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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethylene bis(3-mercaptopropionate)
EC Number:
245-044-3
EC Name:
Ethylene bis(3-mercaptopropionate)
Cas Number:
22504-50-3
Molecular formula:
C8H14O4S2
IUPAC Name:
ethane-1,2-diyl bis(3-sulfanylpropanoate)
Test material form:
liquid

Method

Target gene:
his operon
Species / strain
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
Controls
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

Results and discussion

Test resultsopen allclose all
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

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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).