± 2.0

23 ± 2.3

5000

16 ± 2.2

16 ± 1.2

27 ± 1.0

89 ± 7.4*

25 ± 2.3

18 ± 2.1

13 ± 3.2

27 ± 3.3

93 ± 3.3*

26 ± 3.1

Sodium azide

1 µg/pl.

523 ± 16.0

-

-

600 ± 2.3

-

-

-

-

-

-

DMSO

100 µL/pl.

-

19 ± 1.5

32 ± 1.2

-

-

19 ± 1.5

22 ± 2.5

35 ± 1.5

131 ± 5.2

33 ± 2.6

2-Aminoanthracene

1 or 10 µg/pl.

-

-

-

-

-

98 ± 6.0

98 ± 4.8

279 ± 3.4

934 ± 8.0

221 ± 7.1

9-Aminoacridine

50 µg/pl.

-

169 ± 30.8

-

-

-

-

-

-

-

-

MMS

500 µg/pl.

-

-

-

-

219 ± 6.4

-

-

-

-

-

2-Nitrofluorene

2 µg/pl.

-

-

158 ± 5.2

-

-

-

-

-

-

-

*: thinning of the background lawn

 

Table 4: Genotoxicity test with and without metabolic activation - pre-incubation method (mean ± standard error)

 

Without metabolic activation

With metabolic activation

Dose level

(µg/plate)

TA 1535

Rev/pl.

TA 1537

Rev/pl.

TA 98

Rev/pl.

TA 100

Rev/pl.

Wp2 uvrA

Rev/pl.

TA 1535

Rev/pl.

TA 1537

Rev/pl.

TA 98

Rev/pl.

TA 100

Rev/pl.

Wp2 uvrA

Rev/pl.

Untreated

18 ± 4.0

20 ± 1.5

34 ± 1.2

133 ± 5.6

28 ± 3.2

20 ± 0.3

22 ± 2.7

41 ± 2.6

156 ± 3.5

34 ± 0.9

Acetone

16 ± 1.2

27 ± 2.1

35 ± 2.2

144 ± 7.5

29 ± 1.5

18 ± 1.8

24 ± 3.5

42 ± 1.8

162 ± 6.9

32 ± 2.6

156

21 ± 1.8

26 ± 2.2

34 ± 2.5

144 ± 3.8

29 ± 1.2

20 ± 3.3

24 ± 2.1

38 ± 2.0

152 ± 5.2

30 ± 3.5

313

16 ± 1.7

26 ± 1.5

32 ± 1.9

138 ± 5.8

30 ± 3.0

19 ± 3.2

25 ± 2.7

40 ± 1.0

148 ± 5.5

35 ± 1.2

625

19 ± 1.7

25 ± 1.9

34 ± 2.3

155 ± 3.5

28 ± 2.8

20 ± 2.6

27 ± 0.3

41 ± 1.8

152 ± 6.1

38 ± 2.3

1250

22 ± 0.7

27 ± 2.4

33 ± 2.4

142 ± 6.2

32 ± 1.5

20 ± 0.9

25 ± 2.0

41 ± 3.0

160 ± 5.2

41 ± 0.6

2500

18 ± 2.5

24 ± 1.8

35 ± 1.8

153 ± 4.6

28 ± 1.2

22 ± 2.3

29 ± 0.9

44 ± 2.7

146 ± 5.2

37 ± 1.7

5000

16 ± 1.2

29 ± 0.6

33 ± 2.1

144 ± 6.6*

31 ± 3.5

20 ± 2.6

17 ± 1.0

43 ± 3.5

158 ± 3.0*

38 ± 1.0

Sodium azide

1 µg/pl.

554 ± 11.1

-

-

638 ± 16.2

-

-

-

-

-

-

DMSO

50 µL/pl.

-

22 ± 2.0

-

-

31 ± 2.6

17 ± 1.3

26 ± 1.7

40 ± 3.8

134 ± 4.0

31 ± 2.3

2-Aminoanthracene

1, 2 or 20 µg/pl.

-

-

-

-

-

98 ± 8.7

101 ± 5.5

566 ± 6.0

938 ± 28.8

233 ± 3.9

9-Aminoacridine

50 µg/pl.

-

248 ± 36.6

-

-

-

-

-

-

-

-

MMS

500 µg/pl.

-

-

-

-

217 ± 5.9

-

-

-

-

-

2-Nitrofluorene

2 µg/pl.

-

-

153 ± 6.4

-

-

-

-

-

-

-

*: thinning of the background lawn

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test item did not induce reverse mutation in Salmonella typhimurium under the reported experimental conditions.
Executive summary:

The test material was examined for mutagenic activity by assaying for reverse mutation to Salmonella typhimurium and Escherichia coli according to OECD guideline 471 and in compliance with good laboratory practices (GLP).

 

The five tester strains TA1535, TA1537, TA98, TA100, and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Test item solutions were prepared using acetone.

 

In the toxicity test, the test item was assayed at a maximum dose-level of 5000 µg/plate and 4 lower dose-levels spaced at approximately half-log intervals: 50.0, 158, 500, and 1580 µg/plate.

No precipitation of the test item was observed at the end of the incubation period at any concentration. Slight reduction of revertant colonies was observed with TA100 tester strain at the highest dose level, in the presence of S9 metabolism.

 

Two experiments were performed, one using a plate incorporation method, the other using a pre-incubation method. The test material was assayed at a maximum dose-level of 5000 µg/plate and 4 lower dose-levels, separated by 2-fold dilutions.

Regardless of the incorporation/incubation method, slight toxicity was observed with TA100 tester strain at the highest dose level both in the absence and presence of S9 metabolism. Moreover, no precipitation of the test item was observed at the end of the incubation period at any concentration. Using the plate incorporation method, no increases in revertant numbers were observed at any concentration tested. In the second experiment (i.e., pre-incubation method), the test item did not induce two-fold increases in the number of revertant colonies at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

 

It was concluded that the test item did not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions. Therefore, the test item does not meet the classification criteria of EC Regulation No. 1272/2008 (CLP / EU GHS) and UN GHS for genetic toxicity.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The test material was examined for mutagenic activity by assaying for reverse mutation to Salmonella typhimurium and Escherichia coli according to OECD guideline 471 and in compliance with good laboratory practices (GLP).

 

The five tester strains TA1535, TA1537, TA98, TA100, and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation. Test item solutions were prepared using acetone.

 

Two experiments were performed, one using a plate incorporation method, the other using a pre-incubation method. The test material was assayed at a maximum dose-level of 5000 µg/plate and 4 lower dose-levels, separated by 2-fold dilutions.

Regardless of the incorporation/incubation method, slight toxicity was observed with TA100 tester strain at the highest dose level both in the absence and presence of S9 metabolism. Moreover, no precipitation of the test item was observed at the end of the incubation period at any concentration. Using the plate incorporation method, no increases in revertant numbers were observed at any concentration tested. In the second experiment (i.e., pre-incubation method), the test item did not induce two-fold increases in the number of revertant colonies at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

 

It was concluded that the test item did not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Justification for classification or non-classification

No alerts for the presence of mutagenic properties have been identified for the substance under in vitro gene mutation study in bacteria. According to REACh annex VII requirements, no further mutagenicity studies need be considered for the tonnage band of registration.

No classification for germ cell mutagenicity according to the criteria of Regulation (EC) No. 1272/2008 is required.