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EC number: 204-472-0 | CAS number: 121-46-0
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
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- Density
- Particle size distribution (Granulometry)
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- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
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- Long-term toxicity to aquatic invertebrates
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- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
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- Additional toxicological data
![](https://echa.europa.eu/o/diss-blank-theme/images/factsheets/A-REACH/factsheet/print_toxicological-information.png)
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:
- October 2017 - February 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- 8,9,10-trinorborna-2,5-diene
- EC Number:
- 204-472-0
- EC Name:
- 8,9,10-trinorborna-2,5-diene
- Cas Number:
- 121-46-0
- Molecular formula:
- C7H8
- IUPAC Name:
- bicyclo[2.2.1]hepta-2,5-diene
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and batch number of test material: Air Liquide Electronics Materials. Batch 1590035136 / 1590035137
- Purity: 100% w/w
- Production date: 10.08.2017
- Expiry date: 16.08.2018
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability and homogeneity of the test material and during storage: stable
Method
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- Metabolic activation system: S9 Mix
Type and composition of metabolic activation system:
- source of S9:
S9 fraction, microsome fraction prepared from Sprague Dawley rat liver homogenate, is provided by MOLTOX TM (POB Box 1189 - 157 Industrial Park Dr - Boone, NC 28607 - USA)
- method of preparation of S9 mix:
S9 fraction, microsome fraction prepared from Sprague Dawley rat liver homogenate.
S9 fraction: 10%
MgCl2-6H2O: 8 mM
KCl: 33 mM
Glucose-6-Phosphate Na2: 5 mM
NADP Na2: 4 mM
Phosphate buffer pH 7.4: 0.1 M
- concentration or volume of S9 mix and S9 in the final culture medium:
Preparation of S9-mix 10 % (v/v)
- Sterility controls of S9:
Test item and the corresponding dilutions with S9-mix are added to 2 mL of top agar maintained at 45°C, and poured after homogenization on the bottom agar (20 ml) onto a Petri plate (90 mm in diameter) (n = 3). Plates are incubated for 48 - 72 hours at 37°C and then examined. There should be no bacterial growth on any plate. - Test concentrations with justification for top dose:
- Concentration of the test item: 5 000, 1 500, 500, 150 et 50 µg/plate
- Vehicle / solvent:
- - Vehicle/solvent used to solubilize test item: DMSO
- Vehicle/solvent used to solubilize positive controls: acetone ; DMSO
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- 2-nitrofluorene
- sodium azide
- other: cis-platinum(ii)diamine dichloride ; 9-Aminoacridine ; 2-Anthramine
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: negative control, positive control solvent, positive control, vehicle and five dose tested with and without metabolic activation (-S9-mix) for each strains (5 strains).
ASSAY WITHOUT METABOLIC ACTIVATION
Salmonella Typhimurium strains: for each strain, 0.1 mL (in aqueous or oily vehicle) / 50 µL (in non-aqueous or non-oily vehicule as DMSO) of the bacterial suspension containing 1-9 x10^9 bacteria/mL and 0.1 mL of each dilution of the original solution and 0.5 mL of sterile phosphate buffer are successively added to 2 mL of overlay agar, maintained supercooled at 45°C, containing 10 % (v/v) of a L-Histidine-D-Biotine solution (0.5 mM).
Escherichia coli strain : in a test tube 0.1 mL (in aqueous or oily vehicle) / 50 µL (in non-aqueous or non-oily vehicule as DMSO) of the bacterial suspension containing 1-9 x 10^9 bacteria/mL and 0.1 mL of each dilution of the original solution and 0.5 mL of phosphate buffer are successively added to 2 mL of overlay agar maintained super cooled in 45°C containing 5% (v/v) of nutrient broth n°2 to which are added 5 µL of a L-Tryptophane solution at 2 mg/mL.
Plates are incubated at 37°C over a 48-72 hour period. The number of revertant colonies per plate is counted.
Moreover the following controls are carried out:
- Negative controls: (i) absolute negative control containing no test item corresponding to the spontaneous reversion rate, (i) solvent used to solubilize positive controls ; Acetone ,DMSO, NaCl 0.15M
- Vehicle used to solubilize test item ; DMSO
- Positive control.
ASSAY WITH METABOLIC ACTIVATION
Two protocols can be used:
• either a standard plate incorporation method where the protocol is similar to that described above, except that, 500 µL of S9-mix fraction is quickly added, before pouring the mixture onto the plates ;
• or the pre-incubation assay where the solution of the test item solution with the test strain, and 500 µL of S9-mix fraction are preincubated with shaking for 30 min., at 37°C prior to mixing with the overlay agar and pouring onto the minimal agar plate.
This method is known to increase the detection sensitivity of a number of promutagens like alkaloids, aliphatic N-Nitroso compounds (OECD no 471).
For the first assay direct incorporation method is used.
PRELIMINARY CYTOTOXICITY TESTING (STRAIN TA100)
In a test tube 0.1 mL of the bacterial suspension (1-9 x 10^3 bacteria/mL) and 0.1 mL (in aqueous or oily vehicle) / 50 µL (in non-aqueous or non-oily vehicule as DMSO) of the stock solution and dilutions, are successively added to 2 mL of top agar at 45°C, containing 10 % (v/v) of a solution of L-Histidine-D-Biotine (2.5 mM). After homogenization, the content of the tube is poured onto a Petri plate (90 mm in diameter) containing minimal agar (20 mL). 3 plates per concentration are incubated for 48-72 hours at 370 C, and the colonies counted. A negative control containing the blank alone is run in parallel.
In case bacteriostatic activity is detected, the highest concentration to be retained is that exhibiting a bacteriostatic activity of 75 % or less. The precipitate, if present, should not interfere with the scoring. The following four dilutions studied are distributed according to a semi-logarithmic progression. - Evaluation criteria:
- Ensure that the criteria of validity of the study are well respected namely:
• the bacteriostatic activity of the highest concentration tested shall be equal to or less than 75 %,
• the spontaneous reversion rate of the absolute negative control shall comply with the historical values of the laboratory,
• the spontaneous reversion rate of the solvent shall not be statistically different from absolute negative control,
• the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation shall comply with the historical values of the laboratory.
• Negative and positive values should not show significant difference with the historical values of the laboratory ± 2 standard deviations).
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- • There is no significant difference between the number of spontaneous reversions, the number of reversions obtained for the positive controls (without and with metabolic activation), and the mean of corresponding experimental historical values obtained in the laboratory.
• One can observe in presence of the higher doses tested 5 000 µg/plate and 1 500 µg/plate with metabolic activation and pre-incubation a thinning of the bacterial lawn.
• There is no evidence of any increase in the number of revertant colonies in the presence of the test item stock solution and dilutions (5 000, 1 500, 500, 150 et 50 µg/plate) without and with metabolic activation in (Salmonella lyphimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA) (pKM 101).
• Results are confirmed in an independent experiment.
Any other information on results incl. tables
TA 1535 — Assay no 1 of mutagenic activity
without metabolic activation (-S9-mix)
Serie | Dose/Plate | Plate | Mean | Standard | R | ||
no 1 | no 2 | no 3 | |||||
Negative control | 100 µL | 8 | 8 | 11 | 9.00 | 1.73 | - |
Positive control solvent | 5 µL | 9 | 12 | 16 | 12.33 | 3.51 | - |
Positive control : Sodium azide | 5µg in 5 µL | 1213 | 1226 | 1198 | 1212.33 | 14.01 | 98.30 |
Vehicle | 50 µL | 8 | 11 | 14 | 11.00 | 3.00 | - |
Solution of BCHD | 5000 µg | 14 | 13 | 6 | 11.00 | 4.36 | 1.00 |
1500 µg | 15 | 13 | 16 | 14.67 | 1.53 | 1.33 | |
500 µg | 12 | 12 | 9 | 11.00 | 1.73 | 1.00 | |
150 µg | 12 | 14 | 9 | 11.67 | 2.52 | 1.06 | |
50 µg | 9 | 15 | 7 | 10.33 | 4.16 | 0.94 |
with metabolic activation (10 % S9-mix) — without pre-incubation
Serie | Dose/Plate | Plate | Mean | Standard | R | ||
no 1 | no 2 | no 3 | |||||
Negative control | 100 µL | 7 | 13 | 12 | 10.67 | 3.21 | - |
Positive control solvent | 5 µL | 13 | 10 | 7 | 10.00 | 3.00 | - |
Positive control : Sodium azide | 5µg in 5 µL | 197 | 216 | 180 | 197.67 | 18.01 | 19.77 |
Vehicle | 50 µL | 13 | 16 | 14 | 14.33 | 1.53 | - |
Solution of BCHD | 5000 µg | 10 | 18 | 11 | 13.00 | 4.36 | 0.91 |
1500 µg | 11 | 11 | 11 | 11.00 | 0.00 | 0.77 | |
500 µg | 13 | 10 | 15 | 12.67 | 2.52 | 0.88 | |
150 µg | 14 | 12 | 13 | 13.00 | 1.00 | 0.91 | |
50 µg | 15 | 11 | 15 | 13.67 | 2.31 | 0.95 |
TA 1535 — Assay n02 of mutagenic activity
without metabolic activation (-S9-mix)
Serie | Dose/Plate | Plate | Mean | Standard | R | ||
no 1 | no 2 | no 3 | |||||
Negative control | 100 µL | 10 | 9 | 7 | 8.67 | 1.53 | - |
Positive control solvent | 5 µL | 9 | 13 | 8 | 10.00 | 2.65 | - |
Positive control : Sodium azide | 5µg in 5 µL | 924 | 937 | 917 | 926.00 | 10.15 | 92.60 |
Vehicle | 50 µL | 5 | 12 | 6 | 7.67 | 3.79 | - |
Solution of BCHD | 5000 µg | 8 | 6 | 10 | 8.00 | 2.00 | 1.04 |
1500 µg | 14 | 7 | 9 | 10.00 | 3.61 | 1.30 | |
500 µg | 15 | 11 | 12 | 12.67 | 2.08 | 1.65 | |
150 µg | 11 | 9 | 12 | 10.67 | 1.53 | 1.39 | |
50 µg | 9 | 11 | 10 | 10.00 | 1.00 | 1.30 |
with metabolic activation (10 % S9-mix) — with pre-incubation
Serie | Dose/Plate | Plate | Mean | Standard | R | ||
no 1 | no 2 | no 3 | |||||
Negative control | 100 µL | 10 | 10 | 11 | 10.33 | 0.58 | - |
Positive control solvent | 5 µL | 14 | 11 | 12 | 12.33 | 1.53 | - |
Positive control : Sodium azide | 5µg in 5 µL | 62 | 79 | 74 | 71.67 | 8.74 | 5.81 |
Vehicle | 50 µL | 8 | 8 | 6 | 7.33 | 1.15 | - |
Solution of BCHD | 5000 µg | 2 | 5 | 3 | 3.33 | 1.53 | 0.45 |
1500 µg | 8 | 5 | 3 | 5.33 | 2.52 | 0.73 | |
500 µg | 11 | 5 | 17 | 11.00 | 6.00 | 1.50 | |
150 µg | 8 | 9 | 11 | 9.33 | 1.53 | 1.27 | |
50 µg | 7 | 8 | 11 | 8.67 | 2.08 | 1.18 |
TA 1537 -Assay no 1 of mutagenic activity
without metabolic activation (-S9-mix)
Serie | Dose/Plate | Plate | Mean | Standard | R | ||
no 1 | no 2 | no 3 | |||||
Negative control | 100 µL | 10 | 10 | 6 | 8.67 | 2.31 | - |
Positive control solvent | 5 µL | 10 | 8 | 7 | 8.33 | 1.53 | - |
Positive control : Sodium azide | 5µg in 5 µL | 1810 | 1214 | 1847 | 1623.67 | 355.26 | 194.84 |
Vehicle | 50 µL | 6 | 7 | 8 | 7.00 | 1.00 | - |
Solution of BCHD | 5000 µg | 6 | 6 | 5 | 5.67 | 0.58 | 0.81 |
1500 µg | 5 | 8 | 9 | 7.33 | 2.08 | 1.05 | |
500 µg | 7 | 8 | 7 | 7.33 | 0.58 | 1.05 | |
150 µg | 9 | 10 | 5 | 8.00 | 2.65 | 1.14 | |
50 µg | 10 | 10 | 4 | 8.00 | 3.46 | 1.14 |
with metabolic activation (10 % S9-mix) — without pre-incubation
Serie | Dose/Plate | Plate | Mean | Standard | R | ||
no 1 | no 2 | no 3 | |||||
Negative control | 100 µL | 18 | 17 | 20 | 18.33 | 1.53 | - |
Positive control solvent | 5 µL | 17 | 19 | 22 | 19.33 | 2.52 | - |
Positive control : Sodium azide | 5µg in 5 µL | 94 | 99 | 92 | 95.00 | 3.61 | 4.91 |
Vehicle | 50 µL | 13 | 18 | 15.00 | 2.65 | - | |
Solution of BCHD | 5000 µg | 13 | 17 | 19 | 16.33 | 3.06 | 1.09 |
1500 µg | 12 | 11 | 12 | 11.67 | 0.58 | 0.78 | |
500 µg | 17 | 14 | 14 | 15.00 | 1.73 | 1.00 | |
150 µg | 15 | 13 | 18 | 15.33 | 2.52 | 1.02 | |
50 µg | 20 | 17 | 11 | 16.00 | 4.58 | 1.07 |
TA 1537 — Assay n02 of mutagenic activity
without metabolic activation (-S9-mix)
Serie | Dose/Plate | Plate | Mean | Standard | R | ||
no 1 | no 2 | no 3 | |||||
Negative control | 100 µL | 5 | 5 | 4 | 4.67 | 0.58 | - |
Positive control solvent | 5 µL | 3 | 5 | 11 | 6.33 | 4.16 | - |
Positive control : Sodium azide | 5µg in 5 µL | 368 | 486 | 421 | 425.00 | 59.10 | 67.11 |
Vehicle | 50 µL | 7 | 9 | 12 | 9.33 | 2.52 | - |
Solution of BCHD | 5000 µg | 14 | 5 | 10 | 9.67 | 4.51 | 1.04 |
1500 µg | 17 | 9 | 6 | 10.67 | 5.69 | 1.114 | |
500 µg | 11 | 18 | 5 | 11.33 | 6.51 | 1.21 | |
150 µg | 8 | 10 | 17 | 11.67 | 4.73 | 1.25 | |
50 µg | 6 | 17 | 7 | 10.00 | 6.08 | 1.07 |
with metabolic activation (10 % S9-mix) — with pre-incubation
Serie | Dose/Plate | Plate | Mean | Standard | R | ||
no 1 | no 2 | no 3 | |||||
Negative control | 100 µL | 18 | 12 | 15 | 15.00 | 3.00 | - |
Positive control solvent | 5 µL | 17 | 9 | 23 | 16.33 | 7.02 | - |
Positive control : Sodium azide | 5µg in 5 µL | 31 | 88 | 40 | 53.00 | 30.64 | 3.24 |
Vehicle | 50 µL | 10 | 16 | 17 | 14.33 | 3.79 | - |
Solution of BCHD | 5000 µg | 5 | 2 | 3 | 3.33 | 1.53 | 0.23 |
1500 µg | 7 | 5 | 8 | 6.67 | 1.53 | 0.47 | |
500 µg | 9 | 10 | 11 | 10.00 | 1.00 | 0.70 | |
150 µg | 9 | 14 | 11 | 11.33 | 2.52 | 0.79 | |
50 µg | 13 | 14 | 12 | 13.00 | 1.00 | 0.91 |
TA 98— Assay no 1 of mutagenic activity
without metabolic activation (-S9-mix)
Serie | Dose/Plate | Plate | Mean | Standard | R | ||
no 1 | no 2 | no 3 | |||||
Negative control | 100 µL | 10 | 19 | 19 | 16.00 | 5.20 | - |
Positive control solvent | 5 µL | 19 | 8 | 12 | 13.00 | 5.57 | - |
Positive control : Sodium azide | 5µg in 5 µL | 571 | 509 | 700 | 593.33 | 97.44 | 45.64 |
Vehicle | 50 µL | 16 | 12 | 16 | 14.67 | 2.31 | - |
Solution of BCHD | 5000 µg | 13 | 11 | 16 | 13.33 | 2.52 | 0.91 |
1500 µg | 15 | 19 | 18 | 17.33 | 2.08 | 1.18 | |
500 µg | 15 | 9 | 14 | 12.67 | 3.21 | 0.86 | |
150 µg | 9 | 10 | 12 | 10.33 | 1.53 | 0.70 | |
50 µg | 16 | 15 | 12 | 14.33 | 2.08 | 0.98 |
with metabolic activation (10 % S9-mix) — without pre-incubation
Serie | Dose/Plate | Plate | Mean | Standard | R | ||
no 1 | no 2 | no 3 | |||||
Negative control | 100 µL | 24 | 25 | 20 | 23.00 | 2.65 | - |
Positive control solvent | 5 µL | 25 | 17 | 19 | 20.33 | 4.16 | - |
Positive control : Sodium azide | 5µg in 5 µL | 637 | 682 | 649 | 656.00 | 23.30 | 32.26 |
Vehicle | 50 µL | 22 | 15 | 15 | 17.33 | 4.04 | - |
Solution of BCHD | 5000 µg | 31 | 24 | 21 | 25.33 | 5.13 | 1.46 |
1500 µg | 25 | 27 | 16 | 22.67 | 5.86 | 1.31 | |
500 µg | 13 | 16 | 17 | 18.67 | 3.79 | 1.08 | |
150 µg | 27 | 23 | 23 | 24.33 | 2.31 | 1.40 | |
50 µg | 27 | 20 | 17 | 21.33 | 5.13 | 1.23 |
TA 98 -Assay n02 of mutagenic activity
without metabolic activation (-S9-mix)
Serie | Dose/Plate | Plate | Mean | Standard | R | ||
no 1 | no 2 | no 3 | |||||
Negative control | 100 µL | 11 | 11 | 18 | 13.33 | 4.04 | - |
Positive control solvent | 5 µL | 21 | 11 | 16 | 16.00 | 5.00 | - |
Positive control : Sodium azide | 5µg in 5 µL | 415 | 578 | 478 | 490.33 | 82.20 | 30.65 |
Vehicle | 50 µL | 24 | 14 | 16 | 18.00 | 5.29 | - |
Solution of BCHD | 5000 µg | 8 | 20 | 15 | 14.33 | 6.03 | 0.80 |
1500 µg | 8 | 12 | 18 | 12.67 | 5.03 | 0.70 | |
500 µg | 18 | 15 | 27 | 20.00 | 6.24 | 1.11 | |
150 µg | 12 | 12 | 18 | 14.00 | 3.46 | 0.78 | |
50 µg | 15 | 16 | 13 | 14.67 | 1.53 | 0.81 |
with metabolic activation (10 % S9-mix) — with pre-incubation
Serie | Dose/Plate | Plate | Mean | Standard | R | ||
no 1 | no 2 | no 3 | |||||
Negative control | 100 µL | 21 | 34 | 23 | 26.00 | 7.00 | - |
Positive control solvent | 5 µL | 30 | 32 | 21 | 27.67 | 5.86 | - |
Positive control : Sodium azide | 5µg in 5 µL | 189 | 188 | 244 | 207.00 | 32.05 | 7.48 |
Vehicle | 50 µL | 11 | 21 | 29 | 20.33 | 9.02 | - |
Solution of BCHD | 5000 µg | 9 | 8 | 5 | 7.33 | 2.08 | 0.36 |
1500 µg | 11 | 9 | 15 | 11.67 | 3.06 | 0.57 | |
500 µg | 25 | 24 | 26 | 25.00 | 1.00 | 1.23 | |
150 µg | 25 | 15 | 27 | 22.33 | 6.43 | 1.10 | |
50 µg | 15 | 22 | 27 | 21.33 | 6.03 | 1.05 |
TA 100— Assay n o 1 of mutagenic activity
without metabolic activation (-S9-mix)
Serie | Dose/Plate | Plate | Mean | Standard | R | ||
no 1 | no 2 | no 3 | |||||
Negative control | 100 µL | 64 | 70 | 68 | 67.33 | 3.06 | - |
Positive control solvent | 5 µL | 61 | 77 | 73 | 70.33 | 8.33 | - |
Positive control : Sodium azide | 5µg in 5 µL | 1560 | 1466 | 1282 | 1436.00 | 141.41 | 20.42 |
Vehicle | 50 µL | 62 | 74 | 70 | 68.67 | 6.11 | - |
Solution of BCHD | 5000 µg | 64 | 60 | 76 | 66.67 | 8.33 | 0.97 |
1500 µg | 50 | 63 | 61 | 58.00 | 7.00 | 0.84 | |
500 µg | 56 | 59 | 53 | 56.00 | 3.00 | 0.82 | |
150 µg | 53 | 42 | 56 | 50.33 | 7.37 | 0.73 | |
50 µg | 56 | 54 | 52 | 54.00 | 2.00 | 0.79 |
with metabolic activation (10 % S9-mix) — without pre-incubation
Serie | Dose/Plate | Plate | Mean | Standard | R | ||
no 1 | no 2 | no 3 | |||||
Negative control | 100 µL | 63 | 73 | 85 | 73.67 | 11.02 | - |
Positive control solvent | 5 µL | 63 | 74 | 81 | 72.67 | 9.07 | - |
Positive control : Sodium azide | 5µg in 5 µL | 1202 | 1074 | 1176 | 1150.67 | 67.66 | 15.83 |
Vehicle | 50 µL | 78 | 78 | 77 | 77.67 | 0.58 | - |
Solution of BCHD | 5000 µg | 68 | 76 | 79 | 74.33 | 5.69 | 0.96 |
1500 µg | 71 | 63 | 73 | 69.00 | 5.29 | 0.89 | |
500 µg | 68 | 71 | 74 | 71.00 | 3.00 | 0.91 | |
150 µg | 89 | 65 | 79 | 77.67 | 12.06 | 1.00 | |
50 µg | 63 | 64 | 74 | 67.00 | 6.08 | 0.86 |
TA 100 Assay no2 of mutagenic activity
without metabolic activation (-S9-mix)
Serie | Dose/Plate | Plate | Mean | Standard | R | ||
no 1 | no 2 | no 3 | |||||
Negative control | 100 µL | 41 | 47 | 55 | 47.67 | 7.02 | |
Positive control solvent | 5 µL | 57 | 54 | 76 | 62.33 | 11.93 | |
Positive control : Sodium azide | 5µg in 5 µL | 1101 | 1330 | 1520 | 1317.00 | 209.80 | 21.13 |
Vehicle | 50 µL | 52 | 44 | 45 | 47.00 | 4.36 | |
Solution of BCHD | 5000 µg | 54 | 49 | 49 | 50.67 | 2.89 | 1.08 |
1500 µg | 49 | 45 | 46 | 46.67 | 2.08 | 0.99 | |
500 µg | 51 | 61 | 58 | 56.67 | 5.13 | 1.21 | |
150 µg | 56 | 43 | 51 | 50.00 | 6.56 | 1.06 | |
50 µg | 64 | 54 | 70 | 62.67 | 8.08 | 133 |
with metabolic activation (10 % S9-mix) — with pre-incubation
Serie | Dose/Plate | Plate | Mean | Standard | R | ||
no 1 | no 2 | no 3 | |||||
Negative control | 100 µL | 76 | 88 | 58 | 74.00 | 15.10 | - |
Positive control solvent | 5 µL | 58 | 61 | 59 | 59.33 | 1.53 | - |
Positive control : Sodium azide | 5µg in 5 µL | 367 | 375 | 410 | 384.00 | 22.87 | 6.47 |
Vehicle | 50 µL | 51 | 65 | 63 | 59.67 | 7.57 | - |
Solution of BCHD | 5000 µg | 74 | 54 | 42 | 56.67 | 16.17 | 0.95 |
1500 µg | 44 | 39 | 41 | 41.33 | 2.52 | 0.69 | |
500 µg | 67 | 62 | 71 | 66.67 | 4.51 | 1.12 | |
150 µg | 60 | 53 | 54 | 55.67 | 3.79 | 0.93 | |
50 µg | 58 | 62 | 56 | 58.67 | 3.06 | 0.98 |
E. COLI— Assay no 1 of mutagenic activity
without metabolic activation (-S9-mix)
Serie | Dose/Plate | Plate | Mean | Standard | R | ||
no 1 | no 2 | no 3 | |||||
Negative control | 100 µL | 165 | 166 | 159 | 163.33 | 3.79 | - |
Positive control solvent | 5 µL | 163 | 160 | 161 | 161.33 | 1.53 | - |
Positive control : Sodium azide | 5µg in 5 µL | 492 | 479 | 483 | 484.67 | 6.66 | 3.00 |
Vehicle | 50 µL | 161 | 162 | 157 | 160.00 | 2.65 | - |
Solution of BCHD | 5000 µg | 161 | 159 | 168 | 162.67 | 4.73 | 1.02 |
1500 µg | 150 | 167 | 151 | 156.00 | 9.54 | 0.98 | |
500 µg | 166 | 152 | 152 | 156.67 | 8.08 | 0.98 | |
150 µg | 155 | 163 | 153 | 157.00 | 5.29 | 0.98 | |
50 µg | 154 | 155 | 164 | 157.67 | 5.51 | 0.99 |
with metabolic activation (10 % S9-mix) — without pre-incubation
Serie | Dose/Plate | Plate | Mean | Standard | R | ||
no 1 | no 2 | no 3 | |||||
Negative control | 100 µL | 194 | 176 | 163 | 177.67 | 15.57 | - |
Positive control solvent | 5 µL | 190 | 178 | 163 | 177.00 | 13.53 | - |
Positive control : Sodium azide | 5µg in 5 µL | 576 | 624 | 658 | 619.33 | 41.20 | 3.50 |
Vehicle | 50 µL | 174 | 167 | 183 | 174.67 | 8.02 | - |
Solution of BCHD | 5000 µg | 172 | 160 | 171 | 167.67 | 6.66 | 0.96 |
1500 µg | 161 | 179 | 202 | 180.67 | 20.55 | 1.03 | |
500 µg | 178 | 183 | 183 | 181.33 | 2.89 | 1.04 | |
150 µg | 194 | 216 | 194 | 201.33 | 12.70 | 1.15 | |
50 µg | 177 | 205 | 165 | 182.33 | 20.53 | 1.04 |
E. COLI— Assay no 2 of mutagenic activity
without metabolic activation (-S9-mix)
Serie | Dose/Plate | Plate | Mean | Standard | R | ||
no 1 | no 2 | no 3 | |||||
Negative control | 100 µL | 130 | 123 | 124 | 125.67 | 3.79 | - |
Positive control solvent | 5 µL | 120 | 135 | 123 | 126.00 | 7.94 | - |
Positive control : Sodium azide | 5µg in 5 µL | 424 | 521 | 475 | 473.33 | 48.52 | 3.76 |
Vehicle | 50 µL | 148 | 131 | 123 | 134.00 | 12.77 | - |
Solution of BCHD | 5000 µg | 108 | 180 | 158 | 148.67 | 36.90 | 1.11 |
1500 µg | 116 | 114 | 126 | 118.67 | 6.43 | 0.89 | |
500 µg | 121 | 129 | 124 | 124.67 | 4.04 | 0.93 | |
150 µg | 118 | 115 | 129 | 120.67 | 7.37 | 0.90 | |
50 µg | 130 | 114 | 114 | 119.33 | 9.24 | 0.89 |
with metabolic activation (10 % S9-mix) — with pre-incubation
Serie | Dose/Plate | Plate | Mean | Standard | R | ||
no 1 | no 2 | no 3 | |||||
Negative control | 100 µL | 191 | 189 | 173 | 184.33 | 9.87 | - |
Positive control solvent | 5 µL | 153 | 171 | 165 | 163.00 | 9.17 | - |
Positive control : Sodium azide | 5µg in 5 µL | 575 | 591 | 610 | 592.00 | 17.52 | 3.63 |
Vehicle | 50 µL | 167 | 162 | 163 | 164.00 | 2.65 | - |
Solution of BCHD | 5000 µg | 35 | 93 | 51 | 59.67 | 29.96 | 0.36 |
1500 µg | 80 | 110 | 118 | 102.67 | 20.03 | 0.63 | |
500 µg | 157 | 169 | 162 | 162.61 | 6.03 | 0.99 | |
150 µg | 183 | 162 | 159 | 168.00 | 13.08 | 1.02 | |
50 µg | 186 | 171 | 167 | 174.67 | 10.02 | 1.07 |
Applicant's summary and conclusion
- Conclusions:
- Doses (5 000, 1 500, 500, 150 and 50 µg/plate) prepared from solutions of the test item BCHD-Bicyclo 2,2,1-Hepta 2,5 diene (or BCHD) provided by AIR LIQUIDE ELECTRONICS MATERIALS (Batches 1590035136 & 1590035137), do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA1537, TA98, TA 100 and in Escherichia coli WP2(uvrA3 (pKM 101) without, or with metabolic activation, according to the OECD Guidelines n°471.
Based on this Ames test, the substance BCHD is considered not genotoxic. - Executive summary:
A bacterial reverse mutation test was performed using "Salmonella typhimurium his-" and "Escherichia coli" WP2(uvrA-)(pKM101) according to OECD guideline no 471.
Solutions have been tested for their capacity to induce reverse mutation in four Salmonella typhimurium strains and one Escherichia coli strain. This study was performed in the absence and presence of metabolic activation. Two independent assays were carried out
For assay n°1, various concentrations of solution were put in contact with the strains in the absence and presence of a metabolic activation system (S9-mix 10% (v/v)).
For assay n°2, various concentrations of solution were put in contact with the strains in the absence of metabolic activation and with pre-incubation in the presence of metabolic activation system (S9-mix 10% (v/v)).
For the two assays, negative and positive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental "historical" values obtained in the laboratory.
These results validate the two tests.
There is no evidence of any increase in the number of revertant colonies in the presence of the various concentration of the test item (5 000, 1 500, 500, 150 et 50 µg/plate), without and with metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA ) (pKM 101).
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