Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Start: 30 May 2018 Experimental Completion: 08 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Annex VIII Data Requirement

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Based upon a preliminary study (Experiment 1), the dose range used for the Main Study (Experiment 2) was 1.23, 4.1, 12.3, 41, 123, 410, 1230 and 4100 μg/plate.
Vehicle / solvent:
Tetrahydrofuran
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-aminoanthracene
Details on test system and experimental conditions:
Without Metabolic Activation:
A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.025 mL of the appropriate concentration of test item formulation or solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation:
The procedure was the same as described previously (see 3.3.3.2) except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation & Scoring:
All of the plates were incubated at 37±3°C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (=p<0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Without Metabolic Activation

S9 Mix

Dose
(ug/plate)

No. of Revertant Colonies Per Plate (Mean ± SD)

Base Pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

Solvent Control (THF)

130 ± 4.0

15 ± 2.1

21 ± 3.5

29 ± 10.3

9 ± 6.0

1.23

121 ± 6.6

12 ± 3.5

21 ± 2.9

29 ± 0.0

13 ± 1.2

4.1

127 ± 13.6

9 ± 4.6

21 ± 6.0

29 ± 3.6

12 ± 7.5

12.3

113 ± 5.1

15 ± 6.1

28 ± 2.6*

20 ± 6.0

14 ± 1.2

41

117 ± 5.0

12 ± 3.2

24 ± 2.5

18 ± 3.6

12 ± 9.9

123

83 ± 29.1

12 ± 7.2

25 ± 3.1

0 ± 0.0V

5 ± 3.2

410

0 ± 0.0T

13 ± 3.6

27 ± 4.0

0 ± 0.0T

0 ± 0.0V

1230

0 ± 0.0T

0 ± 0.0T

26 ± 2.0S

0 ± 0.0T

0 ± 0.0T

4100

0 ± 0.0T

0 ± 0.0T

0 ± 0.0V

0 ± 0.0T

0 ± 0.0T

Positive Control

Compound

ENNG

ENNG

ENNG

4NQO

9AA

ug/Plate

3

5

2

0.2

80

Revertants/Plate

1120 ± 253.6

2601 ± 277.8

950 ± 33.1

403 ± 29.9

506 ± 54.0

*p =0.05
S
Sparse bacterial background lawn
VVery weak bacterial background lawn
TToxic – no bacterial background lawn

 

 

With Metabolic Activation

S9 Mix

Dose
(ug/plate)

No. of Revertant Colonies Per Plate (Mean ± SD)

Base Pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

+

Solvent Control (THF)

129 ± 8.5

13 ± 2.0

40 ± 8.5

31 ± 7

18 ± 3.8

1.23

126 ± 28

15 ± 4.2

38 ± 1.7

28 ± 6.4

16 ± 3.2

4.1

139 ± 9.5

12 ± 4.7

40 ± 5.5

26 ± 4.4

18 ± 1.2

12.3

139 ± 3.0

17 ± 4.6

35 ± 3.5

30 ± 9.5

17 ± 4.2

41

126 ± 8.6

13 ± 0.6

33 ± 7.2

29 ± 1.7

19 ± 2.5

123

110 ± 14.7

14 ± 4.0

34 ± 6.9

26 ± 2.1

8 ± 1.5

410

85 ± 11.4S

11 ± 3.5

36 ± 3.5

29 ± 7.5

8 ± 2.1

1230

0 ± 0.0T

5 ± 1.5S

28 ± 5.2

19 ± 3.5S

0 ± 0.0V

4100

0 ± 0.0T

0 ± 0.0T

24 ± 8.0S

15 ± 8.7S

0 ± 0.0T

Positive Control

Compound

2AA

2AA

2AA

BP

2AA

ug/Plate

1

2

10

5

2

Revertants/Plate

1959 ± 61.3

349 ± 13.1

282 ± 32.1

126 ± 7.4

346 ± 33

SSparse bacterial background lawn
VVery weak bacterial background lawn
TToxic – no bacterial background lawn

Applicant's summary and conclusion

Conclusions:
In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item Reaction mass of disodium N,N'-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate did not induce an increase in the frequency of revertant colonies at any of the dose levels used either with or without metabolic activation (S9-mix). Under the conditions of this test Reaction mass of disodium N,N'-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate was considered to be non-mutagenic.
Executive summary:

Introduction

 

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, the ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749) and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

 

Methods

 

Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 andEscherichia colistrain WP2uvrAwere treated with the test item using both the Ames plate incorporation and pre-incubation methods at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.23 to 4100 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was the same as Experiment 1 (1.23 to 4100 μg/plate). Eight test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item following the change in test methodology.

 

Results

 

The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The maximum dose level of the test item in the first experiment was 4100 μg/plate. In the first mutation test (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains (except TA98 dosed in the presence of metabolic activation) initially from 1230 μg/plate in both the absence and presence of metabolic activation.

 

Based on the results of Experiment 1, the same maximum dose level (4100 μg/plate) was employed in the second mutation test (pre-incubation method). In Experiment 2 (pre-incubation method) the test item induced a stronger toxic response with weakened bacterial background lawns initially noted in the absence of S9-mix from 123 μg/plate (TA100 and TA98), 410 μg/plate (TA1535 and TA1537) and 1230 μg/plate (WP2uvrA). In the presence of S9-mix, weakened bacterial background lawns were initially noted from 410 μg/plate (TA100), 1230 μg/plate (TA1535, TA98 and TA1537) and at 4100 μg/plate (WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology.

 

A test item precipitate (greasy in appearance) was noted at and above 1230 μg/plate in both the presence and absence of metabolic activation (S9-mix) in Experiment 1 (plate incorporation method) only. This observation did not prevent the scoring of revertant colonies.

 

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method).

 

Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). A minor statistical value was noted (WP2uvrAat 12.3 μg/plate in the absence of S9-mix (= p ≤0.05)), however this response was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance.

 

Conclusion

 

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item Reaction mass of disodium N,N'-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate did not induce an increase in the frequency of revertant colonies at any of the dose levels used either with or without metabolic activation (S9-mix). Under the conditions of this test Reaction mass of disodium N,N'-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate was considered to be non-mutagenic.