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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Feb - 13 Mar 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Adopted in 1997
Deviations:
yes
Remarks:
2-Aminoanthracene was used as sole indicator of the efficacy of the S9-mix.
GLP compliance:
yes (incl. QA statement)
Remarks:
Slovak National Accreditation Service, Bratislava, Slovak Republic
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium 4,5-dihydroxybenzene-1,3-disulphonate
EC Number:
205-741-5
EC Name:
Disodium 4,5-dihydroxybenzene-1,3-disulphonate
Cas Number:
149-45-1
Molecular formula:
C6H6O8S2.2Na
IUPAC Name:
disodium 4,5-dihydroxybenzene-1,3-disulphonate
Test material form:
solid: particulate/powder

Method

Target gene:
his operon (for S. typhimurium strains)
trp operon (for E. coli strains)
Species / strainopen allclose all
Species / strain / cell type:
other: S. typhimurium TA 97, TA 98, TA 100 and TA 1535
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Range-finding assay: 50, 150, 500, 1500, 2500 and 5000 µg/plate (TA 97, TA 100, and TA 1535 without metabolic activation)
Main (plate incorporation) assay: 50, 150, 500, 1500 and 5000 µg/plate (TA 97, TA 98, TA 100, TA 1535, and WP2uvrA with and without metabolic activation)
Preincubation assay: 15, 50, 150, 500, 1500 and 5000 µg/plate (TA 97, TA 98, TA 100, TA 1535, and WP2uvrA without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: due to good test material solubility in deionized water
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionized water
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-Aminoanthracene
Remarks:
2-AA was used as the sole indicator of the efficacy of the S9-mix. If 2-AA is used, each batch of S9 should also be characterised with a mutagen that requires metabolic activation by microsomal enzymes, e.g., benzo(a)pyrene, dimethylbenzanthracene.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (preincubation); in agar (plate incorporation)
- Cell density at seeding (if applicable): approximately 1 x 10e9 cells per mL

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

NUMBER OF REPLICATIONS: triplicate plates in each of three independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative total growth; the number of revertant colonies per plate were hand counted, not further specified
Evaluation criteria:
The test material is considered positive if the assay is valid and the following conditions are met:
- concentration-related increase over the tested range and reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation
- Mutation factor >2 (mutation factor is determined by [mean revertants (test)/ mean revertants (vehicle)]
A positive result indicates that the test material induces mutations in S. typhimurium or E.coli cells.

The test material for which results do not meet the above criteria is considered non-mutagenic in this test. Negative results indicate that under the test conditions, the test material does not produce mutations in S. typhimurium or E. coli cells.
Statistics:
The mutation frequency at each dose concentration level of the test material was compared to the one observed in negative and positive controls. The statistical analysis was carried out using unpaired T-test.
Mean values and standard deviations were also calculated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 97
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 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:
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
Additional information on results:
HISTORICAL CONTROL DATA (with ranges, means and standard deviation)

- Historical control data: The mean revertants for controls for Salmonella tester strains and E.coli WP2 uvr were within the range of historical control values

Test Period: From 2015 to 2017

Negative Control (Revertants per plate)
Bacterial Strain TA 100 TA 1535 WP2uvrA TA 97 TA 98
Strain S9-mix -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Mean 199 179 21 23 42 47 166 200 25 37
SD 33 30 7 8 13 10 30 32 7 11
Min 131 123 6 11 25 35 114 148 12 19
Max 272 231 39 43 68 70 227 286 43 85


Positive Control (Revertants per plate)
Bacterial Strain TA 100 TA 1535 WP2uvrA TA 97 TA 98
Strain S9-mix -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Mean 718 1056 639 193 480 356 1191 931 243 805
SD 166 454 163 134 274 170 524 355 145 441
Min 440 398 376 34 82 194 400 376 117 256
Max 1148 2888 964 604 912 648 2776 1616 680 1644

Any other information on results incl. tables

Results for AF-378

Range Finding Assay

S9-Mix

Without

Test Item (mg/plate)

Base-pair substitution type

Frameshift type

TA 100

TA 1535

TA 97

VC

230

17

146

50

219

11*

145

150

230

16

164

500

217

22

151

1500

239

16

154

2500

228

22

152

5000

230

20

162

PC (mg/plate)

NaN3(1.5)

NaN3(1.5)

9-AA (75)

776*

573*

833*

VC = Vehicle control; PC = Positive control

NaN3= Sodium azide

9-AA = 9-Aminoacridene

-- = no value

* = different from Vehicle control p<0.05

EXPERIMENT 1 (Standard Plate Test)

S9-Mix

Without

Test Item (mg/plate)

Base-pair substitution type

Frameshift type

WP2uvrA

TA 98

VC

47

20

31

50

41

22

37

150

32*

24

30

500

49

21

37

1500

48

20

36

5000

38*

17*

35

PC (mg/plate)

4-NQO (2.5)

2-NF (3.0)

2-NF (3.0)

405*

126*

179*

S9-Mix

With

Test Item (mg/plate)

Base-pair substitution type

Frameshift type

TA 100

TA 1535

WP2uvrA

TA 98

TA 97

VC

213

14

45

44

26

157

50

215

15

48

43

20*

142

150

209

16

44

45

29

162

500

204

15

35

48

23

172

1500

222

12

31*

42

23

180*

5000

223

13

33

37

21

182*

PC (mg/plate)

2AA (5)

2AA (5)

2AA (5)

2AA (5)

2AA (5)

2AA (5)

1359*

115*

501*

241*

789*

721*

VC = Vehicle control; PC = Positive control

NaN3= Sodium azide

4-NQO = 4-Nitroquinoline-N-oxide

2-NF = 2-Nitrofluorene

9-AA = 9-Aminoacridene

2AA = 2-Aminoanthracene

* = different from Vehicle control p<0.05


EXPERIMENT 2 (Standard Plate Test with Preincubation)

S9-Mix

Without

Test Item (mg/plate)

Base-pair substitution type

Frameshift type

TA 100

TA 1535

WP2uvrA

TA 98

TA 97

VC

221

19

47

19

34

167

15

210

21

43

22

--

160

50

237

19

41

20

35

150*

150

242

21

32*

19

33

132*

500

213

18

49

19

37

147*

1500

213

17

48

23

27*

159

5000

215

17*

38*

18

40

155*

PC (mg/plate)

NaN3(1.5)

NaN3(1.5)

4-NQO (2.5)

2-NF (1.5)

2-NF (3.0)

9-AA (75)

752*

379*

405*

83*

146*

729*

VC = Vehicle control; PC = Positive control

NaN3= Sodium azide

4-NQO = 4-Nitroquinoline-N-oxide

2-NF = 2-Nitrofluorene

9-AA = 9-Aminoacridene

2AA = 2-Aminoanthracene

-- = not tested

* = different from Vehicle control p<0.05

Remarks onstatistical significant results

Salmonella typhimuriumTA100:

Positive control (sodium azide) induced statistically significant increase (p<0.001) of revertant frequency under experimental conditions without activation (MF = 3.37).

Positive control (sodium azide) induced statistically significant increase (p<0.001) of revertant frequency under experimental conditions without activation (MF = 3.41).

 

Salmonella typhimuriumTA1535:

Statistical significance (p<0.05) recognised at test item concentration of 0.05 mg/plate was without biological relevance (mutation factor (MF)=0.65). Positive control (sodium azide) induced statistically significant increase (p<0.001) of revertant frequency under experimental conditions without activation (MF = 33.08). Positive control (sodium azide) induced statistically significant increase (p<0.001) of revertant frequency under experimental conditions without activation (MF = 19.96).

 

Salmonella typhimuriumTA97:

Positive control (9-aminoacridine) induced statistically significant increase (p<0.001) of revertant frequency under experimental conditions without activation (MF = 5.69). In experiments with preincubation the test item was tested in concentration range from 0.015 to 5.0 mg/plate. Statistical significance (p<0.05) recognized at test item concentration of 0.05, 0.15 and 0.5 mg/plate was without biological relevance (MF=0.89, 0.79 and 0.88, respectively). Positive control (9-aminoacridine) induced statistically significant increase (p<0.001) of revertant frequency under experimental conditions without activation (MF = 4.36). In experiment with activation the test item was tested in concentration range from 0.05 to 5.0 mg/plate. Statistical significance (p<0.05) recognized at test item concentration of 1.5 mg/plate and 5 mg/plate was without biological relevance (MF=1.15, and 1.16 respectively).

 

Salmonella typhimuriumTA98:

In the main tests without activation the test item was tested in concentration range from 0.05 to 5.0

mg/plate. The test item did not induce any statistically significant increases of revertant frequency.

Statistical significance (p<0.05) recognized at test item concentration of 5.0 mg/plate was without biological relevance (MF=0.86). Positive control (2NF) induced statistically significant increase (p<0.001) of revertant frequency (MF=6.41and 5.71). In experiments with pre incubation the test item was tested in concentration range from 0.015 to 5.0 mg/plate. Positive control (2-nitrofluorene) induced statistically significant increase (p<0.001) of revertant frequency under experimental conditions without activation (MF = 4.37, and 4.24).

 

E.coliWP 2 uvrA:

In experiments without activation, the test item was tested in concentration range from 0.05 to 5.0

mg/plate. Positive control (4-nitroquinoline 1-oxide) induced statistically significant increase of revertant frequency MF = 8.32). In experiments with preincubation the test item was tested in concentration range from 0.015 to 5.0 mg/plate. Statistical significance (p<0.05) recognized at test item concentration of 0.15 mg/plate and 5.0 mg/plate was without biological relevance (MF=0.67 and 0.81 ,respectively). Positive control induced statistically significant increase (p<0.001) of revertant frequency under experimental conditions without activation (MF = 8.61 Statistical significance (p<0.05)

recognized at test item concentration of 1.5 mg/plate in the first experiment was without biological relevance (MF=0.70).

Applicant's summary and conclusion

Conclusions:
Interpretation of results: the test material AF-378 was negative for mutagenicity (non-mutagenic) with and without metabolic activation.