Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-09-06 to 2016-11-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-hydroxy-5-sulfobenzoic acid dihydrate
Cas Number:
5965-83-3
Molecular formula:
C7H6O6S*2H20
IUPAC Name:
2-hydroxy-5-sulfobenzoic acid dihydrate
Test material form:
solid

Method

Target gene:
his/trp
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Remarks:
uvrA
Metabolic activation:
with and without
Metabolic activation system:
S9 mix, Aroclor induced in male Wistar rats
Test concentrations with justification for top dose:
1st series 5.0, 15.8, 50.0, 158, 500, 1580, and 5000 µg/plate, 10% S9 mix
2nd series: 5.0, 50.0, 500, 1580, and 5000 µg/plate, 30% S9 mix
Vehicle / solvent:
- Solvent used: water
- Justification for choice of solvent/vehicle: The selection of the solvent for this assay was undertaken based on the available information from the preliminary solubility test. Water showed best performance and was thus used for this experiment at a maximum concentration of 100 µL/plate.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Daunomycin
Remarks:
1.0 µg/plate, TA98, without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
2.0 µg/plate, TA100, TA1535, without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
50 µg/plate, TA1537, without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
2.0 µg/plate, E.coli, without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoantrtracene
Remarks:
2.0, 5.0, and 10.0 µg/plate, TA98, TA100, TA1535, TA1537, E.coli, with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 2 - 3 days

SELECTION AGENT (mutation assays):
TA-strains: Histidine lacking medium
E.coli: Tryptophan lacking medium

NUMBER OF REPLICATIONS: 3 per strain/concentraion, 6 per control


Rationale for test conditions:
The test material concentrations used were selected according to the EEC, OECD and Japanese guidelines for this test system. A maximum concentration of 5000 µg/plale was selected, in order that initial treatments were performed up to the maximum recommended concentration according to current regulatory guidelines.
Evaluation criteria:
The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies. The following criteria. based upon the historical controls of the laboratory and statistical considerations, are established (see "Any other information on materials and methods")

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Summary 1st series

Metabolic activation

Test Material

Concentration [µg/plate]

Revertants per plate (Mean ± SD)

TA98

TA100

TA1535

TA1537

E. coli

Without S9 mix

H2O

 

32± 5

109± 10

27± 6

30± 4

36± 10

Test Substance

5.0

32± 9

120± 10

25± 9

29± 10

43± 7

15.8

22± 1

112± 6

23± 7

31± 4

35± 1

50.0

33± 1

120± 15

30± 8

30± 4

37± 6

158

21± 8

116± 12

26± 5

27± 5

36± 9

500

27± 3

112± 7

23± 1

30± 1

35± 4

1580

26± 4

112± 10

24± 3

30± 7

41± 3

5000

20± 3

105± 25

24± 6

29± 2

36± 5

DAUN

1.0

198± 41

 

 

 

 

NaN3

2.0

 

1450± 52

839± 34

 

 

9-AA

50.0

 

 

 

1654± 130

 

NQO

2.0

 

 

 

 

1582± 122

With S9 mix

H2O

 

43± 10

131± 15

28± 9

34± 5

38± 4

Test Substance

5.0

32± 6

132± 16

27± 3

32± 3

50± 3

15.8

32± 2

136± 3

34± 4

33± 4

41± 4

50.0

37± 5

135± 4

23± 9

28± 7

40± 2

158

29± 3

127± 21

28± 9

34± 5

15± 5

500

29± 8

128± 19

32± 4

29± 3

43± 5

1580

44± 5

144± 10

34± 6

30± 5

42± 1

5000

28± 5

115± 17

22± 9

25± 5

38± 8

2-AA

2.0

643± 53

1587± 108

 

 

 

2-AA

5.0

 

 

280± 13

584± 41

 

2-AA

10.0

 

 

 

 

478± 54

c contaminated

NaN3, Sodium azide

2 -AA, 2-Aminoanthracane

9 -AA, 9-Aminoacridine

DAUN, Daunomycin

NQO, 4-Nitroquinoline-N-oxide

Table 2: Summary 2nd series:

Metabolic activation

Test Material

Concentration [µg/plate]

Revertants per plate (Mean ± SD)

TA98

TA100

TA1535

TA1537

E. coli

Without S9 mix

H2O

 

31±8

120± 18

28± 10

26± 2

38± 5

Test Substance

5.0

29± 14

106± 12

26± 4

31± 9

46± 8

50.0

25± 4

108± 2

27± 3

29± 8

44±1

500

25± 2

92± 9

26± 2

30± 7

33± 4

1580

32± 2

102± 11

24± 7

30± 1

44±5

5000

28± 4

108± 3

24± 4

32± 7

36± 4

DAUN

1.0

148± 1

 

 

 

 

NaN3

2.0

 

1327± 56

848±128

 

 

9-AA

50.0

 

 

 

1420± 147

 

NQO

2.0

 

 

 

 

1829± 90

With S9 mix

H2O

 

43± 4

112± 12c

21± 5

33± 7

42± 10

Test Substance

5.0

40± 5

121± 8

17± 5

32± 6

43± 8

50.0

34± 6

121± 14

24± 3

34± 4

31± 8

500

32± 3

125± 20

20± 4

30± 8

45± 5

1580

29± 7

125± 2

19± 4

30± 6

40± 4

5000

34± 8

113± 18

16± 9

25± 7

37± 7

2-AA

2.0

178± 2

 

 

 

 

2-AA

5.0

 

1337± 50

 

 

 

2-AA

10.0

 

 

124± 4

261± 29

139± 5

c contaminated

NaN3, Sodium azide

2 -AA, 2-Aminoanthracane

9 -AA, 9-Aminoacridine

DAUN, Daunomycin

NQO, 4-Nitroquinoline-N-oxide

Applicant's summary and conclusion

Conclusions:
In an in vitro bacterial reverse mutation assay (Ames test) according to OECD Guideline 471 with and with out metabolic activation (S9 mix), the test substance showed no mutagenic potential.
Executive summary:

The investigations for the mutagenic potential of 5-Sulfosalicylic acid dihydrate were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA according to OECD Guideline 471 (reference 7.6.1 -1). The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. In this study, two experimental series were performed. In the two series with S9 mix, 10% S9 mix were used in the 1st and 30% S9 in the 2nd series, respectively. Vehicle and positive control treatments were included and valid for all strains. The mean numbers of revertant colonies all fell within acceptable ranges for vehicle control treatments, and were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

Following test substance treatments of all the tester strains in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed. Furthermore, it was assumed, that the anhydrate form has the same mutagenic potential and thus the result of the test item is also applicable.