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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:
2016-08-25 to 2016-09-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(EC) No 440/2008, 2008-05-30
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998-08
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)
EC Number:
915-758-6
Molecular formula:
C32H18CrN6O8.Na / C32H22CrN10O8.Na / C32H20CrN8O8.Na
IUPAC Name:
Reaction mass of sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No. of test material: N01-131001
- Expiration date of the lot/batch: 2023-03-25

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature


OTHER SPECIFICS: solid, dark brown

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:
phenobarbital and beta naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
In agreement with the recommendations of current guidelines 5 mg/plate or 5 µL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 µL/plate might also be tested in repeat experiments for further clarification/substantiation.
In this study, due to the purity of the test substance 5.3 mg/plate was used as top dose in the 1st Experiment.
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.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
With S9 mix: 2.5 µg/plate, TA 1535, TA 100, TA 1537, TA 98; 60 µg/plate, Escherichia coli WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
Remarks:
Without S9 mix: 5 µg/plate, TA 1535, TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine (NOPD) (
Remarks:
Without S9 mix: 10 µg/plate, TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9 mix: 100 µg/plate, TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9 mix: 5 µg/plate, E. coli WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar

DURATION
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity detected by a decrease in the number of revertants (factor < 0.6) and/or clearing or diminution of the background lawn (= reduced his- or trp- background growth)

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
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
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
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
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
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:
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 333 µg/plate onward with and without S9 mix.

Any other information on results incl. tables

Table 1 SPT - Without metabolic activation

Strain

Test group

Dose (µg/plate)

Mean revertants per plate

Standard deviation

Factor

TA 1535

DMSO

Test item

 

 

 

 

 

MNNG

-

33

100

333

1000

2650

5300

5.0

9.7

9.3

17.0

20.0

25.3

0.0

0.0

5185.0

2.1

2.9

1.0

1.7

2.3

0.0

0.0

420.1

-

1.0

1.8

2.1 P

2.6 P

0.0 B P

0.0 B P

536.4

TA 100

DMSO

Test item

 

 

 

 

 

MNNG

-

33

100

333

1000

2650

5300

5.0

94.0

196.0

441.0

924.0

1506.7

64.0

0.0

3672.3

3.0

28.2

49.2

194.2

78.9

16.8

0.0

226.3

-

2.1

4.7

9.8 P

16.0 P

0.7 P B

0.0 P B

39.0

TA 1537

DMSO

Test item

 

 

 

 

 

AAC

-

33

100

333

1000

2650

5300

100

9.0

15.0

47.0

80.7

27.7

0.0

0.0

2104.3

1.7

4.4

10.4

11.0

6.8

0.0

0.0

217.9

-

1.7

5.2

9.0 P

3.1 P

0.0 P B

0.0 P B

233.8

TA 98

DMSO

Test item

 

 

 

 

 

NOPD

-

33

100

333

1000

2650

5300

10

18.0

48.3

93.0

200.3

376.7

616.7

1120.7

955.3

2.0

3.5

9.0

4.0

17.0

167.0

207.3

16.7

-

2.7

5.2

11.1 P

20.9 P

34.3 P

62.3 P

53.1

E.coli

DMSO

Test item

 

 

 

 

 

4-NQO

-

33

100

333

1000

2650

5300

5

22.7

18.7

19.3

19.7

18.08.2017 15.0

15.7

1440.7

8.1

4.9

2.5

4.0

2.6

4.4

8.1

107.7

-

0.8

0.9

0.9 P

0.8 P

0.7 P

0.7 P

63.6

 


 

Table 2: SPT - With metabolic activation

Strain

Test group

Dose (µg/plate)

Mean revertants per plate

Standard deviation

Factor

TA 1535

DMSO

Test item

 

 

 

 

 

2-AA

-

33

100

333

1000

2650

5300

2.5

9.7

13.7

10.3

16.3

20.7

5.3

0.0

136.3

3.1

3.1

4.2

1.2

3.2

0.6

0.0

10.2

-

1.4

1.1

1.7 P

2.1 P

0.6 P

0.0 P B

14.1

TA 100

DMSO

Test item

 

 

 

 

 

2-AA

-

33

100

333

1000

2650

5300

2.5

82.7

147.7

269.7

873.3

1189.3

1140.0

26.7

2322.7

7.5

9.3

11.5

166.3

22.0

136.9

23.7

210.7

-

1.8

3.3

10.6 P

14.4 P

13.8 P

0.3 P B

28.1

TA 1537

DMSO

Test item

 

 

 

 

 

2-AA

-

33

100

333

1000

2650

5300

2.5

9.7

15.3

18.7

79.0

141.7

6.7

0.0

212.0

2.3

4.7

3.1

4.6

19.9

1.5

0.0

15.6

-

1.6

1.9

8.2 P

14.7 P

0.7 P

0.0 P B

21.9

TA 98

DMSO

Test item

 

 

 

 

 

2-AA

-

33

100

333

1000

2650

5300

2.5

32.7

58.3

116.7

165.3

276.0

504.0

604.0

2476.7

6.7

6.0

13.7

15.8

17.3

68.4

146.9

59.7

-

1.8

3.6

5.1 P

8.4 P

15.4 P

18.5 P

75.8

E.coli

DMSO

Test item

 

 

 

 

 

2-AA

-

33

100

333

1000

2650

5300

60

31.0

37.0

35.0

22.7

24.7

19.7

18.0

94.7

7.5

9.6

2.0

3.1

2.1

4.6

7.0

2.1

-

1.2

1.1

0.7 P

0.8 P

0.6 P

0.6 P

3.1

 


Table 3: Decreased revertant numbers were observed at following concentrations (µg/plate)

Experiment

S9

TA 1535

TA 100

TA 1537

TA 98

E.coli

1st-SPT

Without

2650-5300

5300

2650-5300

-

-

With

2650-5300

5300

5300

-

2650-5300

2nd-SPT

Without

2650

-

2650

-

n.t.

With

-

-

2650

-

n.t.

 

- = no adverse effect observed

n.t. = not tested

 

 

Table 4: Reduced background growth was observed at following concentrations (µg/plate):

Experiment

S9

TA 1535

TA 100

TA 1537

TA 98

E.coli

1st-SPT

Without

2650-5300

2650-5300

2650-5300

-

-

With

5300

5300

5300

-

-

2nd-SPT

Without

2650

2650

2650

-

n.t.

With

-

-

2650

-

n.t.

 

- = no adverse effect observed

n.t. = not tested

 

 

 


 

 

Applicant's summary and conclusion

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