Registration Dossier
Registration Dossier
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EC number: 500-099-5 | CAS number: 37625-56-2
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
An Ames test and a study of mammalian cell clastogenicity are available for CAPA 3050. As the result of the mammalian cell clastogenicity study is not clearly negative, a testing proposal is submitted for a mouse bone marrow micronucleus assay.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 6th to 15th January 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- 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
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine or tryptophan loci
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix prepared from the liver of Aroclor 1254-induced adult male Fischer rats
- Test concentrations with justification for top dose:
- Toxicity test: 17, 50, 167, 500, 1667 and 5000 µg/plate.Mutation experiment: 17, 50, 167, 500, 1667 and 5000 µg/plate.
- Vehicle / solvent:
- Initial solubility tests showed that dimethysulphoxide (DMSO) was a suitable solvent, and it was used to dissolve and dilute the test item throughout the study.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene; all strains with S9
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- Migrated to IUCLID6: without S9; TA 1535 and TA 100
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- Migrated to IUCLID6: without S9; TA 1537
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- Remarks:
- Migrated to IUCLID6: without S9; TA 98
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- Migrated to IUCLID6: without S9; WP2uvrB
- Details on test system and experimental conditions:
- Samples of each bacterial strain were grown up by culturing for 16 h at 37°C in nutrient broth (25 g Oxoid Nutrient Broth No. 2 per litre). Fresh overnight cultures were used in the tests for this study. Samples from cultures were kept for up to 7 days at 4°C to allow relevant checks to be performed.Two experiments were conducted in both the absence and presence of S9 mix. The first test was performed using the Direct Plate Incorporation Method. If the first test was negative, the repeat test was performed using the Pre-Incubation Method. If the the first test was positive, the second test was also performed using the Direct Plate Incorporation Method to confirm the response. Triplicate plates were poured for each exposure level (n=6) and bacterial strain (n=5) in the absence or presence of S9. Aseptic techniquies, conducted under amber light, were used throughout the study. All water used in the preparation of reagents wa sproduced (in-house) by reverse osmosis followed by mixed-bed deionisation and sterilisation by autoclaving.Toxicity tests: S. typhimurium strain TA 100 was used for the toxicity tests, according to the direct plate mutation assay (see below). Plates were incubated at 37°C for 2-3 days. The number of revertant colonies were noted, and the paltes examined microscopically for thinning of the background lawn or microcolonies. Mutation testsDiluted agar (0.6% Difco Bacto-agar, 0.6% NaCl) was autoclaved, and just before use supplemented as follows: For S. typhimurium, sterile 1.0 mM L-histidine.HCl, 1.0 mM biotin solution was added at 50 mL per litre of soft agar. For E. coli, sterile 1.35 mM L-tryptophan solution was added at 10 mL per litre of soft agar. These soft agars were thoroughly mixed and kept in a water bath at 45°C.Direct plate method: 2 mL of soft agar were dispensed into a small, plastic, sterile tube. S9 mix or 0.05 M phosphate buffer, pH 7.4 (0.5 mL) was added, followed by 0.1 mL of bacteria and, finally, the test item solution (0.1 mL). The tube contents (which were continually cooling) were mixed, then poured on to minimal medium plates prepared in-house. The minimal medium plates contained 20 mL of 1.5% BBL Purified Agar in Vogel-Bonner Medium E (Vogel and Bonner, 1956) with 2% glucose.Pre-incubation method: For this assay method, 0.5 mL of S9 mix or 0.05 M phosphate buffer, pH 7.4 was dispensed into a small, plastic, sterile tube followed by 0.1 mL of bacteria and, finally, the test solution (0.1 mL). The tubes were then placed in a shaking incubator at ca 37°C for 20 min. After incubation, 2 mL of soft agar was added to each tube. The tube contents were then mixed and poured onto minimal medium plates (preparation described above).When the soft agar had set, the plates were inverted and incubated at ca 37°C for 2 or 3 days and then examined. The numbers of mutant colonies on each plate were determined using a Sorcerer Colony Counter and captured electronically in a validated software system Ames Study Manager (both from Perceptive Instruments). The plates were also examined microscopically for precipitates and for microcolony growth.
- Evaluation criteria:
- Toxicity test: number of revertant colonies and thinning of the background lawn.Mutation tests: 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 was required before mutagenic activity was suspected. For S. typhimurium strain TA 100, a 1.5-fold increase over the control value was considered indicative of a mutagenic effect.If the mean colony count on the vehicle control plates was less than 10, then a value of 10 was assumed for assessment purposes. In such cases, a minimum count of 20 (representing a 2-fold increase over 10) was required before a response was registered. A concentration-related response was also required for identification of a mutagenic effect. At high concentrations this relationship may be reversed because of, for example, 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. No statistical analysis was performed in this study.
- 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 nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- 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 examined
- Positive controls validity:
- valid
- Additional information on results:
- Toxicity test: No toxicity to TA 100 was evident. CAPA 3050 precipitated at 1667 and 5000 μg per plate.Mutation tests: The vehicle control values were within the normal/historical ranges recorded in the test laboratory and reported in the literature with these strains of S. typhimurium and E. coli. The positive control values were within the normal/historical ranges recorded in the test laboratory for each bacterial strain and activation condition. No evidence of mutagenic activity was obtained with CAPA 3050 in any strain in either test.CAPA 3050 was not toxic to any strain in either test. CAPA 3050 precipitated at 1667 and 5000 μg per plate in both tests.
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):negative with and without metabolic activationNo evidence of mutagenicity was seen under the conditions of this study.
- Executive summary:
CAPA 3050 was tested for mutagenic activity in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and in Escherichia coli WP2uvrA. CAPA 3050 was dissolved and diluted in dimethylsulphoxide. Two independent tests were conducted on agar plates in triplicate in the absence and presence of exogenous metabolic activaiton (S9 mix). The first test was conducted by the direct plate incorporation method, while the second test was conducted by the pre-incubation method. CAPA 3050 was dosed at concentrations ranging from 17 to 5000 μg per plate (the predetermined maximum). Concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the S9 mix. No evidence of mutagenic activity was obtained with any strain in either test. CAPA 3050 was not toxic to the bacteria. CAPA 3050 precipitated at concentrations of 1667 and 5000 μg per plate. It was concluded that CAPA 3050 is not mutagenic in strains of Salmonella typhimurium and Escherichia coli when tested in dimethylsulphoxide in the absence and presence of metabolic activation at concentrations up to the predetermined maximum of 5000 μg per plate. The concentration of 5000 μg per plate also exceeded the limit of solubility of CAPA 3050 in the test system
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 26 September 2014
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- Commission Regulation (EC) No 440/2008 of 30 May 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: chromosomal aberration test in mammalian cells
- Specific details on test material used for the study:
- Name: Capa 3050
Chemical name: 2-Oxepanone, polymer with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol
CAS number: 37625-56-2
EC number: 500-099-5
Batch/Lot number: WCB001095
Description: Colourless clear liquid
Molecular weight: 553.6
Purity: 100%
Expiry date: 08 January 2017
Storage conditions: Controlled room temperature - Target gene:
- Not applicable
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- V79 cells were sourced from ECACC and stored frozen at -80 (short term) or in liquid nitrogen (long term). The stock was checked for mycoplasma infection. No infection of mycoplasma was noted. Trypsin-EDTA (0.25% Trypsin, 1mM EDTA) solution was used for cell detachment to subculture (cells were rinsed with 1X PBS before detachment). The laboratory cultures were maintained in 150 cm2 plastic flasks at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5% CO2 in air. The V79 cells for this study were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 1% (v/v) Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and 10% (v/v) heat-inactivated fetal bovine serum (DMEM-10, culture medium). During the treatments, the serum content of the medium was reduced to 5% (v/v) (DMEM-5).
- Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- 2-2.5 hours prior to harvesting, cell cultures were treated with Colchicine (0.2 μg/mL).
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital / beta-naphthoflavone induced liver S9 fraction from male Wistar rats
- Test concentrations with justification for top dose:
- Concentrations of CAPA 3050 selected for the main assay were based on the results of a preliminary study. Two separate assays were performed. In the first assay, cells were treated for 3 hours in the presence and absence of S9 with a 20-hour harvest. In the second assay, cells were treated for 3 hours in the presence of S9 and for 20 hours in the absence of S9x with a 28-hour harvest. Both assays were performed with a range of concentrations to determine cytotoxicity. Visual examination of the final culture medium was conducted at the beginning and end of the treatments. Measurement of pH and osmolality was also performed at the end of the treatment period. At the scheduled harvest, the number of surviving cells was determined using a haemocytometer.
- Vehicle / solvent:
- DMSO
- Untreated negative controls:
- yes
- Remarks:
- untreated
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Untreated negative controls:
- yes
- Remarks:
- untreated
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Details on test system and experimental conditions:
- 1-3 day old cultures (>50% confluency) were used. Cells were seeded into 92 x 17 mm tissue culture dishes (5 x 105 cells/dish) and incubated for 24 hours at 37°C in 10 mL culture medium (DMEM-10). Duplicate cultures were used for each concentration. After the seeding period, medium was replaced with 9.9 mL treatment medium (DMEM-5) for experiments without metabolic activation or with 9.4 mL treatment medium (DMEM-5) + 0.5 mL S9-mix for experiments with metabolic activation. Cells were treated with different concentrations of CAPA 3050, negative (vehicle) or positive control solutions (treatment volume:100 μL/dish) at 37°C in the absence or presence of S9. After the exposure period, the cultures were washed with DMEM-0 medium (Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine and 1% (v/v) Antibiotic-antimycotic solution). Following washing, 10 mL of fresh culture medium was added into the dishes and cells were incubated further until the scheduled harvest.
Cells were harvested after 20 hours (~1.5 normal cell cycles) or 28 hours (~2 normal cell cycles) from the beginning of treatment. The solubility of CAPA 3050 in the final treatment medium was visually examined at the beginning and end of the treatment for each concentration. pH and osmolality were measured at the end of the treatment period. For the concurrent measurement of cytotoxicity, an extra dish was plated for each sample and treated in the same manner. At the scheduled harvest, the number of surviving cells was determined using a haemocytometer. - Rationale for test conditions:
- Test conditions are consistent with those specified in the guideline.
- Evaluation criteria:
- The assay was considered valid, if the following criteria are met:
- The negative (vehicle) control data are within the laboratory’s normal range for the spontaneous aberration frequency.
- The positive controls induce signficant increases in the aberration frequency.
The test item is considered to have shown clastogenic activity in this study if all of the following criteria are met:
- Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations.
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are not associated with large changes in pH or osmolarity of the treated cultures.
The historical control data for this laboratory were also considered in the evaluation. Evidence of a dose-response relationship (if any) was considered to support the conclusion.
The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed. - Statistics:
- For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations (excluding gaps).
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- ASSAY 1
3-hour treatment with and without metabolic activation were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of CAPA 3050 were 225, 200, 175, 150, 125, 100, 50, 25 and 12.5 μg/mL (without metabolic activation); 2000, 1000, 500, 250 and 125 μg/mL (with metabolic activation). Precipitation of the test material was detected at the end of the treatment period at 2000 and 1000 μg/mL with metabolic activation. There were no large changes in pH or osmolality. Marked cytotoxicity was observed at CAPA 3050 concentrations of 225, 200 and 175 μg/mL without metabolic activation (RICC values of 4, 19 and 33%, respectively). No cytotoxicity was observed at any concentration with metabolic activation. CAPA 3050 concentrations of 175, 150, 125 and 100 μg/mL were chosen for evaluation without metabolic activation; concentrations of 2000, 1000 and 500 μg/mL were chosen for evaluation with metabolic activation. None of these concentrations of CAPA 3050 caused a significant increase in the number of cells with structural chromosome aberrations either with or without metabolic activation when compared with the appropriate negative (vehicle) control values. This assay was considered to be clearly negative.
ASSAY 2
3-hour treatment with metabolic activation and 20-hour treatment without metabolic activation were performed. Sampling was performed 28 hours after the beginning of the treatment. The examined concentrations of CAPA 3050 were 225, 200, 175, 150, 125, 100, 50, 25 and 12.5 μg/mL (without metabolic activation); and 2000, 1000, 500, 250 and 125 μg/mL (with metabolic activation). Precipitation of the test matreial was detected at the end of the treatment period at 2000 and 1000 μg/mL (with metabolic activation). There were no large changes in pH or osmolality. Marked cytotoxicity was observed at CAPA 3050 concentrations of 225, 200, 175, 150 and 125 μg/mL without metabolic activation (RICC values of 8, 13, 28, 46 and 58%, respectively). No cytotoxicity at any concentration with metabolic activation. CAPA 3050 concentrations of 150, 125, 100 and 50 μg/mL were evaluated without metabolic activation; concentrations of 2000, 1000, 500, 250 and 125 μg/mL were evaluated with metabolic activation. None of the CAPA 3050 concentrations caused a significant increase in the number of cells with structural chromosome aberrations with metabolic activation when compared with the appropriate negative (vehicle) control values; confirming the negative result of the first main assay. In the absence of metabolic activation, CAPA 3050 at a concentration of 125 μg/mL caused a statistically significant increase in the number of aberrant cells. However, there was no concentration-response (no other increases were observed including at a higher concentration in this assay). Furthermore, the concurrent control was considered to be low, the result was not significant when compared with the negative control of Assay 1 without metabolic activation. The calculated aberration rate was close to the historical control range. Based on these facts, these data do not meet the criteria for a positive response. - Conclusions:
- Capa 3050 did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the assays with metabolic activation. In the absence of metabolic activation, a marginal increase was seen at one intermediate concentration in Assay 2, however the observed effect did not meet the laboraroty's criteria for a positive response. Capa 3050 is therefore not considered to be clastogenic in this test system.
- Executive summary:
Capa 3050 (dissolved in DMSO) was tested in a chromosome aberration assay in Chinese hamster V79 lung cells at up to cytotoxic concentrations or the limit concentration according to the OECD guideline, in the absence and presence of metabolic activation. Two independent assays were performed, using duplicate cultures of at least 300 well-spread metaphases.
In Assay 1, 3-hour treatments with and without metabolic activation were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The concentrations of CAPA 3050 were 225, 200, 175, 150, 125, 100, 50, 25 and 12.5 μg/mL (without metabolic activation); 2000, 1000, 500, 250 and 125 μg/mL (with metabolic activation). Precipitation was detected at CAPA 3050 concentrations of 2000 and 1000 μg/mL (with metabolic activation). There were no large changes in pH or osmolality. Marked cytotoxicity was observed at concentrations of 225, 200 and 175 μg/mL (without metabolic activation). Concentrations of 175, 150, 125 and 100 μg/mL were chosen for evaluation without metabolic activation; concentrations of 2000, 1000 and 500 μg/mL were chosen for evaluation with metabolic activation. None of these treatment concentrations caused a significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared with the appropriate negative (vehicle) control values. This experiment was considered as clearly negative.
In Assay 2, 3 -hour treatment with metabolic activation and 20 -hour treatment without metabolic activation were performed. Sampling was performed 28 hours after the beginning of the treatment in both cases. Examined concentrations of CAPA 3050 were 225, 200, 175, 150, 125, 100, 50, 25 and 12.5 μg/mL (without metabolic activation); 2000, 1000, 500, 250 and 125 μg/mL (with metabolic activation). Precipitation of CAPA 3050 was detected at 2000 and 1000 μg/mL (with metabolic activation). There were no large changes in pH or osmolality. Marked cytotoxicity was observed at CAPA 3050 concentrations of 225, 200, 175, 150 and 125 μg/mL (without metabolic activation). Concentrations of 150, 125, 100 and 50 μg/mL were evaluated without metabolic activation; concentrations of 2000, 1000, 500, 250 and 125 μg/mL were evaluated with metabolic activation. None of the concentrations of CAPA 3050 caused a significant increase in the number of cells with structural chromosome aberrations with metabolic activation, when compared with the appropriate negative (vehicle) control values. This result confirmed the negative result of the first assay. In the absence of metabolic activation, a statistically significant increase in the number of aberrant cells was seen at the intermediate concentration of 125 μg/mL. However, there was no concentration-response (no other increases were observed including at a higher concentration in the same experiment). Furthermore, the concurrent control was low; the result was not significant when compared with the negative control of Assay 1; and the total number of aberrant cells seen at this concentration was within the historical control range.The result does not meet the laboratory's criteria for a positive response.
The negative (vehicle) control data were within the acceptable range for the spontaneous aberration frequency. The positive control substances caused a statistically significant increase in the number of structural aberrations (excluding gaps) in assyas with or without metabolic activation, thereby demonstrating the sensitivity of the test system.
It is concluded that CAPA 3050 does not show evidence of clastogenicity under the conditions of this study.
Referenceopen allclose all
No evidence of mutagenic activity was obtained with CAPA 3050 in any strain in either test. CAPA 3050 was not toxic to any strain in either test. CAPA 3050 precipitated at 1667 and 5000 μg per plate in both tests.
Summary of findings
µg/plate |
Experiment 1 |
|||||||||
TA1535 |
TA1537 |
TA98 |
TA100 |
WP2uvrA |
||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
-control |
15.0 |
12.0 |
15.3 |
12.0 |
27.7 |
30.0 |
95.7 |
102.0 |
4.7 |
12.0 |
17 |
14.3 |
11.7 |
9.3 |
14.3 |
27.0 |
26.3 |
112.7 |
108.7 |
6.7 |
10.7 |
50 |
8.0 |
5.7 |
11.3 |
13.7 |
30.7 |
29.3 |
106.7 |
109.3 |
6.7 |
8.3 |
167 |
12.3 |
11.7 |
10.0 |
14.0 |
16.3 |
34.0 |
93.0 |
104.3 |
9.3 |
10.3 |
500 |
15.7 |
16.7 |
12.7 |
13.0 |
25.0 |
33.7 |
115.7 |
104.0 |
4.7 |
10.3 |
1667 |
13.3 |
11.3 |
11.3 |
12.0 |
26.3 |
36.7 |
109.0 |
89.7 |
5.7 |
11.7 |
5000 |
12.0 |
13.0 |
12.3 |
13.7 |
31.0 |
30.3 |
120.7 |
95.7 |
8.7 |
8.0 |
+control |
508 |
759 |
3135 |
756 |
712 |
1296 |
1095 |
1928 |
72 |
493 |
µg/plate |
Experiment 2 |
|||||||||
TA1535 |
TA1537 |
TA98 |
TA100 |
WP2uvrA |
||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
-control |
11.0 |
12.3 |
11.7 |
10.3 |
25.7 |
36.0 |
86.3 |
96.7 |
6.0 |
8.0 |
17 |
14.7 |
13.3 |
16.3 |
11.7 |
29.0 |
29.7 |
56.0 |
86.7 |
4.3 |
12.3 |
50 |
12.3 |
12.0 |
8.7 |
13.3 |
19.7 |
33.3 |
80.0 |
94.0 |
3.7 |
10.7 |
167 |
11.7 |
15.7 |
15.0 |
14.0 |
17.7 |
27.0 |
59.3 |
84.0 |
2.3 |
10.3 |
500 |
10.7 |
9.3 |
13.3 |
13.0 |
13.0 |
34.3 |
110.3 |
84.7 |
2.0 |
15.0 |
1667 |
11.3 |
11.7 |
12.0 |
13.0 |
20.0 |
33.3 |
110.7 |
104.0 |
3.0 |
14.3 |
5000 |
11.3 |
11.7 |
7.0 |
10.3 |
20.7 |
41.7 |
102.3 |
93.0 |
3.0 |
8.3 |
+control |
141 |
274 |
1050 |
264 |
733 |
815 |
1041 |
938 |
95.7 |
343 |
Summary of results without metabolic activation
|
Assay 1 (3h / 20h) |
Assay 2 (20h / 28h) |
|||
RICC (%) |
CA (%) |
RICC (%) |
CA (%) |
||
Untreated |
97 |
|
113 |
1.0 |
|
DMSO |
100 |
3.7 |
100 |
1.0 |
|
CAPA 3050 |
225 µg/mL |
15 |
|
15 |
|
200 µg/mL |
28 |
|
20 |
|
|
175 µg/mL |
40 |
4.0 |
34 |
|
|
150 µg/mL |
74 |
4.0 |
50 |
2.7 |
|
125 µg/mL |
83 |
5.0 |
61 |
5.8** |
|
100 µg/mL |
91 |
2.3 |
80 |
3.0 |
|
50 µg/mL |
105 |
|
93 |
2.0 |
|
25 µg/mL |
105 |
|
95 |
|
|
12.5 µg/mL |
108 |
|
100 |
|
|
EMS |
0.4 µL/mL |
|
|
64 |
36.4*** |
1 µL/mL |
102 |
23.5*** |
|
|
|
RICC: relative increase in cell count
CA: cells with chromosomal aberrations (excluding gaps)
***significantly different to controls (p<0.001)
Summary of results with metabolic activation
|
Assay 1 (3h / 20h) |
Assay 2 (3h / 28h) |
|||
RICC (%) |
CA (%) |
RICC (%) |
CA (%) |
||
Untreated |
101 |
|
108 |
|
|
DMSO |
100 |
2.7 |
100 |
1.3 |
|
CAPA 3050 |
2000 µg/mL |
93 |
2.3 |
96 |
1.3 |
1000 µg/mL |
84 |
3.0 |
82 |
1.5 |
|
500 µg/mL |
100 |
3.0 |
100 |
1.0 |
|
250 µg/mL |
102 |
|
109 |
1.3 |
|
125 µg/mL |
107 |
|
111 |
0.7 |
|
CPA |
6 µg/mL |
38 |
89.3*** |
51 |
40.3*** |
RICC: relative increase in cell count
CA: cells with chromosomal aberrations (excluding gaps)
**significantly different to controls (p<0.01); ***p<0.001
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
No data are available. Based on the negative Ames test and equivocal result in the mammalian cell clastogenicity study, a mouse bone marrow micronucleus assay is proposed for CAPA 3050.
Endpoint conclusion
- Endpoint conclusion:
- no study available (further information necessary)
Additional information
Bacterial reverse mutation
CAPA 3050 was tested for mutagenic activity in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and in Escherichia coli WP2uvrA (Riach, 2010). CAPA 3050 was dissolved and diluted in dimethylsulphoxide. Two independent tests were conducted on agar plates in triplicate in the absence and presence of exogenous metabolic activation (S9 mix). The first test was conducted by the direct plate incorporation method, while the second test was conducted by the pre-incubation method. CAPA 3050 was dosed at concentrations ranging from 17 to 5000 μg per plate (the predetermined maximum). Concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the S9 mix. No evidence of mutagenic activity was obtained with any strain in either test. CAPA 3050 was not toxic to the bacteria. CAPA 3050 precipitated at concentrations of 1667 and 5000 μg per plate. It was concluded that CAPA 3050 is not mutagenic in strains of Salmonella typhimurium and Escherichia coli when tested in dimethylsulphoxide in the absence and presence of metabolic activation at concentrations up to the pre-determined maximum of 5000 μg per plate. The concentration of 5000 μg per plate also exceeded the limit of solubility of CAPA 3050 in the test system (Riach, 2010).
Mammalian cell clastogenicity
Capa 3050 (dissolved in DMSO) was tested in a chromosome aberration assay in Chinese hamster V79 lung cells at up to cytotoxic concentrations or the limit concentration according to the OECD guideline, in the absence and presence of metabolic activation. Two independent assays were performed, using duplicate cultures of at least 300 well-spread metaphases.
In Assay 1, 3-hour treatments with and without metabolic activation were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The concentrations of CAPA 3050 were 225, 200, 175, 150, 125, 100, 50, 25 and 12.5 μg/mL (without metabolic activation); 2000, 1000, 500, 250 and 125 μg/mL (with metabolic activation). Precipitation was detected at CAPA 3050 concentrations of 2000 and 1000 μg/mL (with metabolic activation). There were no large changes in pH or osmolality. Marked cytotoxicity was observed at concentrations of 225, 200 and 175 μg/mL (without metabolic activation). Concentrations of 175, 150, 125 and 100 μg/mL were chosen for evaluation without metabolic activation; concentrations of 2000, 1000 and 500 μg/mL were chosen for evaluation with metabolic activation. None of these treatment concentrations caused a significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared with the appropriate negative (vehicle) control values. This experiment was considered as clearly negative.
In Assay 2, 3 -hour treatment with metabolic activation and 20 -hour treatment without metabolic activation were performed. Sampling was performed 28 hours after the beginning of the treatment in both cases. Examined concentrations of CAPA 3050 were 225, 200, 175, 150, 125, 100, 50, 25 and 12.5 μg/mL (without metabolic activation); 2000, 1000, 500, 250 and 125 μg/mL (with metabolic activation). Precipitation of CAPA 3050 was detected at 2000 and 1000 μg/mL (with metabolic activation). There were no large changes in pH or osmolality. Marked cytotoxicity was observed at CAPA 3050 concentrations of 225, 200, 175, 150 and 125 μg/mL (without metabolic activation). Concentrations of 150, 125, 100 and 50 μg/mL were evaluated without metabolic activation; concentrations of 2000, 1000, 500, 250 and 125 μg/mL were evaluated with metabolic activation. None of the concentrations of CAPA 3050 caused a significant increase in the number of cells with structural chromosome aberrations with metabolic activation, when compared with the appropriate negative (vehicle) control values. This result confirmed the negative result of the first assay. In the absence of metabolic activation, a statistically significant increase in the number of aberrant cells was seen at the intermediate concentration of 125 μg/mL. However, there was no concentration-response (no other increases were observed including at a higher concentration in the same experiment). Furthermore, the concurrent control was low; the result was not significant when compared with the negative control of Assay 1; and the total number of aberrant cells seen at this concentration was within the historical control range.The result does not meet the laboratory's criteria for a positive response.
The negative (vehicle) control data were within the acceptable range for the spontaneous aberration frequency. The positive control substances caused a statistically significant increase in the number of structural aberrations (excluding gaps) in assays with or without metabolic activation, thereby demonstrating the sensitivity of the test system.
The study report concludes that CAPA 3050 does not show evidence of clastogenicity under the conditions of this study; however due to the response seen in in the absence of metabolic activation in Assay 2, a testing proposal is provided for a mouse bone marrow micronucleus assay.
Justification for classification or non-classification
There is no evidence for the mutagenicity of CAPA 3050; classification is not required under the CLP Regulation.
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