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:
weight of evidence
Study period:
7th March 2012 to 4th July 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid.
Details on test material:
- Name of test material (as cited in study report): Capa 2043
- Physical state: Colourless liquid.
- Analytical purity: 97%
- Lot/batch No.: WAC000392
- Expiration date of the lot/batch: 17th February 2014
- Storage condition of test material: Stored in the dark at room temperature.
Specific details on test material used for the study:
CAPA 2043 (2-oxepanone, polymer with 1,4-butanediol)

Method

Target gene:
Histidine and tryptophan
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
All S. typhimurium strains contain mutations in the histidine operon, thereby imposing the essential requirement for histidine in the growth medium. Three mutations in the histidine operon are involved:

his G 46 in TA 1535 and TA 100
his C 3076 in TA 1537
his D 3052 in TA 98
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
The strain contains an ochre mutation in the trpE locus and can be mutated to tryptophan-independence either by a base-pair reversion of an A-T base-pair in the trpE locus, or more commonly, by a base-pair substitution within a number of transfer RNA loci elsewhere in the chromosome. The latter mutation causes the original defect to be suppressed (ochre suppression) and involves only base-pair substitutions at G-C base-pairs.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix.
Test concentrations with justification for top dose:
The concentrations used in both the preliminary toxicity test and the mutation experiments were: 17, 50, 167, 500, 1667 and 5000 μg per plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Remarks:
2 μg per plate - TA 1535 and TA 1537, 0.5 μg per plate - TA 98 and TA 100 and 20 μg per plate - E. coli WP2uvrA
Positive control substance:
other: 2-Aminoanthracene
Remarks:
In the presence of S9 mix.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
not specified
Positive controls:
yes
Remarks:
- with S. typhimurium TA 1535 and TA 100
Positive control substance:
sodium azide
Remarks:
In the absence of S9 mix.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Remarks:
80 μg per plate - TA 1537
Positive control substance:
9-aminoacridine
Remarks:
In the absence of S9 mix.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Remarks:
1 μg per plate - TA 98
Positive control substance:
2-nitrofluorene
Remarks:
In the absence of S9 mix.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Remarks:
2 μg per plate - E. coli WP2uvrA
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
In the absence of S9 mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION:
In the first test, the direct plate incorporation test method was used. In the 2nd test, the Pre-incubation test was used.

For the toxicity tests, the plates were placed in an incubator set to maintain a temperature of 37°C for 2 days. The numbers of revertant colonies were noted and the plates carefully examined, microscopically, for thinning of the background lawn of microcolonies. Condition of background lawn was assessed as normal, slightly thin lawn (ST), thin lawn (TL), very thin lawn (VT) or lawn absent (A). Any precipitation of the test item on the plates was noted.

In the mutation tests, diluted agar was autoclaved and supplemented with sterile L-histidine.HCl (1.0 mM)/biotin (1.0 mM) was added at 50 mL per litre of soft agar for S. typhimurium. For E. coli, sterile L-tryptophan (1.35 mM) was added at 10 mL per litre of soft agar. These soft agars were thoroughly mixed and kept in a water bath set to maintain a temperature of 45°C.

In the first experiment using the direct plate incorporation method, soft agar (2 mL) was dispensed into a small plastic sterile tube. S9 mix or phosphate buffer (0.5 mL) was added, followed by the bacteria (0.1 mL) and, finally, the test solution. The tube contents (which were continually cooling) were mixed, then poured on to minimal medium plates prepared in-house. The plates contained BBL Purified Agar (1.5%, 20 mL) in Vogel-Bonner Medium E with glucose (2%, w/v).

In the second experiment using the pre-incubation method, S9 mix or phosphate buffer (0.5 mL) was dispensed into a small plastic sterile tube followed by the bacteria (0.1 mL) and, finally, the test solution. The tubes were then placed for 20 min in a shaking incubator set to maintain a temperature of 37°C. After incubation, soft agar (2 mL) was added to each tube. The tube contents were then mixed and poured onto minimal medium plates.
When the soft agar had set, the plates were inverted and placed in an incubator set to maintain a temperature of 37°C for 3 days and then examined. The numbers of mutant colonies on each plate were determined using a Colony Counter and captured electronically in a validated software system. The plates were also examined microscopically for precipitates and for microcolony growth.

NUMBER OF REPLICATIONS: Triplicate plates were used for each exposure level and bacterial strain in the presence and absence of metabolic activation.

Evaluation criteria:
For S. typhimurium strains TA 1535, TA 1537, and TA 98 and for E. coli WP2uvrA, at least a doubling of the mean concurrent vehicle control value is required before mutagenic activity is suspected. For S. typhimurium strain TA 100, a 1.5-fold increase over the control value is required before mutagenic activity is suspected.

If the mean colony count on the vehicle control plates is less than 10, then a minimum count of 20 (representing a 2-fold increase over 10) is required before a response is registered.

A concentration-related response is also required for identification of a mutagenic effect. At high concentrations this relationship may be reversed because of, toxicity of the test item to the bacteria, specific toxicity of the test item to the mutants, or inhibition of S9 enzymes (where a mutagen requires metabolic activation by the S9 mix). A response should be reproducible in an independent test.
Statistics:
The mean number of mutant colonies, plus standard deviation, was calculated for each set of 3 plates. In addition, the fold-increase over the vehicle control was calculated for all test item and positive control treatments.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no 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
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
In the toxicity tests, no toxicity to the bacteria was observed at any concentration in either the absence or the presence of S9 mix. At the highest concentration of 5000 μg per plate, precipitation was observed in the presence and absence of S9 mix.

In the mutation tests, for all bacterial strains tested, precipitation was observed at the highest concentration of 5000 μg per plate in the plate incorporation test, with and without metabolic activation. In the pre-incubation test method with and without metabolic activation, for strains TA1535, TA1537 and TA98, precipitation and a slight thinning of the background lawn was noted at the highest concentration tested (5000 μg per plate). For strains TA100 and e coli WP2 uvrA, precipitation was observed at this concentration level.

COMPARISON WITH HISTORICAL CONTROL DATA:
The vehicle and positive control values were within the normal/historical ranges recorded in this laboratory.

Any other information on results incl. tables

Summary of results

Mean revertant numbers

Treatment

EXPERIMENT 1 (PLATE INCORPORATION)

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

DMSO

25.7

25.0

83.3

91.7

12.7

15.7

10.3

14.7

11.0

9.7

17 µg

20.7

26.7

105.7

83.3

8.7

11.7

7.7

8.7

7.0

9.0

50 µg

25.3

36.3

86.3

76.0

9..7

8.7

14.0

11.3

8.3

9.0

167 µg

32.7

31.7

103.3

85.3

14.7

12.3

9.0

10.7

7.0

9.0

500 µg

23.7

22.0

91.3

73.3

13.7

13.3

9.78

11.7

8.0

8.3

1667 µg

25.3

27.3

103.0

84.0

13.3

12.7

8.7

9.0

9.3

8.0

5000 µg

21.3

31.3

90.0

85.3

16.0

15.7

9.3

18.7

11.7

5.7

2-NF

521.3

-

-

-

-

-

-

-

-

-

NaN3

-

-

915.0

-

377.7

-

-

-

-

-

9AA

-

-

-

-

-

-

4320

-

-

-

ENNG

-

-

-

-

-

-

-

-

96.3

-

2AAN

-

307.7

-

711.3

-

313.0

-

298.0

-

729.3

Treatment

EXPERIMENT 2 (PREINCUBATION)

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

DMSO

22.3

24.3

90.0

86.7

11.7

13.7

6.3

12.0

5.7

10.0

17 µg

24.7

26.3

90.3

85.7

9.0

9.3

7.7

11.3

6.3

7.3

50 µg

26.3

28.3

83.7

96.7

8.7

16.0

9.7

8.7

7.3

9.0

167 µg

24.0

34.0

81.7

83.7

10.0

8.3

5.3

15.7

7.0

5.7

500 µg

21.0

31.3

87.7

93.0

12.7

10.7

8.0

14.7

8.0

13.3

1667 µg

19.7

26.3

89.7

88.3

10.0

13.3

7.7

14.3

7.0

11.3

5000 µg

13.0

31.7

96.3

92.0

9.7

15.7

7.3

4.7

5.7

6.7

2-NF

462.0

-

-

-

-

-

-

-

-

-

NaN3

-

-

1049

-

368.7

-

-

-

-

-

9AA

-

-

-

-

-

-

5010

-

-

-

ENNG

-

-

-

-

-

-

-

-

407.0

-

2AAN

-

332.3

-

649.3

-

230.3

-

257.3

-

812.3

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the test material Capa 2043, was not considered to be mutagenic when tested in the presence and absence of metabolic activation.
Executive summary:

In a mutagenicity study conducted in accordance with GLP and OECD Guideline 471, CAPA 2043 (2 -oxepanone, polymer with 1,4 -butanediol) was tested in S. typhimurium and E. coli bacterial strains in the presence and absence of metabolic activation (S9 mix). The bacterial strains used were TA1535, TA1537, TA98 and TA100; and E. coli WP2uvrA. Two independent tests were conducted on agar plates in triplicate in the absence and presence of S9 mix, the first of which was conducted by the direct Plate Incorporation Method and the second test was conducted using the Pre-incubation Method, at concentrations of 17, 50, 167, 500, 1667 and 5000 μg per plate (the limit dose for this study). When CAPA 2043 was tested by the direct plate incorporation method, no cytotoxicity was observed. When tested by the pre-incubation method, cytotoxicity to the bacteria was observed as a slight thinning of the background lawn of microcolonies at the highest concentration of 5000 μg per plate. This observation was made in both the absence and the presence of S9 mix in strains TA 1535, TA 1537 and TA 98. CAPA 2043 precipitated at the highest concentration of 5000 μg per plate in all tests. There was no biologically relevant increase in the frequency of revertant colonies of any strain. Appropriate positive controls confirmed the sensitivity of the assay. Under the conditions of this study CAPA was not considered to be mutagenic when tested in the presence and absence of metabolic activation.