Registration Dossier

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:
2017-05-02 till 2017-05-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline-conform study under GLP without deviations

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
IIdentification: 1-((4-Methoxy-2-nitrophenyl)azo)-2-naphthol
CAS No.: 49744-28-7
EINECS / EC No.: 256-458-8
Batch: 3.April 2010 g
Purity: 99.2% (w/w) including isomer
Physical State / Appearance: Dark red powder
Expiry Date: 25 January 2027 (Statement of sponsor)
Storage Conditions: At room temperature
Certificate of Analysis: AZ 1070/Toxd1, dated 25 January 2017
Stability in Solvent: Not indicated by the Sponsor

Method

Species / strain
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-naphthoflavone induced rat liver S9 (experiment I) non- induced hamster liver S9 (experiment II)
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II:
All strains without S9 mix: 33; 100; 333; 1000; 2500; and 5000 µg/plate
All strains with S9 mix: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle:
Solvent used: DMF
Justification for choice of solvent: best suitable solvent, because of its solubility properties and its relative nontoxicity to the bacteria
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
congo red
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene
Details on test system and conditions:
METHOD OF APPLICATION: in agar plate incorporation; pre-incubation

DURATION:
Preincubation period: 30 Minutes
exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3 plates for each concentration including the controls

DETERMINATION OF CYTOTOXICITY: Evident as a reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.
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 WP2 uvrA) 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: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
strain TA 98 with non-induced hamster liver S9
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: reverse mutation assay migrated from the field Test System
Additional information on results:
TEST SPECIFIC CONFOUNDING FACTORS
Effects of pH: none
Water solubility: not soluble at 50 mg/mL
Precipitation: The test item precipitated in the overlay agar in the test tubes from 100 to 5000 µg/plate with and without S9 mix in experiment I and in experiment II from 1000 to 5000 µg/plate without S9 mix and from 333 to 5000 µg/plate with S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I from 2500 to 5000 µg/plate with and without S9 mix and in experiment II from 2500 to 5000 µg/plate without S9 mix and from 1000 to 5000 µg/plate with S9 mix. The undissolved particles had no influence on the data recording.
Other confounding effects: none

COMPARISON WIT HISTORICAL CONTROL DATA: performed, no deviation
ADDITIONAL INFORMATION ON CYTOTOXICITY: no

Any other information on results incl. tables

Summary of Experiment I

Study Name: 1839700

Study Code: Envigo 1839700

Experiment: 1839700 VV Plate

Date Plated: 02.05.2017

Assay Conditions:

Date Counted: 08.05.2017

Metabolic

Activation

Test

Group

Dose Level

(per plate)

TA 1535

Revertant Colony Counts (Mean ±SD)

TA 1537

Revertant Colony Counts (Mean ±SD)

TA 98

Revertant Colony Counts (Mean ±SD)

TA 100

Revertant Colony Counts (Mean ±SD)

WP2 uvrA

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

Without

DMF

 

11 ± 5

8 ± 3

20 ± 6

151 ± 25

35 ± 4

Activation

Untreated

 

8 ± 4

9 ± 3

22 ± 3

160 ± 21

40 ± 6

 

1-((4-Methoxy-2-

3 µg

8 ± 2

9 ± 4

17 ± 4

149 ± 6

38 ± 2

 

nitrophenyl)azo)-

10 µg

10 ± 1

12 ± 3

22 ± 6

144 ± 16

45 ± 9

 

2-naphthol

33 µg

11 ± 1

12 ± 3

25 ± 2

145 ± 22

36 ± 6

 

 

100 µg

8 ± 4

10 ± 4

23 ± 8

158 ± 16

45 ± 8

 

 

333 µg

8 ± 2

10 ± 2

19 ± 3

154 ± 12

39 ± 5

 

 

1000 µg

11 ± 4

13 ± 4

19 ± 8

135 ± 9

33 ± 8

 

 

2500 µg

10 ± 5P

13 ± 1P

19 ± 2P

157 ± 8P

36 ± 5P

 

 

5000 µg

10 ± 1P

11 ± 1P

22 ± 6P M

150 ± 26P

41 ± 11P

 

NaN3

10 µg

1164 ± 122

 

 

2017 ± 102

 

 

4-NOPD

10 µg

 

 

262 ± 7

 

 

 

4-NOPD

50 µg

 

78 ± 5

 

 

 

 

MMS

2.0 µL

 

 

 

 

1030 ± 98

 

 

 

 

 

 

 

 

With

DMF

 

9 ± 2

15 ± 3

24 ± 4

134 ± 16

51 ± 4

Activation

Untreated

 

10 ± 5

13 ± 4

33 ± 9

157 ± 18

53 ± 9

 

1-((4-Methoxy-2-

3 µg

9 ± 1

14 ± 2

30 ± 2

147 ± 8

52 ± 6

 

nitrophenyl)azo)-

10 µg

7 ± 2

17 ± 5

26 ± 5

159 ± 14

50 ± 10

 

2-naphthol

33 µg

11 ± 1

16 ± 4

30 ± 2

161 ± 14

58 ± 7

 

 

100 µg

7 ± 1

16 ± 4

36 ± 7

151 ± 10

53 ± 5

 

 

333 µg

10 ± 4

19 ± 3

34 ± 7

173 ± 15

52 ± 4

 

 

1000 µg

9 ± 3

20 ± 2

30 ± 5

156 ± 28

46 ± 9

 

 

2500 µg

12 ± 2P

19 ± 6P

32 ± 5P

164 ± 8P

53 ± 7P

 

 

5000 µg

11 ± 1P

16 ± 4P M

33 ± 3P M

140 ± 14P M

50 ± 13P

 

2-AA

2.5 µg

409 ± 13

171 ± 4

3450 ± 1007

4360 ± 355

 

 

2-AA

10.0 µg

 

 

 

 

327 ± 57

 

 

 

 

 

 

 

 


Summary of Experiment II

Study Name: 1839700

Study Code: Envigo 1839700

Experiment: 1839700 HV2 Pre

Date Plated: 11.05.2017

Assay Conditions:

Date Counted: 16.05.2017

Metabolic

Activation

Test

Group

Dose Level

(per plate)

TA 1535

Revertant Colony Counts (Mean ±SD)

TA 1537

Revertant Colony Counts (Mean ±SD)

TA 98

Revertant Colony Counts (Mean ±SD)

TA 100

Revertant Colony Counts (Mean ±SD)

WP2 uvrA

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

Without

DMF

 

9 ± 3

9 ± 2

30 ± 7

147 ± 8

33 ± 7

Activation

Untreated

 

9 ± 3

9 ± 2

32 ± 0

193 ± 19

32 ± 1

 

1-((4-Methoxy-2-

33 µg

11 ± 3

10 ± 3

29 ± 6

137 ± 6

44 ± 7

 

nitrophenyl)azo)-

100 µg

7 ± 2

11 ± 1

24 ± 5

153 ± 10

42 ± 12

 

2-naphthol

333 µg

8 ± 2

13 ± 3

30 ± 3

148 ± 7

39 ± 10

 

 

1000 µg

8 ± 2

8 ± 2

22 ± 7

149 ± 3

34 ± 18

 

 

2500 µg

8 ± 2P

8 ± 3P

27 ± 6P

152 ± 13P

26 ± 4P

 

 

5000 µg

7 ± 2P

11 ± 3P

19 ± 4P M

143 ± 1P M

23 ± 5P M

 

NaN3

10 µg

1103 ± 46

 

 

2039 ± 76

 

 

4-NOPD

10 µg

 

 

267 ± 5

 

 

 

4-NOPD

50 µg

 

79 ± 8

 

 

 

 

MMS

2.0 µL

 

 

 

 

1094 ± 36

 

 

 

 

 

 

 

 

With

DMF

 

10 ± 1

23 ± 7

41 ± 1

138 ± 4

43 ± 6

Activation

Untreated

 

7 ± 2

21 ± 9

38 ± 3

126 ± 27

48 ± 6

 

1-((4-Methoxy-2-

3 µg

10 ± 2

28 ± 3

48 ± 6

126 ± 10

44 ± 3

 

nitrophenyl)azo)-

10 µg

12 ± 8

31 ± 10

52 ± 10

148 ± 12

48 ± 4

 

2-naphthol

33 µg

12 ± 1

23 ± 2

61 ± 15

157 ± 7

50 ± 11

 

 

100 µg

15 ± 2

23 ± 3

81 ± 6

170 ± 14

50 ± 21

 

 

333 µg

20 ± 4

25 ± 5

124 ± 17

205 ± 46

49 ± 16

 

 

1000 µg

17 ± 8P

24 ± 7P

104 ± 14P

219 ± 11P

54 ± 3P

 

 

2500 µg

10 ± 2P

21 ± 6P

85 ± 12P M

176 ± 10P M

41 ± 7P M

 

 

5000 µg

16 ± 3P M

25 ± 6P M

78 ± 8P M

196 ± 11P M

36 ± 6P M

 

2-AA

2.5 µg

372 ± 22

269 ± 9

 

2382 ± 92

 

 

2-AA

10.0 µg

 

 

 

 

800 ± 28

 

Congo red

 

 

 

440 ± 48

 

 

 

 

 

 

 

 

 

 

Key to Plate Postfix Codes:              

P: Precipitate

M: Manuel Count

R:  Reduced Background growth

D:  Densely Colored Plate

Key to positive controls:

NaN3: sodium azide

4 -NOPD: 4 -nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

2-AA: 2 -aminoanthracene

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by frameshifts in the genome of strain TA 98 in the presence of non-induced hamster liver S9 mix.
Therefore, 1-((4-Methoxy-2-nitrophenyl)azo)-2-naphthol is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

The test item 1-((4-Methoxy-2-nitrophenyl)azo)-2-naphthol was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) usingSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.

Experiment I was performed with induced rat liver S9 mix as an exogenous metabolic activation system and Experiment II was performed with non-induced hamster liver S9 mix. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:        3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:

All strains without S9 mix:             33; 100; 333; 1000; 2500; and 5000 µg/plate

All strains with S9 mix:                  3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 100 to 5000 µg/plate with and without S9 mix in experiment I and in experiment II from 1000 to 5000 µg/plate without S9 mix and from 333 to 5000 µg/plate with S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I from 2500 to 5000 µg/plate with and without S9 mix and in experiment II from 2500 to 5000 µg/plate without S9 mix and from 1000 to 5000 µg/plate with S9 mix. The undissolved particles had no influence on the data recording.

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 (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

In experiment II an increase in revertant colony numbers was observed following treatment with 1-((4-Methoxy-2-nitrophenyl)azo)-2-naphthol in strain TA 98 in the presence of metabolic activation (non-induced hamster liver S9 mix).The number of colonies exceeded the threshold of twice the number of the corresponding solvent control from 333 to 2500 µg/plate. The induction factor decreased with the increasing precipitation of the test substance and at a concentration of 5000µg/plate the threshold of twice the number of the corresponding solvent control was not reached. This effect may be based on the observed precipitation of the test item which causes a minor bio-availability of the test substance or an overlapping toxic effect.

A minor increase in revertant colony numbers, not reaching the threshold of thrice the number of the corresponding solvent control, was observed in strain TA 1535in the presence of metabolic activation (S9 mix).

 

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.