Registration Dossier
Registration Dossier
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EC number: 260-694-8 | CAS number: 57357-85-4
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Results of an bacterial reverse mutation assay (Ames test) with or without metabolic activation (S9 mix) according to OECD Guideline 471 (BASF SE, 1999) were negative.
Results of a gene mutation study in mammalian cells (HPRT test) according to OECD Guideline 476, with or without metabolic activation (S9 mix) were negative (BASF SE, 2017).
Results of a mammalian cell in vitro assay (MNT) according to OECD Guideline 487 with or without metabolic activation (S9 mix) were negative (BASF SE, 2016).
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2016-03-24 to 2016-11-23
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- 2014-09-26
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch No.of test material: 16/0055-1
- Expiration date of the lot/batch: 2016-10-26
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Mixing before preparation of the test substance preparations.
OTHER SPECIFICS: Liquid, colorless, clear - Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Suitability of cells: The V79 cell line has shown its suitability to detect aneugenic effects in the Micronucleus test in vitro either in the absence and presence of CytB.
- Doubling time: 12 - 14 h
- Methods for maintenance in cell culture if applicable: 1 mL portions of V79 stocks were maintained at -196 °C in liquid nitrogen using 7 % (v/v) dimethyl sulfoxide (DMSO) in culture medium as a cryoprotectant.
- Modal number of chromosomes: 22
MEDIA USED
- Type and identity of media including CO2 concentration: MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with 10 % (v/v) fetal calf serum (FCS), 1 % (v/v) penicillin/streptomycin (10000 IU / 10000 μg/mL), and 1 % (v/v) amphotericine B (250 μg/mL). Cells were grown with 5 % (v/v) CO2.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes - Cytokinesis block (if used):
- Cytochalasin B
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Dose selection according to OECD 487 guideline.
With S9 mix: 6.25, 12.5, 25.0, 50.0, and 100.0 µg/mL
Without S9 mix: 3.13, 6.25, 12.5, 25.0, 50.0, and 100.0 µg/mL - Vehicle / solvent:
- - Solvent used: DMSO 1 % (v/v)
- Justification for choice of solvent/vehicle: DMSO had been demonstrated to be suitable in the V79 in vitro cytogenetic assay and for which historical control data are available. - Untreated negative controls:
- yes
- Remarks:
- DMSO
- Negative solvent / vehicle controls:
- yes
- Remarks:
- with and without S9 mix
- Positive controls:
- yes
- Positive control substance:
- other: ethyl methanesulfonate
- Remarks:
- without S9 mix
- Untreated negative controls:
- yes
- Remarks:
- DMSO
- Negative solvent / vehicle controls:
- yes
- Remarks:
- with and without S9 mix
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with S9 mix
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding: 3 - 5 x10E6 cells/culture
DURATION
- Preincubation period: 20 - 24 h
- Exposure duration: 4 h
- Expression time: 24 - 44 h
- Fixation time: at the end of exposure time.
SPINDLE INHIBITOR: Cytochalasin B
STAIN: Prepared slides were stained with a mixture of 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI; stock: 5 mg/mL; Sigma-Aldrich, Cat.No. D9542) and propidium iodide (PI; stock: 5 mg/mL; Sigma-Aldrich, Cat.No. P4170) in Fluoroshield™ (Sigma-Aldrich, Cat.No. F6182) at a concentration of 0.25 μg/mL each. By the use of the combination of both fluorescence dyes it can be differentiated between DNA (DAPI; excitation: 350 nm, emission: 460 nm) and cytoplasm (PI; excitation: 488 nm, emission: 590 nm).
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: 5x10^4 cells per slide were centrifuged at 600 rpm for 7 min onto labeled slides using a Cytospin centrifuge (Cellspin I, Tharmac, Waldsolms, Germany). At least two slides per flask were prepared. In the case of strongly reduced cell numbers below 1x10^4 cells per flask no slides were prepared. After drying, the slides were fixed in 90 % (v/v) methanol for 10 minutes.
NUMBER OF CELLS EVALUATED: At least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group.
CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The analysis of micronuclei was carried out following the criteria of Countryman and Heddle:
- The diameter of the micronucleus is less than 1/3 of the main nucleus.
- The micronucleus and main nucleus retain the same color.
- The micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell.
- Only binucleated cells clearly surrounded by a membrane were scored.
DETERMINATION OF CYTOTOXICITY
- Method: relative population doubling (RPD)
OTHER EXAMINATIONS:
- Determination of the cytokinesis-block proliferation index (CBPI). - Evaluation criteria:
- Acceptance criteria:
The in vitro micronucleus assay is considered valid if the following criteria are met:
- The quality of the slides allowed the evaluation of a sufficient number of analyzable cells both in the control groups (vehicle/positive) and in at least three exposed test groups.
- Sufficient cell proliferation was demonstrated in the vehicle control.
- The number of cells containing micronuclei in the vehicle control was within the range of our laboratory’s historical negative control data. Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
- The positive control substances both with and without S9 mix induced a distinct, statistically significant increase in the number of micronucleated cells in the expected range.
Assessment criteria:
A test substance is considered to be clearly positive if the following criteria are met:
- A statistically significant increase in the number of micronucleated cells was obtained.
- A dose-related increase in the number of cells containing micronuclei was observed.
- The number of micronucleated cells exceeded both the value of the concurrent vehicle control and the range of our laboratory’s historical negative control data (95 % control limit).
A test substance is considered to be clearly negative if the following criterion is met:
- Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.
- The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95 % control limit). - Statistics:
- The statistical evaluation of the data was carried out using an appropriate statistical analysis. The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions (i.e. one-sided Fisher's exact test, BASF SE).
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- according to OECD guideline 487
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1983-05/revised Draft
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- testing lab
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch No.of test material: 09-0680
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
OTHER SPECIFICS: Yellowish clear liquid - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Remarks:
- uvrA
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- standard plate test: 4, 20, 100, 500, and 1500 µg/plate
preincubation test: 4, 20, 100, 500, and 1000 µg/plate - Vehicle / solvent:
- - Solvent used: DMSO
- Justification for choice of vehicle: DMSO was selected as solvent, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
- No precipitation of the test substance was found. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- with S9 mix
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: N-methyl-N' -nitro-N-nitrosoguanidine
- Remarks:
- without S9 mix
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitro-O-phenylendiamine
- Remarks:
- without S9 mix
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- without S9 mix
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- without S9 mix
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
standard plate test: in agar (plate incorporation)
preincubation test: preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h
TOXICITY
Toxicity detected by a
- decrease in the number of revertants
- clearing or diminution of the background lawn (= reduced his or trp background growth)
- reduction in the titer
NUMBER OF REPLICATIONS: 3 test plates/dose or control - Evaluation criteria:
- Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain with or without S9 mix.
A test substance is generally considered nonmutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other. - Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 500 µg/plate and above
- Vehicle controls validity:
- valid
- Untreated negative 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:
- cytotoxicity
- Remarks:
- 500 µg/plate an above
- Vehicle controls validity:
- valid
- Untreated negative 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:
- cytotoxicity
- Remarks:
- 500 µg/plate and above
- Vehicle controls validity:
- valid
- Untreated negative 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:
- cytotoxicity
- Remarks:
- 500 µg/plate and above
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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:
- cytotoxicity
- Remarks:
- 500 µg/plate and above
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- No increased number of his+ or trp+ revertants was observed in the standard plate test or in the preincubation test with or without S9 mix.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2016-06-06 to 2017-02-20
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 2015-07-28
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: In Vitro Mammalian Cell Gene Mutation Test (HPRT)
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch No.of test material: WE 20PA15
- Expiration date of the batch: 2017-10-26
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Rooom temperature
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Mixing before preparation of the test substance solutions.
OTHER SPECIFICS: Liquid, colorless, clear - Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Doubling time: 12 - 16 h
- Methods for maintenance in cell culture: Stocks of the CHO cell line (1 mL portions) are maintained at -196 °C in liquid nitrogen using 7 % (v/v) DMSO in culture medium as a cryoprotectant. Each batch used for mutagenicity testing was checked for mycoplasma contamination.
- Modal number of chromosomes: 20
MEDIA USED
- Type and identity of media including CO2 concentration: All media were incubated at 5 % CO2 and were supplemented with 1 % (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 μg/mL) and 1 % (v/v) amphotericine B (stock solution: 250 μg/mL).
Culture medium:
Ham's F12 medium containing stable glutamine and hypoxanthine (PAN Biotech; Cat. No. P04-15500) supplemented with 10 % (v/v) fetal calf serum (FCS).
Treatment medium (without S9 mix):
Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10 % (v/v) FCS.
Treatment medium (with S9 mix):
Ham's F12 medium containing stable glutamine and hypoxanthine.
Pretreatment medium ("HAT" medium):
Ham's F12 medium supplemented with hypoxanthine (13.6 x 10-3 mg/mL), aminopterin (0.18 x 10-3 mg/mL),- thymidine (3.88 x 10-3 mg/mL), and 10 % (v/v) FCS.
Selection medium ("TG" medium):
Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 6-thioguanine (10 μg/mL), and 10 % (v/v) fetal calf serum (FCS).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 liver microsomal fraction from male Wistar rats
- Test concentrations with justification for top dose:
- According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested.
1st Experiment
without S9 mix 0, 3.1, 6.3, 12.5, 25.0, 50.0, 100.0, and 200.0 μg/mL
with S9 mix 0, 3.1, 6.3, 12.5, 25.0, 50.0, 100.0, and 200.0 μg/mL
2nd Experiment
without S9 mix 0, 4.7, 9.4, 18.8, 37.5, 75.0, and 150.0 μg/mL
with S9 mix 0, 4.7, 9.4, 18.8, 37.5, 75.0, and 150.0 μg/mL
3rd Experiment
with S9 mix 0, 4.7, 9.4, 18.8, 37.5, 75.0, 150.0, 300.0, and 600.0 μg/mL - Vehicle / solvent:
- - Solvent used: DMSO
- Justification for choice of solvent: It had been demonstrated to be suitable in the CHO/HPRT assay and for which historical data are available. - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: ethyl methanesulfonate
- Remarks:
- without S9 mix
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Remarks:
- with S9 mix
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding: 20x10^6 cells in 40 mL
DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 5 - 7 d and fixation
- Selection time: 7 - 9 d and fixation
- Fixation time: immediately after expression or selection time, respectively.
SELECTION AGENT: Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 6-thioguanine (10 μg/mL) and 10 % (v/v) fetal calf serum (FCS) -> Selection medium ("TG" medium).
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
DETERMINATION OF MUTANT FREQUENCY
- Method: mutant frequency - Evaluation criteria:
- Acceptance criteria
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50 % (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should be within our historical negative control data range (95 % control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
• The positive controls both with and without S9 mix should induce a distinct, statistically significant increase in mutant frequencies in the expected range.
Assessment criteria
A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative/vehicle control value and the range of our laboratory’s historical negative control data (95 % control limit).
Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95 % control limit). - Statistics:
- An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report. The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0. In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using R.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- According to OECD Guideline No. 476 test groups up to a maximum cytotoxicity of 10 % RS were taken in consideration for the assessment of mutagenicity.
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
Referenceopen allclose all
Table 1: Summary table - experimental parts without S9 mix
Exp. |
Exposure/preparation interval [h] |
Test groups [µg/mL] |
Prec.* |
Genotoxicity** Micronucleated cells [%] |
Cytotoxicity |
|
Proliferation index cytostasis (CBPI) [%] |
Relative population doubling (RPD) [%] |
|||||
1 |
4/24 |
Vehicle control(1) |
n.d. |
0.2 |
0.0 |
100.0 |
3.13 |
- |
n.d. |
n.d. |
92.2 |
||
6.25 |
- |
0.4 |
5.4 |
106.8 |
||
12.50 |
- |
0.5 |
12.0 |
89.2 |
||
25.00 |
- |
0.3 |
10.1 |
75.0 |
||
50.00 |
- |
n.d. |
n.d. |
1.2 |
||
100.00 |
- |
n.d. |
n.d. |
-164.6 |
||
Positive control(2) |
n.d. |
1.3 (s) |
11.3 |
100.0 |
||
Positive control(3) |
n.d. |
2.3 (s) |
10.2 |
105.9 |
||
|
||||||
2 |
24/24 |
Vehicle control(1) |
n.d. |
0.2 |
0.0 |
100.0 |
Negative control |
- |
0.6 |
-18.1 |
131.2 |
||
0.78 |
- |
n.d. |
n.d. |
111.4 |
||
1.56 |
- |
n.d. |
n.d. |
95.8 |
||
3.13 |
- |
n.d. |
n.d. |
119.1 |
||
6.25 |
- |
0.5 |
5.5 |
106.8 |
||
12.50 |
- |
0.3 |
12.0 |
75.4 |
||
25.00 |
- |
0.2 |
21.8 |
64.4 |
||
Positive control(2) |
n.d. |
1.6 (s) |
14.1 |
154.8 |
||
Positive control(3) |
n.d. |
2.8 (s) |
16.9 |
150.9 |
||
|
||||||
3 |
24/24 |
Vehicle control(1) |
n.d. |
0.3 |
0.0 |
100.0 |
Negative control |
- |
0.4 |
-14.5 |
137.5 |
||
3.13 |
- |
n.d. |
n.d. |
71.7 |
||
6.25 |
- |
0.3 |
5.2 |
66.2 |
||
12.50 |
- |
0.2 |
9.4 |
66.4 |
||
25.00 |
- |
0.1 |
18.5 |
46.2 |
||
50.00 |
- |
n.d. |
n.d. |
-162.5 |
||
100.00 |
- |
n.p. |
n.p. |
-221.6 |
||
Positive control(2) |
n.d. |
2.9 (s) |
13.1 |
112.5 |
||
* Precipitation in culture medium at the end of exposure period (macroscopic)
** Relative number of binucleated cells with micronuclei per 2000 cells scored per test group
(s) Frequency statistically significant higher than corresponding control values
n.d. Not determined
n.p. No cytospin slides prepared due to strong cytotoxicity
n.s. Not scorable due to strong cytotoxicity
(1) DMSO 1 % (v/v) (2) EMS 500 μg/mL (3) EMS 600 μg/mL
Table 2: Summary table - experimental parts with S9 mix
Exp. |
Exposure/preparation interval [h] |
Test groups [µg/mL] |
Prec.* |
Genotoxicity** Micronucleated cells [%] |
Cytotoxicity |
|
Proliferation index cytostasis (CBPI) [%] |
Relative population doubling (RPD) [%] |
|||||
1 |
4/24 |
Vehicle control(1) |
n.d. |
0.3 |
0.0 |
100.0 |
3.13 |
- |
n.d. |
n.d. |
75.7 |
||
6.25 |
- |
n.d. |
n.d. |
80.0 |
||
12.50 |
- |
0.5 |
0.4 |
83.2 |
||
25.00 |
- |
0.3 |
13.0 |
75.8 |
||
50.00 |
- |
0.4 |
13.1 |
42.6 |
||
100.00 |
- |
n.d. |
n.d. |
-94.6 |
||
Positive control(2) |
n.d. |
1.8 (s) |
22.5 |
61.5 |
||
|
||||||
2 |
4/44 |
Vehicle control (1) |
n.d. |
0.5 |
0.0 |
100.0 |
6.25 |
- |
n.d. |
n.d. |
103.2 |
||
12.50 |
- |
0.6 |
7.8 |
100.8 |
||
25.00 |
- |
0.5 |
8.0 |
86.3 |
||
50.00 |
- |
0.4 |
13.6 |
53.6 |
||
100.00 |
- |
n.s. |
n.s. |
94.1 |
||
Positive control(2) |
n.d. |
3.6 (s) |
-14.7 |
70.2 |
||
* Precipitation in culture medium at the end of exposure period (macroscopic)
** Relative number of binucleated cells with micronuclei per 2000 cells scored per test group
(s) Frequency statistically significant higher than corresponding control values
n.d. Not determined
n.p. No cytospin slides prepared due to strong cytotoxicity
n.s. Not scorable due to strong cytotoxicity
(1) DMSO 1 % (v/v) (2) CPP 0.5 μg/mL
Standard Plate Test:
Standard Plate Test |
TA1535 |
|
|
|
|
|
|
|
|
Without S9 mix |
|
|
|
With S9 mix |
|
|
|
|
Rev. |
Mean |
SD |
Factor |
Rev. |
Mean |
SD |
Factor |
DMSO |
18 22 22 |
21 |
2 |
1.0 |
20 18 21 |
20 |
2 |
1.0 |
4 µg |
19 22 18 |
20 |
2 |
1.0 |
21 18 20 |
20 |
2 |
1.0 |
20 µg |
18 16 19 |
18 |
2 |
0.9 |
16 16 19 |
17 |
2 |
0.9 |
100 µg |
18 21 17 |
19 |
2 |
0.9 |
14 18 17 |
16 |
2 |
0.8 |
500 µg |
18 19 17 |
18 |
1 |
0.9 |
16 18 16 |
17 |
1 |
0.8 |
1500 µg |
13 B 16 B 11 B |
13 |
3 |
0.6 |
14 B 6 B 5 B |
8 |
5 |
0.4 |
MNNG |
1319 1359 1273 |
1317 |
43 |
63.7 |
|
|
|
|
2-AA |
|
|
|
|
126 149 134 |
136 |
12 |
6.9 |
Rev. = Revertant/plate; SD = standard deviation; B = reduced background growth |
||||||||
Standard Plate Test |
TA100 |
|
|
|
|
|
|
|
|
Without S9 mix |
|
|
|
With S9 mix |
|
|
|
|
Rev. |
Mean |
SD |
Factor |
Rev. |
Mean |
SD |
Factor |
DMSO |
101 110 109 |
107 |
5 |
1.0 |
107 115 116 |
113 |
5 |
1.0 |
4 µg |
107 107 94 |
103 |
8 |
1.0 |
100 111 98 |
103 |
7 |
0.9 |
20 µg |
98 96 82 |
92 |
9 |
0.9 |
117 103 97 |
106 |
10 |
0.9 |
100 µg |
104 115 106 |
108 |
6 |
1.0 |
89 104 114 |
101 |
14 |
0.9 |
500 µg |
72 77 84 |
78 |
6 |
0.7 |
126 103 104 |
111 |
13 |
1.0 |
1500 µg |
50 B 31 B 27 B |
36 |
12 |
0.3 |
67 B 66 B 62 B |
65 |
3 |
0.6 |
MNNG |
1693 1606 1200 |
1500 |
263 |
14.1 |
|
|
|
|
2-AA |
|
|
|
|
1352 1287 1328 |
1322 |
33 |
11.7 |
Rev. = Revertant/plate; SD = standard deviation; B = reduced background growth |
||||||||
Standard Plate Test |
TA1537 |
|
|
|
|
|
|
|
|
Without S9 mix |
|
|
|
With S9 mix |
|
|
|
|
Rev. |
Mean |
SD |
Factor |
Rev. |
Mean |
SD |
Factor |
DMSO |
11 10 12 |
11 |
1 |
1.0 |
14 10 11 |
12 |
2 |
1.0 |
4 µg |
11 11 8 |
10 |
2 |
0.9 |
13 10 9 |
11 |
2 |
0.9 |
20 µg |
7 7 9 |
8 |
1 |
0.7 |
8 7 11 |
9 |
2 |
0.7 |
100 µg |
8 9 7 |
8 |
1 |
0.7 |
6 8 10 |
8 |
2 |
.07 |
500 µg |
9 8 5 |
7 |
2 |
0.7 |
10 6 8 |
8 |
2 |
0.7 |
1500 µg |
1 B 1 B 1 B |
1 |
0 |
0.1 |
9 B 8 B 7 B |
8 |
1 |
0.7 |
AAC |
661 550 670 |
627 |
67 |
57.0 |
|
|
|
|
2-AA |
|
|
|
|
170 172 168 |
170 |
2 |
14.6 |
Rev. = Revertant/plate; SD = standard deviation; B = reduced background growth |
||||||||
Standard Plate Test |
TA98 |
|
|
|
|
|
|
|
|
Without S9 mix |
|
|
|
With S9 mix |
|
|
|
|
Rev. |
Mean |
SD |
Factor |
Rev. |
Mean |
SD |
Factor |
DMSO |
28 28 29 |
28 |
1 |
1.0 |
44 42 38 |
41 |
3 |
1.0 |
4 µg |
29 27 25 |
27 |
2 |
1.0 |
42 40 44 |
42 |
2 |
1.0 |
20 µg |
30 24 25 |
26 |
3 |
0.9 |
42 32 35 |
36 |
5 |
0.9 |
100 µg |
29 30 27 |
29 |
2 |
1.0 |
41 42 44 |
42 |
2 |
1.0 |
500 µg |
28 25 20 |
24 |
4 |
0.9 |
41 35 38 |
38 |
3 |
0.9 |
1500 µg |
15 B 12 B 12 B |
13 |
2 |
0.5 |
16 B 9 B 21 B |
15 |
6 |
0.4 |
NOPD |
828 1016 1148 |
997 |
161 |
35.2 |
|
|
|
|
2-AA |
|
|
|
|
1104 951 952 |
1002 |
88 |
24.3 |
Rev. = Revertant/plate; SD = standard deviation; B = reduced background growth |
||||||||
Standard Plate Test |
E.coli WP2 uvrA |
|
|
|
|
|
|
|
|
Without S9 mix |
|
|
|
With S9 mix |
|
|
|
|
Rev. |
Mean |
SD |
Factor |
Rev. |
Mean |
SD |
Factor |
DMSO |
22 24 27 |
24 |
3 |
1.0 |
33 35 33 |
34 |
1 |
1.0 |
4 µg |
26 29 25 |
27 |
2 |
1.1 |
34 37 29 |
33 |
4 |
1.0 |
20 µg |
27 31 29 |
29 |
2 |
1.2 |
33 36 36 |
35 |
2 |
1.0 |
100 µg |
29 22 24 |
25 |
4 |
1.0 |
35 34 33 |
34 |
1 |
1.0 |
500 µg |
22 25 25 |
24 |
2 |
1.0 |
37 31 30 |
33 |
4 |
1.0 |
1500 µg |
11 B 17 B 11 B |
13 |
3 |
0.5 |
27 B 22 B 19 B |
23 |
4 |
0.7 |
ENNG |
665 598 599 |
621 |
38 |
25.5 |
|
|
|
|
2-AA |
|
|
|
|
299 325 389 |
338 |
46 |
10.0 |
Rev. = Revertant/plate; SD = standard deviation; B = reduced background growth |
||||||||
Preincubation Test:
Preincubation Test |
TA1535 |
|
|
|
|
|
|
|
|
Without S9 mix |
|
|
|
With S9 mix |
|
|
|
|
Rev. |
Mean |
SD |
Factor |
Rev. |
Mean |
SD |
Factor |
DMSO |
22 16 20 |
19 |
3 |
1.0 |
18 22 20 |
20 |
2 |
1.0 |
4 µg |
14 15 15 |
15 |
1 |
0.8 |
19 15 11 |
15 |
4 |
0.8 |
20 µg |
12 17 13 |
14 |
3 |
0.7 |
10 17 12 |
13 |
4 |
0.7 |
100 µg |
14 14 14 |
14 |
0 |
0.7 |
13 11 9 |
11 |
2 |
0.6 |
500 µg |
1 B 1 B 2 B |
1 |
1 |
0.1 |
9 B 6 B 7 B |
7 |
2 |
0.4 |
1000 µg |
0 B 0 B 0 B |
|
|
|
0 B 0 B 0 B |
|
|
|
MNNG |
1236 1119 1277 |
1211 |
82 |
62.6 |
|
|
|
|
2-AA |
|
|
|
|
119 149 127 |
132 |
16 |
6.6 |
Rev. = Revertant/plate; SD = standard deviation; B = reduced background growth |
||||||||
Preincubation Test |
TA100 |
|
|
|
|
|
|
|
|
Without S9 mix |
|
|
|
With S9 mix |
|
|
|
|
Rev. |
Mean |
SD |
Factor |
Rev. |
Mean |
SD |
Factor |
DMSO |
112 105 92 |
103 |
10 |
1.0 |
129 121 127 |
126 |
4 |
1.0 |
4 µg |
92 87 73 |
84 |
10 |
0.8 |
92 98 85 |
92 |
7 |
0.7 |
20 µg |
88 81 83 |
84 |
4 |
0.8 |
91 75 77 |
81 |
9 |
0.6 |
100 µg |
69 76 81 |
75 |
6 |
0.7 |
77 94 91 |
87 |
9 |
0.7 |
500 µg |
1 B 2 B 1 B |
1 |
1 |
0.0 |
63 B 57 B 68 B |
63 |
6 |
0.5 |
1000 µg |
0 B 0 B 0 B |
|
|
|
0 B 0 B 0 B |
|
|
|
MNNG |
799 785 683 |
756 |
63 |
7.3 |
|
|
|
|
2-AA |
|
|
|
|
420 459 412 |
430 |
25 |
3.4 |
Rev. = Revertant/plate; SD = standard deviation; B = reduced background growth |
||||||||
Preincubation Test |
TA1537 |
|
|
|
|
|
|
|
|
Without S9 mix |
|
|
|
With S9 mix |
|
|
|
|
Rev. |
Mean |
SD |
Factor |
Rev. |
Mean |
SD |
Factor |
DMSO |
12 10 9 |
10 |
2 |
1.0 |
10 16 11 |
12 |
3 |
1.0 |
4 µg |
10 97 |
9 |
2 |
0.8 |
11 9 9 |
10 |
1 |
0.8 |
20 µg |
9 7 8 |
8 |
1 |
0.8 |
12 10 12 |
11 |
1 |
0.9 |
100 µg |
9 8 7 |
8 |
1 |
0.8 |
10 8 9 |
9 |
1 |
0.8 |
500 µg |
2 B 1 B 3 B |
2 |
1 |
0.2 |
6 B 5 B 7 B |
6 |
1 |
0.5 |
1000 µg |
0 B 0 B 0 B |
|
|
|
0 B 0 B 0 B |
|
|
|
AAC |
721 723 745 |
730 |
13 |
70.6 |
|
|
|
|
2-AA |
|
|
|
|
171 156 135 |
154 |
18 |
12.5 |
Rev. = Revertant/plate; SD = standard deviation; B = reduced background growth |
||||||||
Preincubation Test |
TA98 |
|
|
|
|
|
|
|
|
Without S9 mix |
|
|
|
With S9 mix |
|
|
|
|
Rev. |
Mean |
SD |
Factor |
Rev. |
Mean |
SD |
Factor |
DMSO |
26 28 32 |
29
|
3 |
1.0 |
39 40 32 |
37 |
4 |
1.0 |
4 µg |
22 22 23 |
22 |
1 |
0.8 |
28 27 22 |
26 |
3 |
0.7 |
20 µg |
22 22 17 |
20 |
3 |
0.7 |
22 26 20 |
23 |
3 |
0.6 |
100 µg |
14 16 17 |
16 |
2 |
0.5 |
14 14 10 |
13 |
2 |
0.3 |
500 µg |
2 B 1 B 1 B |
1 |
1 |
0.0 |
7 B 9 B 11 B |
9 |
2 |
0.2 |
1000 µg |
0 B 0 B 0 B |
|
|
|
0 B 0 B 0 B |
|
|
|
NOPD |
1239 1396 1299 |
1311 |
79 |
45.7 |
|
|
|
|
2-AA |
|
|
|
|
936 905 993 |
945 |
45 |
25.5 |
Rev. = Revertant/plate; SD = standard deviation; B = reduced background growth |
||||||||
Preincubation Test |
E.coli WP2 uvrA |
|
|
|
|
|
|
|
|
Without S9 mix |
|
|
|
With S9 mix |
|
|
|
|
Rev. |
Mean |
SD |
Factor |
Rev. |
Mean |
SD |
Factor |
DMSO |
28 21 31 |
27 |
5 |
1.0 |
33 35 37 |
35 |
2 |
1.0 |
4 µg |
27 27 23 |
26 |
2 |
1.0 |
31 21 25 |
26 |
5 |
0. |
20 µg |
28 27 25 |
27 |
2 |
1.0 |
24 20 14 |
19 |
5 |
0.6 |
100 µg |
23 27 22 |
24 |
3 |
0.9 |
13 12 11 |
12 |
1 |
0.3 |
500 µg |
5 B 2 B 1 B |
3 |
2 |
0.1 |
10 B 11 B 7 B |
9 |
2 |
0.3 |
1000 µg |
0 B 0 B 0 B |
|
|
|
9 B 10 B 12 B |
10 |
2 |
0.3 |
ENNG |
789 802 821 |
804 |
16 |
30.2 |
|
|
|
|
2-AA |
|
|
|
|
178 189 205 |
191 |
14 |
5.4 |
Rev. = Revertant/plate; SD = standard deviation; B = reduced background growth |
||||||||
Table 1: Summary of results – experimental parts without S9 mix
Exp. |
Exposure period [h] |
Test groups [µg/mL] |
Prec.* |
Genotoxicity** MFcorr. [per 10E6 cells] |
Cytotoxicity*** |
|
RS [%] |
CE2 [%] |
|||||
1 |
4 |
Vehicle control (1) |
n.d. |
2.15 |
100.0 |
100.0 |
3.1 |
- |
0.82 |
102.5 |
104.3 |
||
6.3 |
- |
1.76 |
84.3 |
97.4 |
||
12.5 |
- |
0.88 |
129.1 |
97.4 |
||
25.0 |
- |
5.07 |
94.2. |
93.1 |
||
50.0 |
- |
0.87 |
72.8 |
99.1 |
||
100.0 |
- |
n.c. (2) |
n.c. (2) |
n.c. (2) |
||
200.0 |
- |
n.c. (2) |
n.c. (2) |
n.c. (2) |
||
Positive control (2) |
n.d. |
105.13 (s) |
41.6 |
83.7 |
||
|
|
|
|
|
|
|
2 |
4 |
Vehicle control (1) |
n.d. |
3.82 |
100.0 |
100.0 |
4.7 |
- |
n.c. (1) |
100.4 |
n.c. (1) |
||
9.4 |
- |
3.27 |
102.5 |
93.5 |
||
18.8 |
- |
3.26 |
100.7 |
82.1 |
||
37.5 |
- |
1.29 |
86.8 |
88.9 |
||
75.0 |
- |
6.25 |
42.2 |
85.5 |
||
150.0 |
- |
3.48 |
2.2 |
120.6 |
||
Positive control (2) |
n.d. |
162.00 (s) |
96.2 |
76.3 |
||
* Macroscopically visible precipitation in culture medium at the end of exposure period
** Mutant frequency MFcorr. mutant colonies per 10E6 cells corrected with the CE2 value
*** Cloning efficiency related to the respective vehicle control
(s) Mutant frequency statistically significant higher than corresponding control values (p ≤ 0.05)
n.c. (1) Culture was not continued since a minimum of only four analysable concentrations is required
n.c. (2) Culture was not continued due to strong cytotoxicity
n.d. Not determined
(1) DMSO 1 % (v/v) (2) EMS 400 μg/mL (3) DMBA 1.25 μg/mL
Table 2: Summary of results – experimental parts with S9 mix
Exp. |
Exposure period [h] |
Test groups [µg/mL] |
Prec.* |
Genotoxicity** MFcorr. [per 10E6 cells] |
Cytotoxicity*** |
|
RS [%] |
CE2 [%] |
|||||
1 |
4 |
Vehicle control (1) |
n.d. |
0.43 |
100.0 |
100.0 |
3.1 |
- |
0.86 |
99.1 |
99.6 |
||
6.3 |
- |
0.49 |
86.0 |
88.0 |
||
12.5 |
- |
4.09 (s) |
93.0 |
94.0 |
||
25.0 |
- |
2.93 |
89.3 |
87.6 |
||
50.0 |
- |
3.45 |
71.3 |
86.8 |
||
100.0 |
- |
n.c. (2) |
n.c. (2) |
n.c. (2) |
||
200.0 |
- |
n.c. (2) |
n.c. (2) |
n.c. (2) |
||
Positive control (3) |
n.d. |
262.77 (s) |
92.4 |
80.3 |
||
|
||||||
2 |
4 |
Vehicle control (1) |
n.d. |
4.95 |
100.0 |
100.0 |
4.7 |
- |
n.c. (1) |
99.3 |
n.c. (1) |
||
9.4 |
- |
n.c. (1) |
123.2 |
n.c. (1) |
||
18.8 |
- |
1.43 |
100.7 |
94.6 |
||
37.5 |
- |
0.44 |
100.5 |
102.3 |
||
75.0 |
- |
2.12 |
100.6 |
127.5 |
||
150.0 |
- |
2.65 |
30.6 |
101.8 |
||
Positive control (3) |
n.d. |
152.22 (s) |
91.3 |
91.4 |
||
|
||||||
3 |
4 |
Vehicle control (1) |
n.d. |
0.71 |
100.0 |
100.0 |
4.7 |
- |
0.39 |
66.2 |
91.1 |
||
9.4 |
- |
2.75 |
139.9 |
91.1 |
||
18.8 |
- |
1.81 |
73.5 |
98.6 |
||
37.5 |
- |
1.83 |
75.9 |
78.2 |
||
75.0 |
- |
0.85 |
52.6 |
83.9 |
||
150.0 |
+ |
14.29 |
6.0 |
70.0 |
||
300.0 |
+ |
n.c. (2) |
n.c. (2) |
n.c. (2) |
||
600.0 |
+ |
n.c. (2) |
n.c. (2) |
n.c. (2) |
||
Positive control (3) |
n.d. |
181.93 (s) |
55.7 |
59.3 |
||
* Macroscopically visible precipitation in culture medium at the end of exposure period
** Mutant frequency MFcorr. mutant colonies per 10E6 cells corrected with the CE2 value
*** Cloning efficiency related to the respective vehicle control
(s) Mutant frequency statistically significant higher than corresponding control values (p ≤ 0.05)
n.c. (1) Culture was not continued since a minimum of only four analysable concentrations is required
n.c. (2) Culture was not continued due to strong cytotoxicity
n.d. Not determined
(1) DMSO 1 % (v/v) (2) EMS 400 μg/mL (3) DMBA 1.2 5μg/mL
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
The substance (+)-3 -aminomethylpinan was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium (TA 1535, TA 100, TA 1537, and TA 98) and Escherichia coli (E. coli WP2 uvrA), in a reverse mutation assay (Ames test) according to OECD guideline 471 (BASF SE, 1999). The following two tests were conducted. A standard plate test (SPT) at a dose range of 4 - 5000 µg/plate and a preincubation test (PIT) at a dose range of 4 - 1000 µg/plate, both with and without metabolic activation (Aroclor-induced ratliver S9 mix). No precipitation of the test substance was found. A bacteriotoxic effect was observed under all test conditions. No increase in the number of his- or trp- revertants was observed in the standard plate test or in the preincubation test either with or without S9 mix.
According to the results of the present study, the test substance (+)-3-Amiriomethylpinan is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the chosen experimental conditions.
The substance (+)-3-Aminomethylpinan was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro according to OECD guideline 476 (BASF SE, 2017). Three independent experiments were carried out, with and/or without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested. Test groups printed in bold type were evaluated for gene mutations:
1st Experiment
without S9 mix 0, 3.1, 6.3, 12.5, 25.0, 50.0,100.0, and 200.0 μg/mL
with S9 mix 0, 3.1, 6.3, 12.5, 25.0, 50.0, 100.0, and 200.0 μg/mL
2nd Experiment
without S9 mix 0, 4.7, 9.4, 18.8, 37.5, 75.0, and 150.0 μg/mL
with S9 mix 0, 4.7, 9.4, 18.8, 37.5, 75.0, and 150.0 μg/mL
3rd Experiment
with S9 mix 0, 4.7, 9.4, 18.8, 37.5, 75.0, 150.0, 300.0, and 600.0 μg/mL
Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations. In all experiments in the absence and presence of metabolic activation at least the highest concentrations tested for gene mutations were clearly cytotoxic, except in the 2nd experiment in the presence of metabolic activation. Thus, this experimental part of the study was repeated, designated 3rd experiment.
Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in three experiments performed independently of each other.
Thus, under the experimental conditions of this study, the test substance (+)-3 aminomethylpinan is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.
The substance (+)-3-Aminomethylpinan was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity) according to OECD guideline 487 (BASF SE, 2016). Three independent experiments were carried out with and/or without the addition of liver S9 mix from induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested. The test groups printed in bold type were evaluated for cytogenetic damage.
1st Experiment
4 hours exposure, 24 hours harvest time, without S9 mix
0, 3.13, 6.25, 12.5, 25.0, 50.0, and 100.0 μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix
0, 3.13, 6.25, 12.50, 25.0, 50.0, and 100.0 μg/mL
2nd Experiment
24 hours exposure, 24 hours harvest time, without S9 mix
0, 0.78, 1.56, 3.13, 6.25, 12.5, and 25.0 μg/mL
4 hours exposure, 44 hours harvest time, with S9 mix
0, 6.25, 12.5, 25.0, 50.0, and 100.0 μg/mL
3rd Experiment
24 hours exposure, 24 hours harvest time, without S9 mix
0, 3.13, 6.25, 12.5, 25.0, 50.0, and 100.0 μg/mL
A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, ethyl methanesulfonate (EMS) and cyclophosphamide (CPP), led to the expected increase in the number of cells containing micronuclei. Cytotoxicity indicated by clearly reduced cell count (indicated by relative population doubling) or proliferation index (CBPI) was observed at least at one applied test substance concentration in all experimental parts of this study, except in the 2nd experiment without S9 mix. This experimental part was repeated, designated 3rd experiment.
On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.
Thus, under the experimental conditions described, (+)-3-Aminomethylpinan is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.
Justification for classification or non-classification
The available experimental in vitro test data with the test substances (+)-3-aminomethylpinan are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the test substance (+)-3-aminomethylpinan is not considered to be classified for genetic toxicity under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.
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