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:
8 May 2012 to 1 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

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
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
METI Reverse Mutagenicity Test on Bacteria, Methods of Testing New Chemical Substances (Section 5.1-1 to 5.1-11): Japanese Act on Evaluation of Chemical Substances and Regulation of Their Manufacture, Act No. 117, Ministerial Ordinance No.1-3 (April 2004)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
944127-07-1
IUPAC Name:
944127-07-1
Test material form:
other: solid (unspecified)
Details on test material:
- Appearance: White Solid
- Storage conditions of the test material: Ambient (18 to 36 °C)

Method

Target gene:
Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other: S. typhimurium: all strains possess rfa and uvrB; TA98 and TA100 also possess the R-factor plasmid pKM101. E. coli strain possesses the uvrA mutation and the R-factor plasmid pKM101.
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction prepared from induced rat liver
Test concentrations with justification for top dose:
Initial toxicity-mutation assay: 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Confirmatory mutation assay: 0, 93.8, 187.5, 375, 750, 1500, 3000 and 5000 µg/plate for strain TA 100
Confirmatory mutation assay: 0, 187.5, 375, 750, 1500, 3000 and 5000 µg/plate for strains TA 98, TA 1535, TA 1537 and WP2uvrA
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: DMSO is one of the organic vehicles compatible with this test system. The test material was found to form a solution in DMSO at 500 mg/mL and was stable at 15 and 50000 μg/mL after 4 hours.
Controls
Untreated negative controls:
no
Remarks:
Sterility control plates were observed for microbial colonies. Viable count plates were observed to determine the number of colony forming units per mL of each bacterial suspension.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: pre-incubation
100 µL of the appropriate bacterial culture, 100 µL of the test material or control material solution and 500 µL of the S9 mix or PBS were transferred into sterile test tubes and were kept in an incubator shaker for approximately 20 ± 2 minutes at 37 ± 1 °C. After this period, 2 mL of soft agar containing histidine-biotin / tryptophan was added to each of the tubes and the constituents were overlaid onto VB agar plates. After the soft agar had set, the plates were incubated.

DURATION
- Exposure duration: 67 hours at 37 ± 1 °C under yellow light

NUMBER OF REPLICATIONS
- Initial toxicity-mutation assay: duplicate
- Confirmatory mutation assay: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Examined for effects on the background lawn of bacterial growth
Evaluation criteria:
The conditions necessary for determining a positive result are as follows:
There should be a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test material either in the absence or presence of the metabolic activation system.

- Strains TA98, TA1535, and TA1537
Data sets will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 3.0 times the mean vehicle control value.

- Strain TA100 and WP2uvrA
Data sets will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2.0 times the mean vehicle control value.

A response that does not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) will not be evaluated as positive.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: S. typhimurium TA98, TA1535, TA1537 and E. coli WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
other: Viable counts of all the tester strains were within the required range of 1 to 2 x10⁹ CFU/mL. The most concentrated test material dilution, the Sham (PBS) and S9 mixes were found to be sterile.
Positive controls validity:
valid
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:
other: Viable counts of all the tester strains were within the required range of 1 to 2 x10⁹ CFU/mL. The most concentrated test material dilution, the Sham (PBS) and S9 mixes were found to be sterile.
Positive controls validity:
valid
Additional information on results:
INITIAL TOXICITY-MUTATION ASSAY
The mean number of revertant colonies/plate in the DMSO control was within the range of the in-house spontaneous revertant counts for all the tester strains
The test material did not cause any precipitation on the basal agar plates up to 5000 µg/plate. No toxicity was observed up to up to 1500 µg/plate as the intensity of the bacterial background lawn was comparable to the vehicle control in the presence and absence of metabolic activation. However, at the top dose of 5000 µg/plate, there was slight to moderate thinning of the bacterial background lawn in the presence and absence of metabolic activation.
The test material was found to be toxic to all the five tester strains at the top dose of 5000 μg/mL when compared to the vehicle control, both in the presence and absence of metabolic activation. For the strain TA 100, there was a greater than 2-fold increase in the mean number of revertant colonies compared to the vehicle control at the dose of 1500 μg/plate both in the presence and absence of metabolic activation.
Positive control chemicals tested simultaneously produced more than a 3-fold increase in the mean numbers of revertant colonies for all the strains when compared to the respective vehicle control plates. No toxicity was observed in the positive controls as the intensity of the bacterial background lawn of all the tester strains was comparable to that of the respective vehicle control plates.

CONFIRMATORY MUTATION ASSAY
Summary results from the confirmatory toxicity-mutation assay are presented in Table 1.
The mean number of revertant colonies/plate in the DMSO control was within the range of the in-house spontaneous revertant counts for all the tester strains.
The test material did not cause any precipitation on the basal agar plates up to 5000 µg/plate. The toxicity profile of the test material varied with the tester strains in the confirmatory mutation assay.
Strains TA 98, TA 1535 and TA 1537 did not exhibit any toxicity up to 1500 µg/plate as the intensity of the bacterial background lawn was comparable to the vehicle control in the presence and absence of metabolic activation. However, TA 98 exhibited slight thinning at 3000 µg/plate, and moderate thinning of the background lawn at the top dose of 5000 µg/plate, in the presence and absence of metabolic activation. Similarly, strains TA 1535 and TA 1537 exhibited moderate thinning of the background lawn at and above 3000 µg/plate, in the presence and absence of metabolic activation.
Strains TA 100 and WP2uvrA (pKM101) did not exhibit any toxicity up to 3000 µg/plate as the intensity of the bacterial background lawn was comparable to the vehicle control in the presence and absence of metabolic activation. However, at the top dose of 5000 µg/plate, there was slight thinning of the bacterial background lawn in the presence and absence of metabolic activation for these 2 tester strains.

There was no positive mutagenic response observed in the strains TA 98, TA 1535, TA 1537 and WP2uvrA (pKM101) in any of the tested doses either in the presence or in the absence of metabolic activation. However, there was ≥ 2-fold increase in the mean number of revertant colonies for the strain TA 100 at 1500 and 3000 μg/plate in the presence of metabolic activation and at 750, 1500 and 3000 μg/plate in the absence of metabolic activation compared to the vehicle control.

Positive control chemicals tested simultaneously produced more than a 3-fold increase in the mean numbers of revertant colonies for all the strains when compared to the respective vehicle control plates. No toxicity was observed in the positive controls as the intensity of the bacterial background lawn of all the tester strains was comparable to that of the respective vehicle control plates.

S-9 HOMOGENATE
The S-9 homogenate was found to be sterile, active and the protein content of the S-9 homogenate was 26.6 mg/mL.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Summary Results of the Confirmatory Mutation Assay

+/- S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

-

-

-

-

-

-

-

Solvent

93.8

187.5

375

750

1500

3000

5000

116

119

135

199

231***

256***

287***

52*

14

-

13

12

11

10

4*

2*

133

-

133

139

145

161

175

65*

26

-

25

24

32

30

15**

4*

11

-

11

10

9

8

3*

2*

+

+

+

+

+

+

+

+

Solvent

93.8

187.5

375

750

1500

3000

5000

105

111

124

139

180

255***

266***

57**

16

-

16

13

14

11

3*

2*

141

-

154

168

168

178

192

80**

27

-

24

30

33

38

15**

6*

11

-

10

9

9

9

2*

2*

                                                         Positive Controls

-

Name

SA

SA

4NQO

2NF

9AA

Concentration (µg/plate)

1

1

4

2

50

Mean no. colonies/plate

554

127

589

236

101

+

Name

2AA

2AA

2AA

2AA

2AA

Concentration (µg/plate)

4

4

30

4

4

Mean no. colonies/plate

871

137

556

542

109

*Cytotoxicity observed as a >50 % reduction in revertants compared to the vehicle control

**Cytotoxicity observed as a reduced background lawn

***Mutagenic (positive) result at ≥2-fold increase in revertants as compared to the vehicle control

SA = sodium azide

4NQO = 4-nitroquinoline-1-oxide

2NF = 2-nitrofluorene

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive in strain TA 100 with and without metabolic activation

Under the conditions of this study, the test material is considered to be mutagenic for the S. typhimurium tester strain TA 100 in both the presence and absence of S9 activation.
Executive summary:

The test material was assessed for mutagenic potential in the bacterial reverse mutation assay in accordance with the standardised guidelines OECD 471, EU Method B.13/14, USA EPA OPPTS 870.5100 and the Japanese METI Reverse Mutagenicity Test on Bacteria under GLP conditions.

The study was conducted using TA 98, TA 100, TA 1535 and TA 1537 strains of Salmonella typhimurium and the WP2uvrA (pKM101) strain of Escherichia coli in two phases. In the first phase, an initial toxicity-mutation test was performed. The second phase was an independent confirmatory mutation test. The bacterial tester strains were exposed to the test material in dimethyl sulphoxide in the presence and absence of a metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver) using a pre-incubation procedure.

In the initial toxicity-mutation assay, cultures were exposed to the test material in duplicate at 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, along with the vehicle and appropriate positive controls.

The test material did not cause any precipitation on the basal agar plates up to 5000 μg/plate. No toxicity was observed up to 1500 μg/plate. However, the test material was found to be toxic to all five tester strains at the top dose of 5000 μg/mL, both in the presence and absence of metabolic activation when compared to the vehicle control.

There was no positive mutagenic response observed in the strains TA 98, TA 1535, TA 1537 and WP2uvrA (pKM101) in any of the tested doses either in the presence or in the absence of metabolic activation.

However, TA 100 resulted in a greater than 2-fold increase in the mean number of revertant colonies compared to the vehicle control at 1500 μg/plate both in the presence and absence of metabolic activation.

Based on these initial findings, in the confirmatory mutation assay, cultures were exposed in triplicate at 93.8, 187.5, 375, 750, 1500, 3000 and 5000 μg/plate test doses for the strain TA 100 and 187.5, 375, 750, 1500, 3000 and 5000 μg/plate test doses for the strains TA 98, TA 1535, TA 1537 and WP2uvrA (pKM101) along with the vehicle and appropriate positive controls. The mean and standard deviation of revertant colonies were calculated for each test dose and the controls for all the tester strains.

The test material did not cause any precipitation on the basal agar plates up to 5000 μg/plate. The toxicity profile of the test material varied with the tester strains in the confirmatory mutation assay. Strains TA 98, TA 1535 and TA 1537 exhibited toxicity as evidenced by the intensity of the bacterial background lawn at concentrations of 1500 µg/plate and higher, in the presence and absence of metabolic activation. Strains TA 100 and WP2uvrA (pKM101) exhibited toxicity at concentrations of 3000 μg/plate and higher, in the presence and absence of metabolic activation.

There was no positive mutagenic response observed in the strains TA 98, TA 1535, TA 1537 and WP2uvrA (pKM101) in any of the tested doses either in the presence or in the absence of metabolic activation. However, there was a 2-fold increase in the mean number of revertant colonies for the strain TA 100 at 1500 and 3000 μg/plate in the presence of metabolic activation and at 750, 1500 and 3000 μg/plate test doses in the absence of metabolic activation compared to the vehicle control.

There was a more than 3-fold increase in the mean numbers of revertant colonies in the positive controls, demonstrating the sensitivity of the assay.

Under the conditions of this study, the test material is considered to be mutagenic for the S. typhimurium tester strain TA 100 in both the presence and absence of S9 activation.