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Genetic toxicity in vitro

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Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
Currently, do data on toxicokinetics/metabolism is available for this category. Based on structural features (e.g. sterical hindrance) it is however assumed, that ester cleavage would not be fast and complete, especially since the substances contain up to 6 ester functions, which are in addition sterically shielded. Therefore, it seems more reasonable to base the category hypothesis on structural similarity.
In addition, it is not clear yet, whether the strength of the effects vary in a predictable manner, or if no relevant variations occur. However, there are variations in structure (number of ester bonds and consequently number of free -SH groups) and physicochemical properties (especially water solubility and log Kow). It is assumed that these variations will also be reflected by variations in effect levels. Therefore, scenario 4 is the working hypothesis for the time being.
More data points within the category are needed to further strengthen the category hypothesis. The scenario selection will be re-evaluated after the studies are finished.
This currently selected scenario covers the category approach for which the read-across hypothesis is based on structural similarity. For the REACH information requirement under consideration, the property investigated in studies conducted with different source substances is used to predict the property that would be observed in a study with the target substance if it were to be conducted. Similar properties are observed for the different source substances; this may include absence of effects for every member of the category.
There are expected to be differences in strength of the effects forming a regular pattern. The prediction will be based on a worst-case approach. The read-across is a category approach based on the hypothesis that the substances in this category share structural similarities with common functional groups. This approach serves to use existing data on acute toxicity, repeated-dose toxicity, and reproductive toxicity endpoints for substances in this category.
The hypothesis corresponds to Scenario 4 of the RAAF. The substances GDMP, TMPMP, PETMP, and Di-PETMP are esters of a common acid, 3-mercaptopropionic acid (3-MPA). The key functionality of the substances within this category is the presence of free SH-groups. It is hypothesised that the strength of effects correlates with the number of SH-groups. In addition, differences in bioavailability are expected to influence the strength of effects.
For details, please refer to the category document attached to Iuclid section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
E. coli WP2
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
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:
valid
Positive controls validity:
valid
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:
valid
Positive controls validity:
valid
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:
valid
Positive controls validity:
valid
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:
valid
Positive controls validity:
valid
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:
valid
Positive controls validity:
valid
Conclusions:
GDMP and PETMP showed a no signs of genotoxicity in an bacterial reverse mutation assay. Based on the category approach Di-PETMP is considered to be non genotoxic.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-04-23 to 2012-05-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
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:
Liver post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254. S9 was collected from 20 - 30 rats.
Test concentrations with justification for top dose:
5000 µg GDMP/plate based on cytotoxicity (scarce background lawn and reduction of the number of revertants)
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-amino-anthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48-72 h

SELECTION AGENT (mutation assays): his-free medium
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Table 1: Plate incorporation test -without metabolic activation

S9 Mix

Conc.

(µg/ plate)

Number of revertants (mean number of colonies per plate, n=3)

TA98

TA100

TA1535

TA1537

TA1537

WP2 uvr A

0

40.7

157.3

22.0

5.0

5.7

29.3

31.6

32.0

179.3

19.0

4.3

4.7

54.3

100

37.7

140.0

19.3

2.3

3.3

53.0

316

37.3

168.0

14.0

5.0

4.3

51.7

1000

29.3

171.3

20.7

2.3

3.3

49.3

3160

27.3

140.7

18.0

5.3

5.3

43.7

5000

10.3#

87.0#

9.3#

1.0#

2.0#

12.7#

Pos controls
‑S9

Name

2-NF

NaN3

NaN3

9-AA

2-NF

NQO

Conc. (µg/plate)

10

10

10

100

10

5

No. of revertants per plate

134.7

865.7

118.0

140.7

174.3

249.3

2-NF   2-Nitrofluorene

NQO   4-Nitroquinoline-1-oxide

9-AA   9-Aminoacridine

NaN3   Sodium azide

#        scarce background lawn

 

Table 2: Plate incorporation test -with metabolic activation

S9 Mix

Conc.

(µg/ plate)

Number of revertants (mean number of colonies per plate, n=3)

TA98

TA100

TA1535

TA1537

TA1537

WP2 uvr A

+

0

45.0

177.0

15.7

6.7

5.3

54.3

+

31.6

37.7

163.3

16.0

4.7

6.7

42.3

+

100

39.7

153.7

14.0

2.0

6.3

52.7

+

316

35.3

161.0

19.7

5.0

5.3

50.7

+

1000

26.7

166.3

22.3

1.7

4.3

53.0

+

3160

35.3

161.3

18.0

3.7

4.3

52.0

+

5000

15.0#

91.3#

7.7#

1.0#

1.7#

16.3#

Pos controls
+S9

Name

BaP

NaN3

NaN3

9-AA

2-NF

NQO

Conc. (µg/plate)

5

2

2

5

5

2

No. of revertants per plate

120.7

869.0

125.7

142.7

170.3

211.0

BaP    Benzo[a]pyrene

2-AA   2-Aminoanthracene

#        scarce background lawn

 

 

 

Table 3: Pre-incubation test -without metabolic activation

S9 Mix

Conc.

(µg/ plate)

Number of revertants (mean number of colonies per plate, n=3)

TA98

TA100

TA1535

TA1537

TA1537

WP2 uvr A

0

35.3

153.7

17.7

4.7

5.7

47.0

31.6

33.3

153.7

16.0

6.3

5.3

47.7

100

23.7

155.0

15.7

5.0

5.3

51.3

316

35.7

1296.7

17.7

5.3

5.0

51.7

1000

24.7

137.0

26.7

5.7

6.3

48.0

3160

4.07

14137

22.7

5.0

5.7

43.7

5000

9.3#

59.0#

4.3#

1.0#

1.7#

9.3#

Pos controls
‑S9

Name

2-NF

NaN3

NaN3

9-AA

2-NF

NQO

Conc. (µg/plate)

10

10

10

100

10

5

No. of revertants per plate

175.7

945.3

127.7

127.0

230.1

224.7

2-NF   2-Nitrofluorene

NQO   4-Nitroquinoline-1-oxide

9-AA   9-Aminoacridine

NaN3   Sodium azide

#        scarce background lawn

 

Table 4: Pre-incubation test -with metabolic activation

S9 Mix

Conc.

(µg/ plate)

Number of revertants (mean number of colonies per plate, n=3)

TA98

TA100

TA1535

TA1537

TA1537

WP2 uvr A

+

0

36.7

161.7

16.7

2.7

4.7

56.0

+

31.6

27.7

148.3

19.7

6.0

7.0

39.0

+

100

28.0

141.0

18.7

4.7

5.0

45.7

+

316

35.3

163.7

11.7

5.7

4.7

30.7

+

1000

23.7

152.0

19.7

4.7

4.7

50.3

+

3160

22.7

168.7

15.0

3.0

5.3

46.3

+

5000

10.7#

43.7#

6.7#

1.0#

1.7#

8.3#

Pos controls
+S9

Name

BaP

NaN3

NaN3

9-AA

2-NF

NQO

Conc. (µg/plate)

5

2

2

5

5

2

No. of revertants per plate

178.0

980.7

173.3

113.0

227.7

255.3

BaP    Benzo[a]pyrene

2-AA   2-Aminoanthracene

#        scarce background lawn

 

Conclusions:
GDMP is negative in the Ames test, with and without metabolic activation.
Executive summary:

GDMP was examined in the 5 Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 and in the Escherichia coli strain WP2 uvr A in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation 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.
GDMP was completely dissolved in dimethylsulfoxide (DMSO). The vehicle DMSO served as the negative control.



Preliminary test
GDMP was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA 100. Ten concentrations ranging from 0.316 to 5000 μg/plate were tested. Cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at the top concentration of 5000 μg GDMP/plate.
Hence, 5000 μg GDMP/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test, respectively.



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



In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at the top concentration of 5000 μg GDMP/plate, in all Salmonella typhimurium strains and in the Escherichia coli strain WP2 uvr A.



No mutagenic effect (no increase in revertant colony numbers as compared with control counts) was observed for GDMP, tested up to a cytotoxic concentration of 5000 μg/plate, in the Salmonella typhimurium and in the Escherichia coli test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 - 26 July 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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)
Version / remarks:
adopted 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9, induced with phenobarbitone/ß-naphthoflavone (80/100 mg per kg per day)
Test concentrations with justification for top dose:
50 to 5000 µg/plate

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: established vehicle for substances with poor water solubility
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
mitomycin C
other: (without metabolic activation)
Positive control substance:
benzo(a)pyrene
other: 1,8-Dihydroxyanthraquinone, 2-aminoanthracene (with metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Preincubation period: n.a.
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 per concentration per experiment
Evaluation criteria:
The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunnett's method of linear regression
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:
valid
Positive controls validity:
valid
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:
valid
Positive controls validity:
valid
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:
valid
Positive controls validity:
valid
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:
valid
Positive controls validity:
valid
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at 5000 µg/plate


RANGE-FINDING/SCREENING STUDIES:
The test material was non-toxic to the strain of Salmonella used (TA100).

COMPARISON WITH HISTORICAL CONTROL DATA:
pos. and neg. controls within historical range

Table 1: Experiment 1 - Without Metabolic Activation

S9 Mix

Test substance concentration (µg/ plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

0

82

25

383

18

12

50

77

22

352

15

10

150

75

22

359

17

7

500

80

21

365

16

9

1500

71

21

346

18

9

5000

74 P

33 P

325 P

18 P

7 P

Pos controls
‑S9

Name

ENNG

ENNG

MMC

4NQO

9AA

Conc. (µg/plate)

3

5

0.5

0.2

80

No. of revertants per plate

270

444

1186

171

1287

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

MMC  Mitomycin C

P        Precipitate

 

 

Table 2: Experiment 1 - With Metabolic Activation

S9 Mix

Test substance concentration (µg/ plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

+

0

87

13

375

46

19

+

50

84

9

365

37

13

+

150

80

10

356

35

18

+

500

77

13

345

30

14

+

1500

62

14

319

24

13

+

5000

51 P

16 P

233 P

32 P

8 P

Pos controls +S9

Name

2AA

2AA

DAN

BP

2AA

Conc. (µg/plate)

1

2

10

5

2

No. of revertants per plate

1853

351

784

225

329

2AA    2-Aminoanthracene

BP     Benzo(a)pyrene

DAN   1,8-Dihydroxyanthraquinone

P        Precipitate

 

 

Table 3: Experiment 2 - Without Metabolic Activation

S9 Mix

Test substance concentration (µg/ plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

0

76

22

353

15

10

50

81

23

368

14

7

150

69

29

373

14

8

500

78

30

351

18

9

1500

90

30

343

23

8

5000

94 P

36 P

280 P

24 P

4 P

Pos controls
‑S9

Name

ENNG

ENNG

MMC

4NQO

9AA

Conc. (µg/plate)

3

5

0.5

0.2

80

Avg. no. of revertants per plate

473

449

998

110

847

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

MMC  Mitomycin C

P        Precipitate

 

 

Table 4: Experiment 2 - With Metabolic Activation

S9 Mix

Test substance concentration (µg/ plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

+

0

87

15

376

30

12

+

50

75

15

395

23

15

+

150

76

17

384

22

11

+

500

69

17

382

22

9

+

1500

60

16

338

19

6

+

5000

54 P

12 P

157 P

21 P

9 P

Pos controls +S9

Name

2AA

2AA

DAN

BP

2AA

Conc. (µg/plate)

1

2

10

5

2

No. of revertants per plate

2950

168

748

152

264

2AA    2-Aminoanthracene

BP     Benzo(a)pyrene

DAN   1,8-Dihydroxyanthraquinone

P        Precipitate

 

 

 

Conclusions:
Interpretation of results: negative

PETMP is negative in the Ames test, with and without metabolic activation.
Executive summary:

The genotoxic potential of the test item PETMP was assessed in a Bacterial reverse mutation assay according to OECD Guideline 471 and EU method B13/14.Salmonella typhimuriumstrains TA1535, TA1537, TA102, TA98 and TA100 were treated with solutions of the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (DMSO) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A white, cloudy precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the

bacterial strains, with any dose of the test material, either with or without metabolic activation.

The test material was considered to be non-mutagenic under the conditions of this test.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Negative Ames tests are available for GDMP and PETMP.
A negative chromosomal aberration assay is available for PETMP; an additional chromosomal aberration assay is planned for GDMP.
A negative mouse lymphoma assay is available for PETMP; no additional tests are planned for this endpoint.
The entirety of eventually available genotoxicity data in combination with the absence of structural alerts for genotoxicity in this category will be used to strengthen the weight of the evidence for non-genotoxicity.

Justification for classification or non-classification

Based on the category approach, no positive result in an in vitro study was observed. Therefore no in vivo study on the substance was triggered and the GHS classification criteria are not fulfilled.