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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:
experimental study
Adequacy of study:
key study
Study period:
from 30 March 2012 to 04 September 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to EU / OECD guidelines and GLP

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:
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
19/07/2011
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda
EC Number:
939-368-0
Cas Number:
1322-93-6
Molecular formula:
Not applicable (a generic molecular formula can not be provided for this specific UVCB substance)
IUPAC Name:
Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Supragil WP
- Storage condition of test material: In dark at room temperature

Method

Target gene:
Histidine gene
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 from the liver of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
The dose-levels selected for the preliminary tests were 10, 100, 500, 1000, 2500 and 5000 µg/plate (expressed in terms of active ingredient i.e the three forms of sodium isopropylnaphtalenesulphonate: mono, di and tri => equivalent to 13.3, 133, 665, 1330, 3325 and 6650 µg/plate in terms of registered substance (100% UVCB)).
The selected treatment-levels were 312.5, 625, 1250, 2500 and 5000 µg/plate (expressed in terms of active ingredient i.e the three forms of sodium isopropylnaphtalenesulphonate: mono, di and tri => equivalent to 415.6, 831.3, 1662.5, 3325 and 6650 µg/plate in terms of registered substance (100% UVCB)) for the five strains in both mutagenicity experiments, with and without S9 mix.
Vehicle / solvent:
Vehicle(s)/solvent(s) used: water for injection
- Justification for choice of solvent/vehicle: the test item was not soluble in any of the vehicles usually used for in vitro tests.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Anthramine
Remarks:
With S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: All experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method

DURATION
- Preincubation period: 60 minutes, 37°C
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATES: three plates/dose-level

DETERMINATION OF CYTOTOXICITY
- Method: The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

OTHER: SCORING METHOD: automated
Positive controls:
Without S9 mix:
- sodium azide (NAN3) for strains TA 1535 and TA 100 (1 µg/plate),
- 9-Aminoacridine (9AA) for strain TA 1537 (50 µg/plate),
- 2-Nitrofluorene (2NF) for strain TA 98 (0.5 µg/plate),
- Mitomycin C (MMC) for strain TA 102 (0.5 µg/plate).

With S9 mix:
- 2-Anthramine (2AM) for strains TA 1535, TA 1537, TA 98 (2 µg/plate) and TA102 (10 µg/plate),
- Benzo(a)pyrene (BAP) for strain TA 100 (5 µg/plate).
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See tables 7.6.1/1 and 7.6.1/2
Cytotoxicity / choice of top concentrations:
other: In the second experiment with S9 mix (pre-incubation method), a moderate to strong toxicity was noted in the five strains used at the dose-level of 5000 µg/plate (See table 7.6.1/2).
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: In the second experiment with S9 mix (pre-incubation method), a moderate to strong toxicity was noted in the five strains used at the dose-level of 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES:
To assess the toxicity of the test item to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA 98, TA 100 and TA 102 strains, with and without S9 mix. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.
No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels.
No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted towards the three strains used, either with or without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: The control data reported in these report are in the range of the historical control data observed in the laboratory. The study was therefore considered valid.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/1:First experiment (direct plate incorporation) - Mean revertant colony counts

 

TA 1535

TA 1537

TA 98

TA 100

TA 102

Conc.
(µg/plate)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

0*

10

14

No

7

7

No

20

29

No

115

140

No

294

468

No

312.5

14

16

No

6

6

No

26

28

No

130

141

No

280

559

No

625

10

11

No

4

7

No

15

31

No

144

164

No

348

465

No

1250

12

14

No

5

10

No

18

29

No

125

136

No

318

508

No

2500

14

10

No

6

6

No

19

27

No

132

162

No

312

466

No

5000

7

12

No

8

7

No

18

23

No

122

154

No

276

403

No

NAN3

718

-

-

-

-

-

-

-

-

513

-

-

-

-

-

2AM

-

215

-

-

108

-

-

828

-

-

-

-

-

3415

-

9AA

-

-

-

277

-

-

-

-

-

-

-

-

-

-

-

2NF

-

-

-

-

-

-

107

-

-

-

-

-

-

-

-

BAP

-

-

-

-

-

-

-

-

-

-

776

-

-

-

-

MMC

-

-

-

-

-

-

-

-

-

-

-

-

2554

-

-

 

 

*solvent control with water for injection

MA: metabolic activation

NaN3: sodium azide

9AA :9-Aminoacridine

2NF:2-Nitrofluorene

MMC:Mitomycin C

2AM:2-Anthramine

BAP:Benzo(a)pyrene

 

Table 7.6.1/2:Second experiment (direct plate incorporation without S9 mix and preincubation with S9 mix) - Mean revertant colony count

 

 

TA 1535

TA 1537

TA 98

TA 100

TA 102

Conc.
(µg/plate)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

0*

18

15

No

8

14

No

24

29

No

134

142

No

332

585

No

312.5

9

13

No

6

16

No

25

31

No

130

168

No

328

654

No

625

17

9

No

10

18

No

25

35

No

148

145

No

392

609

No

1250

13

13

No

6

8

No

29

30

No

138

141

No

379

465

No

2500

19

10

No

12

6

No

19

36

No

135

124

No

357

348

No

5000

11

19

Yes (Mt)

7

15

Yes (Mt)

19

39

Yes (Mt)

112

101

Yes (Mt)

368

180

Yes (St)

NAN3

610

-

-

-

-

-

-

-

-

485

-

-

-

-

-

2AM

-

83

-

-

186

-

-

859

-

-

-

-

-

3394

-

9AA

-

-

-

353

-

-

-

-

-

-

-

-

-

-

-

2NF

-

-

-

-

-

-

103

-

-

-

-

-

-

-

-

BAP

-

-

-

-

-

-

-

-

-

-

510

-

-

-

-

MMC

-

-

-

-

-

-

-

-

-

-

-

-

2766

-

-

 

 

*solvent control with water for injection

MA: metabolic activation

NaN3: sodium azide

9AA :9-Aminoacridine

2NF:2-Nitrofluorene

MMC:Mitomycin C

2AM:2-Anthramine

BAP:Benzo(a)pyrene

Mt: Moderate toxicity

St: Strong toxicity

Applicant's summary and conclusion

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

Under these experimental conditions, the test item Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
Executive summary:

This study was performed to investigate the potential of the test item, reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda, to induce reverse mutation in Salmonella typhimurium. The study was performed according to OECD guideline no. 471 and EC guideline n° B13/14 and in compliance with the Principles of Good Laboratory Practice. 

 

A preliminary toxicity test was performed to define the dose-levels of reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post‑mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the pre-incubation method (60 minutes,37°C).

Five strains of bacteria Salmonella typhimuriumTA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose‑level). After 48 to 72 hours of incubation at37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The test item was dissolved in water for injections.

 

Since the test item was freely soluble and non-cytotoxic in the preliminary test, the highest dose‑level selected for the main experiments was 5000 µg/plate, according to the criteria specified in the international guidelines.The selected treatment-levels were 312.5, 625, 1250, 2500 and 5000 µg/plate (doses expressed in terms of active ingredient i.e the three forms of sodium isopropylnaphtalenesulphonate: mono, di and tri => equivalent to 415.6, 831.3, 1662.5, 3325 and 6650 µg/plate in terms of registered substance (100% UVCB)) for the five strains in both mutagenicity experiments, with and without S9 mix. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered to be valid. 

No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels. No noteworthy toxicity was noted at any dose-levels in the absence of S9 mix (in either experiments) or in the presence of S9 mix when using the direct plate incorporation method (first experiment). In the second experiment with S9 mix (pre-incubation method), a moderate to strong toxicity was noted in the five strains used at the dose-level of 5000 µg/plate.The test item did not induce any noteworthy increase in the number of revertants, either with or without S9 mix, in any of the five strains.

In conclusion, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, in the absence or in the presence of a rat metabolising system therefore reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda is not classified according to Annex VI of the Directive 67/548/CEE and according to EU Regulation 1272/2008 (CLP).