Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Studies of bacterial reverse mutation (Ames test), clastogenicity and mammalian cell mutation are available for the submission substance 2 -oxepanone, polymer with 1,4-butanediol (CAPA 2043).

Link to relevant study records

Referenceopen allclose all

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
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
Specific details on test material used for the study:
CAPA 2043 (2-oxepanone, polymer with 1,4-butanediol)
Target gene:
Histidine and tryptophan
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
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.
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.

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

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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
23rd April 2012 - 2nd October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
CAPA 2043 (2-oxepanone, polymer with 1,4-butanediol)
Target gene:
Not applicable (chromosomal aberration study)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The cell line used was Chinese hamster ovary cells, CHO 10 B4 cells. The cell line is mycoplasma tested (in house) on a regular basis. The cells were grown as monolayers, and have a generation time of approximately 12-14 h. The modal chromosome number has been determined for these cells to be 21. The cells were incubated at 35-38°C.
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Colcemid was added to all cultures at a final concentration of 0.1 μg/mL
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
In Test 1, nine concentrations were tested, the highest being 4000 μg/mL.
In Test 2 in the presence of S9 mix, the following concentrations were used: 125, 250, 500, 750, 1000, 1500, 2000 and 2500 μg/mL
In Test 2 in the absence of S9 mix, the following concentrations were used: 12.5, 25, 50, 100, 150, 200, 250 and 300 μg/mL
Vehicle / solvent:
Dimethylsulphoxide.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
concentration range of 20-50 μg/mL
Positive control substance:
cyclophosphamide
Remarks:
Used with S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
concentration range of 10-40 μg/mL
Positive control substance:
methylmethanesulfonate
Remarks:
Used without S9 mix.
Details on test system and experimental conditions:
Cells were trypsinised from stock flasks at passage numbers 11 (Test 1) and 18 (Test 2), and resuspended in fresh culture medium at densities of 0.1 x 10E6 or 0.05 x 10E6 cells/mL. The cell suspensions (5 mL) were dispensed into 25 cm2 tissue culture flasks. The high and low cell densities were for cultures harvested at 24 or 48 h post treatment, respectively. Test cultures were established from the stock flask 20-24 h before testing. All tests were conducted both in the presence and in the absence of S9 mix. Treatment with test item or vehicle control substances were performed on duplicate cell cultures. The positive controls were tested using single cultures. Cultures to be treated in the presence of S9 mix were washed with serum-free medium before treatment. Immediately before dosing, exposure medium was prepared in sterile containers. Cultures, established 20-24 hours before testing, were treated for 6 hours in the presence and 6 hours or 22 hours in the absence of S9 mix. Cultures were harvested at 24 hours (Test 1 and 2) or 48 hours (Test 2) post treatment. Colcemid was added to all cultures at a final concentration of 0.1 μg/mL. Culturing the cells in medium containing colcemid for 2 h accumulated cells in metaphase (the stage of cell division at which chromosomes can be examined using light microscopy). Mitotic cells were harvested by gently tapping flasks to release these cells from the monolayer. Cells were sedimented by centrifugation and treated with hypotonic solution (trisodium citrate, 1%, w/v), for 15 min at room temperature. The cells were then fixed (after sedimentation as before) using 4 mL of freshly prepared fixative (methanol:acetic acid, 3:1, v/v). After sedimentation two further fixative changes were conducted. Monolayer cells were trypsinised, counted and discarded. This provided a quantitative measure of toxicity. For both tests, 3 slides per culture were prepared. All slides were marked with the study number and assigned a unique coded number from a computer-generated sequence. Slides were prepared by dropping the cell suspension onto clean grease-free slides. The slides were allowed to air dry overnight and were then stained with Giemsa (5%, v/v), then made permanent by mounting coverslips with DPX mountant.

NUMBER OF REPLICATIONS: Testing was conducted in duplicate

NUMBER OF CELLS EVALUATED: 100
Evaluation criteria:
Interpretation of Toxicity:
A concentration was considered to be toxic if the cell count was reduced to less than 50% of the mean vehicle control culture values or if consistent evidence of changes to cell morphology was observed.

Interpretation of Clastogenicity:
The results for test item and positive control-treated cultures were evaluated by comparison with the concurrent vehicle control cultures and with historical negative control data. A negative response was recorded if responses from the test item treated cultures were within the 95% confidence levels for the historical negative control data. The response at a single dose was classified as significant if the percentage of aberrant cells was consistently greater than the 99% confidence levels for the historical negative control data.
A test was positive if the response in at least one acceptable dose level was significant by the criteria described above. A test item was positive if Test 1 was positive, as described above or if one of the tests was positive and the other test gave indications of activity. These indications may be suspicious levels of aberrant cells (between 95% and 99% confidence levels). Experiments that met in part the criteria for a positive response, or marginally met all the criteria, were classed as inconclusive.
Statistics:
No statistical analysis was performed.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
For the toxicity testing, in Test 1, in the presence of S9 mix, reduced cell counts (below 50% of the vehicle control cultures) were noted in the cultures treated with 2000 and 4000 μg/mL, with no metaphase cells present for assessment in the cultures treated with 4000 μg/mL. In the absence of S9 mix (6 h treatment), toxicity was noted at 250-4000 μg/mL, with no metaphase cells present for assessment in the cultures treated with 1000-4000 μg/mL.
In Test 2, presence of S9 mix, reduced cell counts were noted in the cultures treated with 1500-2500 μg/mL. In the absence of S9 mix (22 h treatement, 24 h harvest), toxicity was noted in the cultures treated with 200-300 μg/mL, with reduced cell counts in both cultures treated with 300 μg/mL and one culture treated with 250 μg/mL. The duplicate culture treated with 250 μg/mL and the cultures treated with 200 μg/mL were classed as toxic from culture observations. In the cultures harvested at 48 h, toxicity was noted in the cultures treated with 250 and 300 μg/mL, with reduced cell counts in the cultures treated with 300 μg/mL.

Chromosomal Aberrations Results:
Cultures treated with the following dose levels of Capa 2043 were selected for assessment of chromosomal aberrations:
Test 1, Presence of S9 Mix: 500, 1000 and 2000 μg/mL
Test 1, Absence of S9 Mix: 62.5, 125 and 250 μg/mL
Test 2, Presence of S9 Mix: 1000, 1500 and 2000 μg/mL
Test 2, Absence of S9 Mix, 24 h Harvest: 150, 250 and 300 μg/mL
Test 2, Absence of S9 Mix, 48 h Harvest: 200, 250 and 300 μg/mL

All cultures treated with Capa 2043 had levels of structural aberrations within the 95% confidence levels for a negative response. An extra assessment of polyploidy was carried out on the cultures treated in the absence of S9 mix and harvested at 48 h. All the cultures treated with Capa 2043 had levels of polyploidy within the 95% confidence levels for a negative response.

Summary of results

Treatment

Frequency of aberrations (excluding gaps)

Test 1

Test 2

6/24h

22/24h

6/24h

22/48h

-S9

+S9

-S9

+S9

-S9

DMSO 1%

0.0%

0.0%

0.0%

0.5%

0.0%

62.5 µg/mL

0.0%

-

-

-

-

125 µg/mL

0.0%

-

-

-

-

150 µg/mL

-

-

0.0%

-

-

200 µg/mL

-

-

-

-

0.0%

250 µg/mL

0.0%

-

0.0%

-

0.0%

300 µg/mL

-

-

0.0%

-

0.0%

500 µg/mL

-

0.0%

-

-

-

1000 µg/mL

-

0.0%

-

0.0%

-

1500 µg/mL

-

-

-

0.0%

-

2000 µg/mL

-

0.0%

-

1.0%

-

MMS 20 µg/mL

2%

-

-

-

0%

MMS 30 µg/mL

14%

-

8%

-

9%

MMS 40 µg/mL

-

-

20%

-

-

CPA 40 µg/mL

-

3%

-

0%

-

CPA 50 µg/mL

-

8%

-

11%

-

Conclusions:
Under the conditions of this study, Capa 2043 was not clastogenic when tested in Chinese hamster ovary cells.
Executive summary:

An in vitro mammalian chromosome aberration assay was conducted with CAPA 2043 (2 -oxepanone, polymer with 1,4 -butanediol) using Chinese hamster ovary (CHO) cells in acordance with OECD Guideline 473 and EU Method B10 and in compliance with GLP. Two independent tests were conducted in duplicate with appropriate negative and positive controls. The vehicle control was DMSO and the positive controls were cyclophosphamide and methyl methanesulphonate. Both tests were conducted in the presence and absence of exogenous metabolic activation (S9 mix). Cultures, established 20 -24 hours before testing, were treated for 6 hours in the presence of S9 and for 6 or 22 hours in the absence of S9. Cultures were harvested at 24 hours (Test 1 and 2) or 48 hours (Test 2) post treatment. CAPA 2043 was toxic to CHO cells in both the presence and absence of S9 mix and was tested up to the limit concentration of 4000 μg/mL (equivalent to 10 mM) in Test 1. Toxicity was noted at 2000 and 4000 μg/mL in the presence of S9 mix and at 250 -4000 μg/mL in the absence of S9 mix. In Test 2, Capa 2043 was tested up to 2500 μg/mL in the presence of S9 mix and to 300 μg/mL in the absence of S9 mix. In the presence of S9, toxicity was noted in cultures treated with 1500 -2500 μg/mL and in the absence of S9 mix toxicity was noted in the cultures treated with 200-300 μg/mL (24 -hour harvest) and 250 and 300 μg/mL (48 -hour harvest). There was no evidence that CAPA 2043 induced structural chromosomal aberrations in either the presence or absence of S9 mix. Capa 2043 did not induced polyploidy in the absence of S9 mix in cultures harvested 48 hours post treatment. Capa 2043 was not clastogenic in cultured CHO cells under the conditions of this study.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
3rd July 2012 to 30th October 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
CAPA 2043 (2-oxepanone, polymer with 1,4-butanediol)
Target gene:
Thymidine kinase (tk)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The cells grown in suspension culture, have a generation time of about 11 h, have a stable, near-diploid chromosome number and have a high cloning
efficiency in serum-enriched cloning medium.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Initial toxicity testing: 0.4, 1.2, 4, 12, 40, 120, 400, 1200 and 4000 μg/mL

Mutagenicity testing:
Assay 1 (4 hour exposure in the absence of S9 mix): 40, 80, 120, 160, 200, 240, 280 and 320 μg/mL
Assay 2 (4 hour exposure in the presence of S9 mix): 250,500, 750, 1000, 1250, 1750, 2500 and 3500 μg/mL
Assay 3 (24 hour exposure in the absence of S9 mix): 120, 160, 200, 240, 280, 320, 360, 400 and 440 μg/mL
Assay 4 (4 hour exposure in the presence of S9 mix): 750, 1000, 1250, 1500, 1750, 2000 and 2250 μg/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
250 μg/mL
Positive control substance:
ethylmethanesulphonate
Remarks:
-S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
10 μg/mL
Positive control substance:
methylmethanesulfonate
Remarks:
+S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
2.5 and 10 μg/mL
Positive control substance:
3-methylcholanthrene
Remarks:
+S9
Details on test system and experimental conditions:
METHOD OF APPLICATION:
In the mutation tests with 4 hours exposure, on the day of the test (Day 0), samples of cell culture (in 5 mL R10P) were dispensed to sterile tubes containing R0P (3.9 mL). Freshly prepared S9 mix or R0P (1 mL) was added to each tube followed by the test formulation (0.1 mL). Vehicle control cultures received DMSO (0.1 mL). Positive control cultures received the appropriate solution (0.1 mL). The final reaction mixture in all cultures contained 10 mL of cells, at a population density estimated at 6.0 x 10E5 cells/mL, in R5P medium. All tubes were placed on a 10 r.p.m. rotating drum, inside an incubator set to maintain a temperature of 37°C, for 4 h. After this, the cells were gently sedimented by centrifugation at 200 g for 5 min and resuspended in R10P medium (20 mL). This step was repeated to give a cell density estimated at 3 x 10E5/mL. The cells were returned to the rotating drum and allowed to express their genetic lesions for 2 days. Cell numbers were adjusted, after counting, to an estimated 3 x 10E5 cells/mL on Day 1. In the extended treatment peiod with 24 hour exposure, samples of cell culture (in 10 mL R10P) were dispensed to sterile tubes containing R0P (7.8 mL) on day 0. R50P (R0P:serum, 50:50, v/v) (2 mL) was added to each tube followed by the test formulation (0.2 mL). Vehicle control cultures received DMSO (0.2 mL). Positive control cultures received the appropriate solution (0.2 mL). The final reaction mixture in all cultures contained 20 mL of cells, at a population density estimated at 3 x 10E5 cells/mL, in R10P medium.
All tubes were incubated on the rotating drum (as described above) for 24 h. After this (on Day 1), the cells were gently sedimented by centrifugation at 200 g for 5 min and were then resuspended in R10P medium (20 mL). This step was repeated. Cell counts were made and the densities adjusted (where higher) to give an estimated 3 x 10E5 cells/mL. The cells were returned to the rotating drum and allowed to express their genetic lesions for 2 days. Cell numbers were adjusted, after counting, to an estimated 3 x 10E5 cells/mL on Day 2. On Day 2 (4 h exposure) or Day 3 (24 h exposure), cell counts were determined. The cell counts over the 2 or 3 days of the experiments provided a measure of suspension growth. This in turn provided a measure of RSG. This was used when choosing dose levels to carry through to final assessment, as no other measures of toxicity were known at the time the decision was required. The treated cultures from the 5 highest concentrations of Capa 2043 giving satisfactory cell survival were selected for final assessment in the assays in the absence of S9 mix. In the presence of S9 mix, the reaction of Capa 2043 and S9 mix lowered the pH of the medium. In both assays in the presence of S9 mix, the 4 highest concentrations giving a pH change of ≤1 were selected for assessment. The cultures were then assessed for expression of genetic damage. This was determined by performing parallel cloning assays for cloning efficiency and mutant selection. For the cloning efficiency assay, each culture was diluted into cloning medium to give an estimated 8 cells/mL. Two 96-well dishes were filled with 200 μL cell culture per well, so giving an estimated 1.6 cells per well. For the mutant selection assay, TFT stock solution was added to cloning medium to give a final concentration of 3 μg/mL. Into this medium, the cell cultures were diluted to give an estimated 1 x 10E4 cells/mL. Two 96-well dishes were filled with 200 μL cell culture per well, so giving an estimated 2000 cells per well. All dishes were placed in an incubator set to maintain a humid atmosphere of 5% CO2:95% air (v/v) at 37°C until the colonies were fully developed (at least 9 days for cloning efficiency assay, at least 12 days for mutant selection assay). The plates were scored using a dissecting microscope fitted to a light box with dark field illumination. The number of empty wells in each plate in the cloning efficiency assay was counted. When scoring the mutant selection assay, separate counts were made of the numbers of wells containing large type and small type colonies. Large colonies are defined as covering greater than ¼ of the floor of the well, while small colonies cover less than ¼ of the well

NUMBER OF REPLICATIONS
In the mutagenicity testing, duplicate cultures were treated. Vehicles controls were conducted in quadruplicate.

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth; relative total growth; mutant fractions and colony size fractions
Evaluation criteria:
Toxicity: Mutagenic responses that occur at RTG values below 10%, while being statistically significant, are recognised as having questionable biological significance. If the test item was toxic, results should have been obtained from concentrations resulting in RTG values down to 20%, if a conclusion of nonmutagenic was to be reached.

Mutagenicity - Positive result:
An experiment was positive if one or more concentrations showed a biologically significant increase in mutant fraction and there was significant linear trend. An experiment may also be positive in the absence of a linear trend if there was mitigating evidence. Additional comparisons that can aid interpretation of results includ comparison of the induced mutant fraction with the historical maximum for difference between vehicle control; comparison of the mutant fraction of a treated group with the historical range of vehicle control values. A test item was positive if 2 positive experiments out of 2 were recorded within the same activation condition. Test items that gave a negative response in the standard exposure in the absence of S9 mix, but gave a positive response in the extended exposure, were liable to a confirmatory experiment with the extended exposure.

Mutagenicity - Negative result:
A test item was defined as non-mutagenic, provided data were obtained in both the absence and the presence of S9 mix that accompanied one or more of the following:
• the predetermined maximum concentration of 5000 μg/mL or 10 mM, whichever is lower
• the highest practicable concentration limited by the solubility or pH of the test item
• RTG in the range 10-20%.
Circumstances when a chemical may be determined to be non-mutagenic when there was no treatment showing an RTG value between 10 and 20% occur when there is no evidence of mutagenic activity in a series of data points within 100% to 20% RTG and there was at least one data point between 20 and 25% RTG or there was a data point between 10 and 1%
Statistics:
The results for each experiment were subjected to a test for linear trend in mutant fraction with concentration of Capa 2043 by the recommended UKEMS method.
Key result
Species / strain:
mouse lymphoma L5178Y cells
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
For Assays 1 and 2, no evidence of mutagenic activity was detected. All mean IMF values were well below the minimum of 126 mutants per million, required to indicate a relevant increase. As this indicated Capa 2043 was not mutagenic, the second experiment in the absence of S9 mix was conducted with the 24hour exposure. In this assay, no evidence of mutagenic activity was detected. All mean IMF values were well below the minimum of 126 mutants per million, required to indicate a relevant increase, and the test for linear trend in mutant fraction with concentration of Capa 2043 was not reported, as the slope was negative.

All the assessed concentrations of Capa 2043 initially precipitated on dosing, apparently redissolving during the exposure period. It is therefore likely that the high concentration, while being limited by pH change, is close to the limit of Capa 2043’s solubility in the test system. The test substance was considered to be non-mutagenic in the assay.

For Assays 3 and 4 (absence and presence of S9 mix for 24 and 4 hours respectively), no evidence of mutagenic activity was detected in the absence of S9 mix. All mean IMF values were well below the minimum of 126 mutants per million, required to indicate a relevant increase. The test for linear trend in mutant fraction with concentration of Capa 2043 (240-360 μg/mL) was not significant (P=0.67).Based on these findings, the test substance was considered to be non-mutagenic.

In the presence of S9 mix, no evidence of mutagenic activity was detected. All mean IMF values were well below the minimum of 126 mutants per million, required to indicate a relevant increase, and the test for linear trend in mutant fraction with concentration of Capa 2043 was not significant (P=0.62). All the assessed concentrations of Capa 2043 initially precipitated on dosing, apparently redissolving during the exposure period. It is therefore likely that the high concentration, while being limited by pH change, is close to the limit of Capa 2043’s solubility in the test system. The test substance was conisdered to be non-mutagenic in the assay.

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity study was performed, the results of which showed that Capa 2043 was moderately toxic to the cells in the absence of S9 mix. A concentration of 120 μg/mL did not cause a substantial reduction in cell growth in either the 4 h or the 24 h exposure systems, while all concentrations of 400 μg/mL and higher caused complete killing. In the presence of S9 mix, toxicity was lower, with no substantial reduction in cell growth at a concentration of 1200 μg/mL. The highest concentration of 4000 μg/mL caused complete killing. A reduction in pH was noted at the end of the exposure period in the 2 highest concentrations. In the absence of S9 mix, Capa 2043 precipitated at the 2 highest concentrations of 1200 and 4000 μg/mL. In the presence of S9 mix, precipitation was observed on dosing at 1200 and 4000 μg/mL. At the completion of the 4 h exposure period, precipitation was visible only at 4000 μg/mL.



Mutation Test in the Absence of S9 Mix (4 hour Exposure): Assay 1

Chemical

Concentration (μg/mL)

Relative Total Growth %

Mutant Fraction (x 10 -6)

IMF (Induced

Mutant

Fraction x 10-6)

Ratio of Small to

Large Colonies

DMSO

(100μL added)

100

78

N/A

1.04

EMS

250

73

643

565

0.45

MMS

10

49

889

811

1.67

Capa 2043

40

80

120

160

200

240

280

320

NPS

122

104

81

59

18

NPT

NPT

NPS

63

77

57

93

122

NPT

NPT

NPS

-

-

-

16

45

NPT

NPT

NPS

1.10

2.75

2.63

1.35

1.75

NPT

NPT

NPT = Not plated – Toxic

NPS = Not plated – surplus.

Mutation Test in the Presence of S9 Mix (4 hour Exposure): Assay 2

Chemical

Concentration (μg/mL)

pH after 4 hours

Relative Total Growth %

Mutant Fraction (x 10-6)

IMF (Induced

Mutant

Fraction x 10-6)

Ratio of Small to

Large Colonies

DMSO

(100μL added)

7.78

100

74

N/A

1.69

3-MC

2.5

-

62

805

731

0.82

10

-

42

974

900

1.29

Capa 2043

250

500

750

1000

1250

1750

2500

3000

-

-

-

-

7.12

6.91

6.41

5.74

NPS

NPS

95

83

83

69

NP-pH

NPT

NPS

NPS

81

86

89

65

NP-pH

NPT

NPS

NPS

7

12

15

-

NP-pH

NPT

NPS

NPS

1.40

0.71

0.82

1.00

NP-pH

NPT

NPT = Not plated – Toxic

NPS = Not plated – surplus.

NP-pH = Not plated – excessively low pH

Mutation Test in the Absence of S9 Mix (24 hour Exposure): Assay 3

Chemical

Concentration (μg/mL)

Relative Total Growth %

Mutant Fraction (x 10-6)

IMF (Induced

Mutant

Fraction x 10-6)

Ratio of Small to

Large Colonies

DMSO

(200μL added)

100

69

N/A

0.45

EMS

100

64

894

826

0.33

MMS

5

33

1297

1228

1.58

Capa 2043

120

160

200

240

280

320

360

400

440

NPS

NPS

NPS

72

63

44

27

9

NPT

NPS

NPS

NPS

61

60

79

92

94

NPT

NPS

NPS

NPS

-

-

10

23

25

NPT

NPS

NPS

NPS

0.90

0.76

0.83

0.48

0.88

NPT

NPT = Not plated – Toxic

NPS = Not plated – surplus.

Mutation Test in the Presence of S9 Mix (4 hour Exposure): Assay 4

Chemical

Concentration (μg/mL)

pH after 4 hours

Relative Total Growth %

Mutant Fraction (x 10-6)

IMF (Induced

Mutant

Fraction x 10-6)

Ratio of Small to

Large Colonies

DMSO

(100μL added)

7.53

100

81

N/A

1.08

3-MC

2.5

-

61

701

619

1.15

10

-

43

1041

960

1.04

Capa 2043

750

1000

1250

1500

1750

2000

2250

7.11

6.99

6.84

6.78

6.62

6.55

6.42

NPS

NPS

68

64

71

53

NP-pH

 

NPS

NPS

109

80

99

82

NP-pH

 

-

-

27

-

17

1

NP-pH

 

-

-

1.44

1.18

0.72

0.69

NP-pH

NPS = Not plated – surplus.

NP-pH = Not plated – excessively low pH

Conclusions:
Under the conditions of this study, Capa 2043 was not mutagenic in mouse lymphoma L5178Y cells, in either the absence or the presence of S9 mix.
Executive summary:

In a study compliant with GLP and conducted in accordance with OECD guideline 476 and EC Directive 2000/32/EC B.17, CAPA 2043 was assayed for mutagenic potential in the mouse lymphoma L5178Y cell line. Tests were conducted in the presence and absence of exogenous metabolic activation (S9 mix). A preliminary toxicity study was conducted which indicated that CAPA 2043 was of moderate toxicity in the absence of S9 mix.CAPA 2043 exhibited less toxicity in the presence of S9 mix, however the pH of the treatment medium was lowered in the presence of S9 mix to the extent that change in pH became a limiting factor. In the mutagenicity assay, four independent assays were performed. Assays were conducted in the presence or absence of S9 mix, with an exposure period of either 4 or 24 hours and concentrations ranging from 80 to 2000μg/mL. Duplicate cultures were carried through the experiments for each treatment point. Vehicle control cultures were also included and were tested in quadruplicate. No evidence of mutagenic activity was obtained in any experiment. In the absence of S9 mix (4 h and 24 h exposure period), results were obtained from concentrations extending into the toxic range. In the presence of S9 mix, results were obtained at the permissible limit of pH change (≤1). The concentrations tested in the presence of S9 mix were close to the limit of solubility of CAPA 2043 in the test system. In conclusion, under the conditions of this study, CAPA 2043 was not mutagenic in mouse lymphoma L5178Y cells, in either the absence or the presence of S9 mix.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a GLP/OECD Test Guideline 471 study to determine the mutagenic potential of 2-Oxepanone, polymer with 1,4-butanediol (CAPA 2043) in bacterial cells in vitro (Riach, 2012), negative results were obtained in Salmonella typhimurium strains TA 100, TA 98, TA 1537 and TA 1535 and Escherichia coli strain WP2 uvrA, in the presence and in the absence of metabolic activation (S9 mix). The substance did not induce structural chromosomal aberrations in either the presence or absence of metabolic activation (S9 mix) and did not induce polyploidy in the absence of S9 mix in cultures harvested 48 hours post treatment.

The potential for 2 -oxepanone, polymer with 1,4-butanediol (CAPA 2043) to cause clastogenicity in Chinese Hamster Ovary cells was investigated in an in vitro chromosome aberration assay conducted according to OECD Test Guideline 473 (Murie, 2012). The substance did not induce structural chromosomal aberrations in either the presence or absence of metabolic activation (S9 mix) and did not induce polyploidy in the absence of S9 mix in cultures harvested 48 hours post treatment. Based on the study, 2 -oxepanone, polymer with 1,4-butanediol was not clastogenic in Chinese hamster ovary cells in vitro.

The potential for 2 -oxepanone, polymer with 1,4-butanediol (CAPA 2043) to induce gene mutations in mouse lymphoma L5187Y cells in vitro was investigated in a gene mutation study conducted according to OECD Test Guideline 476 (Riach, 2012). In the study, four independent assays were performed in the presence or absence of metabolic activation (S9 mix) with exposure periods of 4 or 24 hours and concentrations ranging from 80 to 2000 μg/mL. The test substance CAPA 2043 was not mutagenic in mouse lymphoma cells, in either the absence or the presence of metabolic activation (S9 mix) in any of the assays performed. Based these findings, 2-Oxepanone, polymer with 1,4-butanediol is not mutagenic in mammalian cells in vitro.

Justification for classification or non-classification

2-Oxepanone, polymer with 1,4-butanediol is not mutagenic in a suitable battery of studies in bacterial and mammalian cells in vitro. The substance does not meet the criteria for classification for germ cell mutagencity according to the CLP Regulation.