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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
end on 22-SEP-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Uronium hydrogen sulphate
EC Number:
244-343-6
EC Name:
Uronium hydrogen sulphate
Cas Number:
21351-39-3
IUPAC Name:
amino(hydroxy)methaniminium hydrogen sulfate
Details on test material:
- Name of test material (as cited in study report): Aduct Urea-Sulfuric
- Physical state: not indicated
- Stability under test conditions: no data
- Storage condition of test material: At room temperature

Method

Species / strain
Species / strain / cell type:
other: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100, and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
- pre-experiment (experiment I): 3 – 5000 μg/plate
- experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties.
Controls
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9-mix: sodium azide (TA1535, TA100: 10μg/pl); 4-nitro-o-phenylene-diamine (TA1537, TA98: 10-50µg/pl); methyl methane sulfonate (TA102: 3μL/pl) - With S9-mix: 2-aminoanthracene (all strains: 2.5-10µg/pl)
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation test (experiment I) and pre-incubation test (experiment II)

DURATION
- Preincubation period: 60 minutes at 37°C
- Exposure duration: at least 48 hours at 37 °C in the dark

NUMBER OF REPLICATES: for each strain and dose level, including the controls three plates were used.

DETERMINATION OF CYTOTOXICITY
- Method: background growth
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results
Species / strain:
other: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100, and TA 102
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:
valid
Positive controls validity:
valid
Additional information on results:
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Aduct Urea-Sulfuric at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct in-crease in induced revertant colonies.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH, effects of osmolality, evaporation from medium, water solubility: no data.
- Precipitation: no precipitation of the test item occurred up to the highest investigated dose.

RANGE-FINDING/SCREENING STUDIES: reported as experiment I

COMPARISON WITH HISTORICAL CONTROL DATA:
In experiment II, the data in the untreated control with metabolic activation of strain TA 102 were slightly above our historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.
No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: result of pre-experiment (experiment I)

Metabolic activation

Test group

Dose level (per plate)

Revertant colony counts (Mean ± SD)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Without Activation

water

 

14 ± 5

7 ± 1

32 ± 3

145 ± 9

437 ± 36

untreated

 

17 ± 7

8 ± 2

35 ± 7

137 ± 3

420 ± 13

Aduct Urea-Sulfuric

3 µg

15 ± 2

7 ± 1

30 ± 7

143 ± 6

416 ± 6

10 µg

14 ± 4

7 ± 2

34 ± 3

136 ± 13

424 ± 14

33 µg

12 ± 4

9 ± 3

32 ± 10

125 ± 6

425 ± 30

100 µg

10 ± 2

6 ± 1

31 ± 5

135 ± 20

445 ± 6

333 µg

13 ± 3

6 ± 1

29 ± 6

131 ± 19

396 ± 38

1000 µg

15 ± 2

6 ± 1

29 ± 9

125 ± 7

410 ± 37

2500 µg

13 ± 0

9 ± 1

29 ± 3

134 ± 6 

407 ± 15

5000 µg

14 ± 3

9 ± 1

32 ± 6

135 ± 13

415 ± 18

NaN3

10 µg

1606 ± 83

 

 

1790 ± 162

 

4-NOPD

10 µg

 

 

298 ± 8

 

 

4-NOPD

50 µg

 

65 ± 8

 

 

 

MMS

3 µL

 

 

 

 

2268 ± 290

With Activation

water

 

20 ± 1

16 ± 5

42 ± 12

166 ± 10

561 ± 38

untreated

 

21 ± 5

12 ± 4

31 ± 3

161 ± 13

539 ± 61

Aduct Urea-Sulfuric

3 µg

21 ± 5

13 ± 1

46 ± 5

152 ± 3

574 ± 26

10 µg

21 ± 3

13 ± 3

46 ± 10

147 ± 14

571 ± 14

33 µg

17 ± 2

14 ± 4

41 ± 6

158 ± 22

569 ± 96

100 µg

23 ± 5

18 ± 2

36 ± 12

151 ± 7

624 ± 27

333 µg

25 ± 2

15 ± 5

43 ± 5

141 ± 13

615 ± 34

1000 µg

21 ± 6

13 ± 3

30 ± 4

150 ± 8

594 ± 25

2500 µg

20 ± 1

18 ± 3

42 ± 1

155 ± 17

627 ± 8

5000 µg

24 ± 2

16 ± 2

38 ± 3

155 ± 20

625 ± 11

2-AA

2.5 µg

473 ± 43

511 ± 45

2491 ± 156

4053 ± 143

 

2-AA

10 µg

 

 

 

 

2304 ± 92

Table 2: result of experiment II

Metabolic activation

Test group

Dose level (per plate)

Revertant colony counts (Mean ± SD)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Without Activation

water

 

17 ± 4

16 ± 3

37 ± 13

168 ± 24

418 ± 37

untreated

 

17 ± 4

15 ± 8

31 ± 5

157 ± 28

436 ± 24

Aduct Urea-Sulfuric

33 µg

17 ± 5

24 ± 8

34 ± 3

148 ± 11

449 ± 29

100 µg

16 ± 1

17 ± 4

34 ± 8

160 ± 23

450 ± 24

333 µg

17 ± 4

16 ± 3

41 ± 3

161 ± 9

460 ± 10

1000 µg

19 ± 4

20 ± 6

35 ± 9

145 ± 11

418 ± 56

2500 µg

13 ± 4

13 ± 4

34 ± 3

143 ± 18

444 ± 4

5000 µg

17 ± 8

19 ± 2

31 ± 3

145 ± 5

442 ± 39

NaN3

10 µg

1780 ± 54

 

 

1244 ± 225

 

4-NOPD

10 µg

 

 

369 ± 14

 

 

4-NOPD

50 µg

 

78 ± 6

 

 

 

MMS

3 µL

 

 

 

 

2472 ± 404

With Activation

water

 

24 ± 6

26 ± 3

37 ± 9

195 ± 8

585 ± 77

untreated

 

34 ± 12

26 ± 6

44 ± 8

185 ± 4

659 ± 21

Aduct Urea-Sulfuric

33 µg

26 ± 7

27 ± 3

36 ± 2

211 ± 17

686 ± 25

100 µg

22 ± 2

28 ± 5

33 ± 6

193 ± 14

634 ± 15

333 µg

25 ± 1

22 ± 7

35 ± 11

200 ± 8

686 ± 3

1000 µg

19 ± 5

30 ± 1

45 ± 6

183 ± 8

644 ± 12

2500 µg

28 ± 5

22 ± 5

42 ± 8

190 ± 5

593 ± 36

5000 µg

25 ± 10

24 ± 9

39 ± 4

171 ± 16

554 ± 20

2-AA

2.5 µg

400 ± 63

357 ± 13

1950 ± 96

2862 ± 128

 

2-AA

10 µg

 

 

 

 

2361 ± 277

Applicant's summary and conclusion

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

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Executive summary:

In a reverse gene mutation assay in bacteria (OECD 471, GLP), strains TA 1535, TA 1537, TA 98, TA 100, and TA 102 of S. typhimurium were exposed to Aduct Urea-Sulfuric (Uronium hydrogen sulphate) at concentrations of 33; 100; 333; 1000; 2500; and 5000 μg/plate (experiment II) in the presence and absence of mammalian metabolic activation (pre-incubation). 

 

Aduct Urea-Sulfuric was tested up to limit concentration (5000 µg/plate) and no precipitation was observed.

The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Aduct Urea-Sulfuric at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

The positive controls induced the appropriate responses in the corresponding strains.

 

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.