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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is considered to be mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the presence of metabolic activation.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 Sep 2016 to 16 Sep 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 Jul 1997
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
No. L142, 30 May 2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Aug 1998
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test substance: Orasol Orange 251
- Test substance No: 16/0111-1
- Source and lot/batch No.of test material: batch 002-141306
- Expiration date of the lot/batch: 15 Jun 2020
- Purity: ca. 98.8%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stability of the test substance under storage conditions is guaranteed until 15 Jun 2020
- Solubility and stability of the test substance in the solvent/vehicle: the test substance is soluble in DMSO used as vehicle. Test substance precipitation was found from about 333 μg/plate onward with and without S9 mix.
- Stability of test substance in the vehicle: not determined analytically, because the test substance was administered immediately after preparation and is usually stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: the test substance was dissolved in dimethyl sulfoxide (DMSO); to achieve a clear solution of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and was shaken thoroughly
Target gene:
S. typhimurium: his
E. coli: trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from phenobarbital i.p. and β-naphthoflavone orally induced rats
Test concentrations with justification for top dose:
First Experiment
0; 33; 100; 333; 1000; 2500 and 5000 µg/plate (with and without S9 mix)

Second experiment
0; 1000; 2000; 2500; 3000; 4000 and 5000 µg/plate (with S9 mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available
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
other: 2-aminoantracene (2-AA); N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 4-nitro-o-phenylenediamine (NOPD)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): Fresh cultures of bacteria were grown up to late exponential or early stationary phase of growth (approximately 10^9 cells per mL)

DURATION
- Exposure duration: 48-72 h

NUMBER OF REPLICATIONS: 3 test plates per dose or per control

STRAINS TESTED
- First experiment: TA 1535, TA 100, TA 1537, TA 98 and WP2 uvrA (with and without S9 mix)
- Second experiment: TA 98 (with S9 mix)

DETERMINATION OF CYTOTOXICITY
Toxicity detected by a:
- decrease in the number of revertants (factor ≤ 0.6)
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)
Evaluation criteria:
ACCEPTANCE CRITERIA
Generally, the experiment was considered valid if the following criteria were met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain
- The sterility controls revealed no indication of bacterial contamination
- The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above
- Fresh bacterial culture containing approximately 10^9 cells per mL were used.

ASSESSMENT CRITERIA
The test substance was considered positive in this assay if the following criteria were met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance was generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 100, TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test depending on the strain and test conditions from about 1000 μg/plate onward
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
Slight increase in the number of his+ revertants at a concentration of 333 μg/plate (factor 1.7)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test depending on the strain and test conditions from about 1000 μg/plate onward
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
1st Exp.: Increase in his+ revertants at conc. of 2500 and 5000 μg/plate (factors 3.2 and 2.9, resp.); 2nd Exp.: Increase in his+ revertants at conc. of 1000, 2000, 2500, 3000, 4000 and 5000 μg/plate (factors 3.6, 3.8, 3.7, 2.8, 3.0 and 2.7 resp.)
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
cytotoxicity
Remarks:
A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test depending on the strain and test conditions from about 1000 μg/plate onward
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 333 μg/plate onward with and without S9 mix.

Strain

Test group

Dose (µg/plate)

Mean revertants per plate

Std Dev

Factor

TA 1535

DMSO

-

9.7

2.1

-

Test item

33

12.0

6.6

1.2

100

11.3

6.4

1.2

333

10.7

4.6

1.1

1000

12.0

1.7

1.2

2500

1.7

1.2

0.2

5000

0.0

0.0

0.0

MNNG

5.0

5185.0

420.1

536.4

TA 100

DMSO

-

94.0

3.0

-

Test item

33

85.3

5.5

0.9

100

106.3

24.5

1.1

333

112.3

13.6

1.2

1000

23.7

4

0.3

2500

1.7

1.2

0

5000

0

0

0

MNNG

5.0

3672.3

226.3

39.1

TA 1537

DMSO

-

9

1.7

-

Test item

33

11.3

4.6

1.3

100

14.3

2.3

1.6

333

14.3

0.6

1.6

1000

18.7

2.5

2.1

2500

9

1

1

5000

0

0

0

AAC

100

2104.3

217.9

233.8

TA 98

DMSO

-

18

2

-

Test item

33

20

1.7

1.1

100

27.3

7.1

1.5

333

30.7

8

1.7

1000

14

1.7

0.8

2500

1.7

1.2

0.1

5000

0

0

0

NOPD

10

955.3

16.7

53.1

E. coli

DMSO

-

22.7

8.1

-

Test item

33

30.3

6.4

1.3

100

25.3

2.9

1.1

333

24

5.3

1.1

1000

26

8.2

1.1

2500

11.7

2.5

0.5

5000

14.7

4

0.6

4-NQO

-

18

2

-

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a GLP compliant study, performed according to OECD guideline 471, the mutagenic potential of the test substance was evaluated in the bacterial strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A with and without metabolic activation (rat liver S9 mix). Concentrations from 33 up to 5000 μg/plate were tested in triplicate in the standar plate test. Precipitation of the test substance was found from about 333 μg/plate onward with and without S9 mix. A bacteriotoxic effect was observed depending on the strain and test conditions from about 1000 μg/plate onward. A biologically relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100 and TA 98 or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test using tester strains TA 1535, TA 100, TA 1537 and E. coli WP2 uvrA with and without S9 mix. Furthermore, using tester strain TA 98 without metabolic activation no relevant increase in the number of his+ revertants was observed in standard plate test. Using tester strains TA 98, a reproducible increase in the number of his+ revertants exceeding a factor of 2 compared to the concurrent vehicle control was observed after the addition of a metabolizing system. Increase at concentrations of 2500 and 5000 μg/plate in the 1st Experiment and at concentrations of 1000 up to 5000 μg/plate in the 2nd Experiment was observed using tester strain TA 98 with S9 mix. Under the experimental conditions of this study, the test substance is mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the presence of metabolic activation.

Justification for classification or non-classification

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