Registration Dossier
Registration Dossier
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EC number: 204-075-2 | CAS number: 115-25-3
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Nov 2000 - Feb 2001
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 001
- Report date:
- 2001
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- S. typhimurium tester strain TA97a using ICR 191 as positive control was used as a substitute for strains TA97 and/or TA1537.
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Octafluorocyclobutane
- EC Number:
- 204-075-2
- EC Name:
- Octafluorocyclobutane
- Cas Number:
- 115-25-3
- Molecular formula:
- C4F8
- IUPAC Name:
- octafluorocyclobutane
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source: E. I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A.
- Purity: 99.99921%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: As supplied by the sponsor, a compressed gas in a low-pressure gas cylinder. The cylinder was stored at ambient conditions.
- Stability under storage conditions: stable; no evidence of instability was observed.
- Stability under test conditions: stable under the conditions of the study; no evidence of instability was observed.
- Solubility and stability of the test substance in the vehicle: stable; no evidence of instability was observed.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Dilution of H-24619 (test material) in filtered, house-line air in order to generate chamber atmosphere. The test substance was metered into the air stream with a Brooks model 1355 Sho-Rate Rotameter® and the resulting mixture (~6 L/min total flow rate) was carried into the exposure chamber.
The test gas was generated from the vapor headspace of the cylinder. The cylinder was stored and operated at ambient conditions (Trial 1 and Assay 2 of Trial 2) or was heated with a Variac® -controlled heat tape to ~32ºC (Trial 2, assay 1). Heat was applied to the cylinder to maintain an adequate vapor head pressure to accommodate the 6-L/min flow volume of H-24619 (test material) required for the 100% target exposure level.
OTHER SPECIFICS
- measurement: Chamber concentrations were determined nominally using calibrated rotameters and confirmed employing gas chromatographic techniques.
- other information: Pre- and post-treatment period analysis of the test substance dilutions indicated the test substance was stable under the conditions of the study and during the treatment period.
Method
- Target gene:
- hisD6610 (for S.typhimurium TA97a)
hisD3052 (for S.typhimurium TA98)
hisG46 (for S.typhimurium TA100)
hisG46 (for S.typhimurium TA1535)
trpE (for E.coli WP2)
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium, other:
- Remarks:
- Salmonella typhimurium TA97a, TA98, TA100, TA1535
- Additional strain / cell type characteristics:
- other: S.typhimurium TA97a, TA98, TA100: deletion of uvrB; rfa; pKM101 S.typhimurium TA1535: deletion of uvrB; rfa
- Remarks:
- S. typhimurium tester strains were obtained from Dr. Bruce Ames, Berkeley, CA, USA.
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- other: E.coli WP2: deletion of uvrA; pKM101
- Remarks:
- E. coli WP2 uvrA(pKM101) was obtained from the National Collection of Industrial Bacteria, Torrey Research Station, Scotland, UK.
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9 :
The Aroclor®-induced rat liver S9 was purchased from MOLTOX™.
- method of preparation of S9 mix :
The exogenous metabolic activation system was a cofactor-supplemented post-mitochondrial fraction, (i.e., 9000 x g; homogenate of 1 g wet liver weight in 3 mL of an approximately 0.15 M KCl solution) prepared from the livers of young male Sprague Dawley rats treated with the enzyme-inducing agent Aroclor® 1254 (500 mg/kg intraperitoneal) as a single dose 5 days prior to sacrifice. The Aroclor®-induced rat liver S9 (purchased from MOLTOX™) was characterized for protein content and metabolic activity by the vendor
- concentration or volume of S9 mix and S9 in the final culture medium:
The amount of Aroclor®-1254 induced rat liver S9 in the exogenous metabolic activation system was 4.0 mg S9 protein (~10% [v/v])/ mL. The cofactor-supplement concentrations in the exogenous metabolic activation system were 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate (as a sodium salt), 4 mM NADP+ (as a sodium salt), and 100 mM sodium phosphate buffer pH 7.4.
Treatments with activation were conducted by adding 0.5 mL of S9 mix to 2 mL of top agar and 0.1 mL of a bacteria culture. These components were briefly mixed and poured onto a minimal glucose agar plate (25-30 mL).
- quality control of S9:
To confirm the sterility of the exogenous metabolic activation system, an aliquot was plated on nutrient agar capable of supporting the growth of viable bacteria. - Test concentrations with justification for top dose:
- Target concentrations: 0, 20, 40, 60, 80 and 100%
Trial 1:
- Without metabolic activation: 0, 25.6, 38.4, 57.2, 74.5, 89.0 %
- With metabolic activation: 0, 26.0, 40.6, 57.9, 68.4, 90.5 %
Trial 2:
- Without metabolic activation: 0, 26.7, 41.0, 54.5, 68.6, 90.0 %
- With metabolic activation: 0, 14.5, 42.0, 28.9, 67.5, 85.2 % - Vehicle / solvent:
- - Vehicle used: Filtered house-line air was the test substance diluent and negative control.
- Justification for choice of vehicle: The substance is a gaz
Controls
- Untreated negative controls:
- yes
- Remarks:
- Filtered house-line air was the test substance diluent and negative control.
- Positive controls:
- yes
- Positive control substance:
- 9,10-dimethylbenzanthracene
- 2-nitrofluorene
- sodium azide
- N-ethyl-N-nitro-N-nitrosoguanidine
- other: Acridine mutagen (ICR 191) and 2-aminoanthracene
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 6 different strains with an without metabolic activation for each concentration. Triplicates (3 plates per concentration, strain and ±S9).
- Number of independent experiments : 2 trials, with 6 different concentrations with an without metabolic activation and 6 different strains for each trial.
METHOD OF TREATMENT:
Treatments with activation were conducted by adding 0.1 mL of positive control (to positive control plates only), 0.5 mL of S9 mix, and 0.1 mL (containing approximately 1 x 108 bacteria) of an overnight culture to 2 mL of top agar [0.6% agar (w/v) and 0.6% NaCl (w/v)] supplemented with 0.05 mM L-histidine and 0.05 mM D-biotin for S. typhimurium strains or 0.05 mM L-tryptophan for the E. coli strain. These components were briefly mixed and poured onto a minimal glucose agar plate (25-30 mL, 0.2% [w/v] glucose with Davis salts, purchased from MOLTOX™).
Treatments in the absence of the metabolic activation system were identical to those in the presence of the exogenous metabolic activation system with the exception that 0.5 mL of sterile buffer (PBS) was used as a replacement for the 0.5 mL volume of the exogenous metabolic activation system. - Statistics:
- Data for each tester strain were evaluated independently. For each tester strain, the mean number of revertants and the standard deviation at each concentration in the presence of and absence of the exogenous metabolic activation system were calculated.
Results and discussion
Test results
- Key result
- Species / strain:
- other: Salmonella typhimurium TA97a, TA98, TA100, TA1535 and Escherichia coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
Any other information on results incl. tables
TA97a - Trial 1
A. WITHOUT METABOLIC ACTIVATION |
||
Mean Concentration (%)(±S.D.) |
Mean of revertants (±S.D.) |
Observations |
0.0 |
106 (6) |
T0,P0 |
25.6 (1.6) |
97 (3) |
T0,P0 |
38.4 (2.2) |
91 (3) |
T0,P0 |
57.2 (0.8) |
76 (3) |
T0,P0 |
74.5 (3.4) |
72 (6) |
T0,P0 |
89.0 (2.0) |
34 (2) |
T0,P0 |
ICR 191 -2μg/plate |
933 (42) |
N |
B. WITH METABOLIC ACTIVATION |
||
Mean Concentration (%)(±S.D.) |
Mean of revertants (±S.D.) |
Observations |
0.0 |
148 (12) |
T0,P0 |
26.0 (1.6) |
153 (19) |
T0,P0 |
40.6 (2.5) |
128 (11) |
T0,P0 |
57.9 (1.6) |
129 (10) |
T0,P0 |
68.4 (1.8) |
72 (8) |
T0,P0 |
90.5 (4.0) |
25 (5) |
T0,P0 |
DMBA -20μg/plate |
1069 (78) |
N |
TA97a – Trial 2
A. WITHOUT METABOLIC ACTIVATION |
||
Mean Concentration (%)(±S.D.) |
Mean of revertants (±S.D.) |
Observations |
0.0 |
135 (13) |
T0,P0 |
26.7 (0.8) |
128 (9) |
T0,P0 |
41.0 (2.0) |
125 (11) |
T0,P0 |
54.5 (1.1) |
98 (3) |
T0,P0 |
68.6 (1.8) |
115 (26) |
T0,P0 |
90.0 (5.4) |
33 (4) |
T0,P0 |
ICR 191 -2μg/plate |
830 (42) |
N |
B. WITH METABOLIC ACTIVATION |
||
Mean Concentration (%)(±S.D.) |
Mean of revertants (±S.D.) |
Observations |
0.0 |
113 (11) |
T0,P0 |
14.5 (16.2) |
R |
- |
42.0 (0.9) |
142 (28) |
T0,P0 |
28.9 (29.4) |
R |
- |
67.5 (2.4) |
59 (22) |
T0,P0 |
85.2 (1.8) |
30 (3) |
T0,P0 |
DMBA -20μg/plate |
677 (14) |
N |
TA98 - Trial 1
A. WITHOUT METABOLIC ACTIVATION |
||
Mean Concentration (%)(±S.D.) |
Mean of revertants (±S.D.) |
Observations |
0.0 |
22 (4) |
T0,P0 |
25.6 (1.6) |
26 (2) |
T0,P0 |
38.4 (2.2) |
20 (5) |
T0,P0 |
57.2 (0.8) |
16 (1) |
T0,P0 |
74.5 (3.4) |
5 (3) |
T0,P0 |
89.0 (2.0) |
1 (1) |
T0,P0 |
2NF -25μg/plate |
1662 (77) |
N |
B. WITH METABOLIC ACTIVATION |
||
Mean Concentration (%)(±S.D.) |
Mean of revertants (±S.D.) |
Observations |
0.0 |
23 (7) |
T0,P0 |
26.0 (1.6) |
23 (1) |
T0,P0 |
40.6 (2.5) |
19 (9) |
T0,P0 |
57.9 (1.6) |
17 (6) |
T0,P0 |
68.4 (1.8) |
9 (2) |
T0,P0 |
90.5 (4.0) |
4 (1) |
T0,P0 |
2AA -2μg/plate |
766 (86) |
N |
TA98 – Trial 2
A. WITHOUT METABOLIC ACTIVATION |
||
Mean Concentration (%)(±S.D.) |
Mean of revertants (±S.D.) |
Observations |
0.0 |
30 (10) |
T0,P0 |
26.7 (0.8) |
37 (6) |
T0,P0 |
41.0 (2.0) |
41 (8) |
T0,P0 |
54.5 (1.1) |
29 (7) |
T0,P0 |
68.6 (1.8) |
29 (7) |
T0,P0 |
90.0 (5.4) |
4 (4) |
T0,P0 |
2NF -25μg/plate |
100 (64) |
N |
B. WITH METABOLIC ACTIVATION |
||
Mean Concentration (%)(±S.D.) |
Mean of revertants (±S.D.) |
Observations |
0.0 |
26 (2) |
T0,P0 |
14.5 (16.2) |
R |
- |
42.0 (0.9) |
28 (11) |
T0,P0 |
28.9 (29.4) |
R |
- |
67.5 (2.4) |
20 (13) |
T0,P0 |
85.2 (1.8) |
8 (3) |
T0,P0 |
2AA -2μg/plate |
976 (255) |
N |
TA100 - Trial 1
A. WITHOUT METABOLIC ACTIVATION |
||
Mean Concentration (%)(±S.D.) |
Mean of revertants (±S.D.) |
Observations |
0.0 |
100 (3) |
T0,P0 |
25.6 (1.6) |
83 (3) |
T0,P0 |
38.4 (2.2) |
90 (5) |
T0,P0 |
57.2 (0.8) |
75 (6) |
T0,P0 |
74.5 (3.4) |
56 (8) |
T0,P0 |
89.0 (2.0) |
16 (3) |
T0,P0 |
NAAZ -2μg/plate |
744 (28) |
N |
B. WITH METABOLIC ACTIVATION |
||
Mean Concentration (%)(±S.D.) |
Mean of revertants (±S.D.) |
Observations |
0.0 |
115 (8) |
T0,P0 |
26.0 (1.6) |
122 (6) |
T0,P0 |
40.6 (2.5) |
105 (16) |
T0,P0 |
57.9 (1.6) |
97 (8) |
T0,P0 |
68.4 (1.8) |
71 (9) |
T0,P0 |
90.5 (4.0) |
13 (4) |
T0,P0 |
2AA -2μg/plate |
1315 (197) |
N |
TA100 – Trial 2
A. WITHOUT METABOLIC ACTIVATION |
||
Mean Concentration (%)(±S.D.) |
Mean of revertants (±S.D.) |
Observations |
0.0 |
131 (5) |
T0,P0 |
26.7 (0.8) |
135 (20) |
T0,P0 |
41.0 (2.0) |
130 (17) |
T0,P0 |
54.5 (1.1) |
123 (13) |
T0,P0 |
68.6 (1.8) |
109 (17) |
T0,P0 |
90.0 (5.4) |
46 (3) |
T0,P0 |
NAAZ -2μg/plate |
862 (93) |
N |
B. WITH METABOLIC ACTIVATION |
||
Mean Concentration (%)(±S.D.) |
Mean of revertants (±S.D.) |
Observations |
0.0 |
159 (24) |
T0,P0 |
14.5 (16.2) |
138 (20) |
T0,P0 |
42.0 (0.9) |
122 (14) |
T0,P0 |
28.9 (29.4) |
102 (3) |
T0,P0 |
67.5 (2.4) |
77 (12) |
T0,P0 |
85.2 (1.8) |
34 (2) |
T0,P0 |
2AA -2μg/plate |
1034 (16) |
N |
TA1535 - Trial 1
A. WITHOUT METABOLIC ACTIVATION |
||
Mean Concentration (%)(±S.D.) |
Mean of revertants (±S.D.) |
Observations |
0.0 |
15 (1) |
T0,P0 |
25.6 (1.6) |
18 (3) |
T0,P0 |
38.4 (2.2) |
16 (6) |
T0,P0 |
57.2 (0.8) |
18 (3) |
T0,P0 |
74.5 (3.4) |
8 (3) |
T0,P0 |
89.0 (2.0) |
3 (1) |
T0,P0 |
NAAZ -2μg/plate |
625 (36) |
N |
B. WITH METABOLIC ACTIVATION |
||
Mean Concentration (%)(±S.D.) |
Mean of revertants (±S.D.) |
Observations |
0.0 |
13 (3) |
T0,P0 |
26.0 (1.6) |
17 (7) |
T0,P0 |
40.6 (2.5) |
15 (2) |
T0,P0 |
57.9 (1.6) |
14 (3) |
T0,P0 |
68.4 (1.8) |
11 (3) |
T0,P0 |
90.5 (4.0) |
3 (2) |
T0,P0 |
2AA -2μg/plate |
236 (28) |
N |
TA100 – Trial 2
A. WITHOUT METABOLIC ACTIVATION |
||
Mean Concentration (%)(±S.D.) |
Mean of revertants (±S.D.) |
Observations |
0.0 |
12 (1) |
T0,P0 |
26.7 (0.8) |
15 (3) |
T0,P0 |
41.0 (2.0) |
17 (2) |
T0,P0 |
54.5 (1.1) |
21 (2) |
T0,P0 |
68.6 (1.8) |
17 (7) |
T0,P0 |
90.0 (5.4) |
3 (1) |
T0,P0 |
NAAZ -2μg/plate |
609 (142) |
N |
B. WITH METABOLIC ACTIVATION |
||
Mean Concentration (%)(±S.D.) |
Mean of revertants (±S.D.) |
Observations |
0.0 |
24 (10) |
T0,P0 |
14.5 (16.2) |
R |
- |
42.0 (0.9) |
22 (6) |
T0,P0 |
28.9 (29.4) |
R |
- |
67.5 (2.4) |
13 (2) |
T0,P0 |
85.2 (1.8) |
6 (1) |
T0,P0 |
2AA -2μg/plate |
202 (27) |
N |
Escherichia coliWP2uvrA(pKM101)- Trial 1
A. WITHOUT METABOLIC ACTIVATION |
||
Mean Concentration (%)(±S.D.) |
Mean of revertants (±S.D.) |
Observations |
0.0 |
136 (15) |
T0,P0 |
25.6 (1.6) |
142 (19) |
T0,P0 |
38.4 (2.2) |
11 (3) |
T0,P0 |
57.2 (0.8) |
81 (9) |
T0,P0 |
74.5 (3.4) |
40 (12) |
T0,P0 |
89.0 (2.0) |
2 (2) |
T0,P0 |
ENNG -2μg/plate |
1526 (50) |
N |
B. WITH METABOLIC ACTIVATION |
||
Mean Concentration (%)(±S.D.) |
Mean of revertants (±S.D.) |
Observations |
0.0 |
212 (19) |
T0,P0 |
26.0 (1.6) |
175 (28) |
T0,P0 |
40.6 (2.5) |
122 (13) |
T0,P0 |
57.9 (1.6) |
95 (7) |
T0,P0 |
68.4 (1.8) |
33 (10) |
T0,P0 |
90.5 (4.0) |
0 (0) |
T0,P0 |
2AA -25μg/plate |
1461 (116) |
N |
Escherichia coliWP2uvrA(pKM101)– Trial 2
A. WITHOUT METABOLIC ACTIVATION |
||
Mean Concentration (%)(±S.D.) |
Mean of revertants (±S.D.) |
Observations |
0.0 |
122 (23) |
T0,P0 |
26.7 (0.8) |
113 (13) |
T0,P0 |
41.0 (2.0) |
88 (9) |
T0,P0 |
54.5 (1.1) |
91 (6) |
T0,P0 |
68.6 (1.8) |
87 (7) |
T0,P0 |
90.0 (5.4) |
36 (9) |
T0,P0 |
ENNG -2μg/plate |
1221 (319) |
N |
B. WITH METABOLIC ACTIVATION |
||
Mean Concentration (%)(±S.D.) |
Mean of revertants (±S.D.) |
Observations |
0.0 |
124 (7) |
T0,P0 |
14.5 (16.2) |
R |
- |
42.0 (0.9) |
119 (14) |
T0,P0 |
28.9 (29.4) |
R |
- |
67.5 (2.4) |
78 (6) |
T0,P0 |
85.2 (1.8) |
54 (7) |
T0,P0 |
2AA -25μg/plate |
1488 (224) |
N |
Applicant's summary and conclusion
- Conclusions:
- Under the conditions of this study, no evidence of mutagenic activity was detected in either trial with the test substance.
Based on the findings, H-24619 (octafluorocyclobutane) is concluded to be negative for the induction of mutagenicity in the bacterial reverse mutation test in Salmonella typhimurium and Escherichia coli in the presence and absence of the Aroclor-induced rat liver S9. - Executive summary:
A bacterial reverse mutation test was performed according to OECD guideline No 471 on the test material H-24619 (octafluorocyclobutane). The experiment include 2 trials, with 6 different concentrations with an without metabolic activation and 6 different strains for each trial. Strains used were S. typhimurium TA97a, TA98, TA100, TA102, TA1535 and E. coli WP2 uvrA (pKM101). The test was conducted with and without metabolic activation (Aroclor-induced rat liver S9).
Under the conditions of this study, no evidence of mutagenic activity was detected in either trial with the test substance.
Based on the findings, H-24619 (octafluorocyclobutane) is concluded to be negative for the induction of mutagenicity in the bacterial reverse mutation test in Salmonella typhimurium and Escherichia coli in the presence and absence of the Aroclor-induced rat liver S9.
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