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EC number: 939-946-2 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
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- Endpoint summary
- Stability
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- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2011-04-18 to 2011-05-16
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted in compliance with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive and done to a valid guideline
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 011
- Report date:
- 2011
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
Test material
- Reference substance name:
- reaction mass of (3R,5R)-3-chloro-5-(trichloromethyl)cyclopentene and (3S,5S)-3-chloro-5-(trichloromethyl)cyclopentene and (3R,4R)-3-chloro-4-(trichloromethyl)cyclopentene and (3S,4S)-3-chloro-4-(trichloromethyl)cyclopentene
- EC Number:
- 939-946-2
- Molecular formula:
- C6H6Cl4
- IUPAC Name:
- reaction mass of (3R,5R)-3-chloro-5-(trichloromethyl)cyclopentene and (3S,5S)-3-chloro-5-(trichloromethyl)cyclopentene and (3R,4R)-3-chloro-4-(trichloromethyl)cyclopentene and (3S,4S)-3-chloro-4-(trichloromethyl)cyclopentene
- Test material form:
- not specified
Constituent 1
- Specific details on test material used for the study:
- EC Number: 939-946-2
Method
Species / strain
- Species / strain / cell type:
- lymphocytes: human
- Details on mammalian cell type (if applicable):
- Blood samples were obtained from a healthy donor not receiving medication. For this study, blood was collected from a female donor (37 years old).
Blood samples were drawn by venous puncture and collected in heparinized tubes by Dr. V. Theodor (64380 Rossdorf, Germany). The tubes were sent to Harlan CCR to initiate cell cultures within 24 h after blood collection. If necessary, the blood was stored before use at 4 °C.
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- between 15.4 and 2370 microgram/mL
- Vehicle / solvent:
- DMSO
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- A preliminary cytotoxicity test was performed to determine the concentrations to be used in the mutagenicity assay. Cytotoxicity is characterized by the percentages of mitotic suppression in comparison to the controls by counting 1000 cells per culture in duplicate. The experimental conditions in this pre-test phase were identical to those required and described for the mutagenicity assay. The pre-test phase was performed with 10 concentrations of the test item and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 h (with and without S9 mix). The preparation interval was 22 h after start of the exposure.
Blood cultures were set up in bulk within 24 hrs after collection in 75 cm² cell culture flasks. The culture medium was DMEM:F12 (Dulbecco's modified eagle medium / Ham's F12 medium; mixture 1:1; already supplemented with 15 mM HEPES and 200 mM L-glutamine. The antibiotic solution contained 10,000 U/mL penicillin and 10,000 Ng/mL streptomycin. Additionally, the medium was supplemented with the mitogen Phytohemagglutinin (PHA, final concentration 3 Ng/mL, 10 % FBS (fetal bovine serum), the anticoagulant heparin.
About 72 h after seeding for each test group 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 NL S9 mix per mL medium were used. Concurrent solvent and positive controls were performed. After 4 h the cells were spun down by gentle centrifugation for 5 minutes (approx. 900 x g). The supernatant with the dissolved test item was discarded and the cells were re-suspended in "saline G". The washing procedure was repeated once as described.
The "saline G" solution was composed as follows (per litre):
NaCl 8000 mg
KCl 400 mg
glucose•H2O 1100 mg
Na2HPO4•2H20 192 mg
KH2PO4 150 mg
pH was adjusted to 7.2
After washing the cells were re-suspended in complete culture medium and cultured until preparation. All cultures were incubated at 37 °C in a humidified atmosphere with 5.5 % CO2 (94.5 % air). - Evaluation criteria:
- The slides were evaluated (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides, except for three values in the presence of S9 mix, where 50 metaphases were scored. Only metaphases with 46 ± 1 centromer regions were included in the analysis. To describe a cytotoxic effect, the mitotic index (% cells in mitosis) was determined.
A test item is classified as non-mutagenic if:
the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the laboratory historical control data
no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as mutagenic if:
the number of induced structural chromosome aberrations is not in the range of the laboratory historical control data
and either a concentration-related or a significant increase of the number of structuralchromosome aberrations is observed.
However, both biological and statistical significance should be considered together. If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications - Statistics:
- Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05)
Results and discussion
Test results
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Remarks:
- induced structural chromosomal aberrations in human lymphocytes in vitro
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 51.2% at highest evaluated concentration in absence of S9 mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures
Any other information on results incl. tables
TABLE 1 Doses applied in the Chromosome Aberration Assay with Test Item
Preparation |
Exposure |
Concentrations in µg/mL |
|||||||||
Without S9 mix |
|||||||||||
|
|||||||||||
22 hrs |
4 hrs |
15.4 |
26.9 |
47.2 |
82.5 |
144.4 |
252.7P |
442.9P |
773.9P |
1354.3P |
2370.0P |
With S9 mix |
|||||||||||
22 hrs |
4 hrs |
15.4 |
26.9 |
47.2 |
82.5 |
144.4 |
252.7P |
442.9P |
773.9P |
1354.3P |
2370.0P |
Evaluated
experimental points are shown in bold characters
P Precipitation
was observed at the end of treatment
TABLE 2 Summary of Results
Summary of results of the chromosomal aberration study with test item
Preparation |
Test item |
Mitotic indices |
Aberrant cells |
interval |
concentration |
in % |
in % |
|
in µg/mL |
of control |
incl. gaps* |
excl. gaps* |
carrying exchanges |
Exposure period 4 hrs without S9 mix |
|||||
22 hrs |
Solvent control1 |
100.0 |
2.5 |
2.0 |
0.0 |
|
Positive control2 |
62.3 |
8.5 |
8.0S |
0.5 |
|
144.4 |
78.8 |
2.5 |
2.5 |
0.5 |
|
252.7P |
72.4 |
3.5 |
2.5 |
0.0 |
|
1354.3P |
51.2 |
10.0 |
10.0S |
2.0 |
Exposure period 4 hrs with S9 mix |
|||||
22 hrs |
Solvent control1 |
100.0 |
1.0 |
1.0 |
0.0 |
|
Positive control3 |
65.2 |
12.5 |
12.5S |
2.0 |
|
15.4 |
72.8 |
11.5 |
11.0S |
0.5 |
|
26.9# |
89.8 |
45.0 |
44.0S |
0.0 |
|
47.2 |
82.2 |
16.0 |
16.0S |
2.0 |
|
82.5# |
102.9 |
52.0 |
52.0S |
5.0 |
|
144.4# |
101.6 |
61.0 |
61.0S |
4.0 |
* Including cells carrying exchanges
# Evaluation of 50 metaphases per culture
P Precipitation occurred at the end of treatment
S Aberration frequency statistically significant higher than corresponding control values
1 DMSO 0.5 % (v/v)
2 EMS 825.0 µg/mL
3 CPA 7.5 µg/mL
TABLE 3 Toxicity – Main Experiment (Cytotoxicity of Test Item to the Cultures of Human Lymphocytes)
In the Main Experiment the mitotic index in two cultures (1000 cells per culture) was determined.
Concentration |
Exposure time |
Preparation interval |
Mitotic cells |
% of |
Without S9 mix |
||||
Solvent control |
4 hrs |
22 hrs |
18.9 |
100.0 |
15.4 |
4 hrs |
22 hrs |
19.7 |
104.2 |
26.9 |
4 hrs |
22 hrs |
18.4 |
97.3 |
47.2 |
4 hrs |
22 hrs |
17.7 |
93.9 |
82.5 |
4 hrs |
22 hrs |
16.5 |
87.3 |
144.4 |
4 hrs |
22 hrs |
14.9 |
78.8 |
252.7P |
4 hrs |
22 hrs |
13.7 |
72.4 |
442.9P |
4 hrs |
22 hrs |
12.9 |
68.4 |
773.9P |
4 hrs |
22 hrs |
9.3 |
49.3 |
1354.3P |
4 hrs |
22 hrs |
9.7 |
51.2 |
2370.0P |
4 hrs |
22 hrs |
7.7 |
40.6 |
With S9 mix |
||||
Solvent control |
4 hrs |
22 hrs |
19.1 |
100.0 |
15.4 |
4 hrs |
22 hrs |
13.9 |
72.8 |
26.9 |
4 hrs |
22 hrs |
17.2 |
89.8 |
47.2 |
4 hrs |
22 hrs |
15.7 |
82.2 |
82.5 |
4 hrs |
22 hrs |
19.7 |
102.9 |
144.4 |
4 hrs |
22 hrs |
19.4 |
101.6 |
252.7P |
4 hrs |
22 hrs |
11.5 |
60.2 |
442.9P |
4 hrs |
22 hrs |
13.9 |
72.8 |
773.9P |
4 hrs |
22 hrs |
0.0 |
0.0 |
1354.3P |
4 hrs |
22 hrs |
4.9 |
25.7 |
2370.0P |
4 hrs |
22 hrs |
0.0 |
0.0 |
Experimental groups
evaluated for cytogenetic damage are shown in bold characters
* Mean
value of two cultures in %
P Precipitation
occurred at the end of treatment
TABLE 4 Main
Experiment-Mitotic
Index;
(Preparation
Interval 22 hrs with and without S9 Mix)
Treatment |
Conc. |
S9 |
Exposure |
Polyploid |
Mitotic Indices* |
||||
group |
per mL |
mix |
period/ |
cells |
Absolute |
Mean |
%** |
||
|
|
|
Recovery (hrs) |
|
1 |
2 |
|
|
|
Solv. control# |
0.5 % |
- |
4 / 18 hrs |
No polyploidies observed |
18.6 |
19.1 |
18.9 |
100.0 |
|
Pos. control## |
825.0 µg |
- |
4 / 18 hrs |
" |
11.5 |
12.0 |
11.8 |
62.3 |
|
Test item |
144.4 µg |
- |
4 / 18 hrs |
" |
14.8 |
14.9 |
14.9 |
78.8 |
|
" |
252.7 µg |
- |
4 / 18 hrs |
" |
14.7 |
12.6 |
13.7 |
72.4 |
|
" |
1354.3 µg |
- |
4 / 18 hrs |
" |
11.1 |
8.2 |
9.7 |
51.2 |
|
Solv. control# |
0.5 % |
+ |
4 / 18 hrs |
No polyploidies observed |
16.8 |
21.4 |
19.1 |
100.0 |
|
Pos. control### |
7.5 µg |
+ |
4 / 18 hrs |
" |
10.8 |
14.1 |
12.5 |
65.2 |
|
Test item |
15.4 µg |
+ |
4 / 18 hrs |
" |
11.6 |
16.2 |
13.9 |
72.8 |
|
" |
26.9 µg |
+ |
4 / 18 hrs |
" |
14.1 |
20.2 |
17.2 |
89.8 |
|
" |
47.2 µg |
+ |
4 / 18 hrs |
" |
15.5 |
15.9 |
15.7 |
82.2 |
|
" |
82.5 µg |
+ |
4 / 18 hrs |
" |
19.7 |
19.6 |
19.7 |
102.9 |
|
" |
144.4 µg |
+ |
4 / 18 hrs |
" |
20.8 |
18.0 |
19.4 |
101.6 |
* The mitotic index was determined in a sample of 1000 cells per culture of each test group in %
** For the positive control groups and the test item groups, the relative values of the mitotic index are related to the solvent controls
# DMSO
## EMS
### CPA
TABLE 5 Structural Chromosome Aberrations Main Experiment;
(Preparation Interval 22 hrs without S9 Mix:
Exposure Period 4 hrs)
Slide |
Cells |
% Aberrant cells |
Aberrations |
|
|||||||||||||||||||||||||||||
no. |
scored |
incl. |
excl. |
carrying ex-changes |
Gaps |
Chromatid type |
Chromosome type |
Other |
|
||||||||||||||||||||||||
|
|
gaps* |
gaps* |
|
g |
ig |
b |
f |
d |
ex |
ib |
if |
id |
cx |
ma |
cd |
|||||||||||||||||
|
|
||||||||||||||||||||||||||||||||
Solvent control: DMSO 0.5 % |
Without S9 mix |
||||||||||||||||||||||||||||||||
1 |
100 |
|
|
|
0 |
0 |
2 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||||||||||||||||
2 |
100 |
|
|
|
1 |
0 |
2 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||||||||||||||||
1 + 2 |
200 |
2.5 |
2.0 |
0.0 |
1 |
0 |
4 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||||||||||||||||
Positive control: EMS 825.0 µg / mL |
|
||||||||||||||||||||||||||||||||
1 |
100 |
|
|
|
0 |
0 |
6 |
1 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
|||||||||||||||||
2 |
100 |
|
|
|
1 |
0 |
7 |
2 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
|||||||||||||||||
1 + 2 |
200 |
8.5 |
8.0 |
0.5 |
1 |
0 |
13 |
3 |
0 |
1 |
0 |
1 |
0 |
0 |
0 |
0 |
|||||||||||||||||
Test item: 144.4 µg / mL |
|
||||||||||||||||||||||||||||||||
1 |
100 |
|
|
|
0 |
0 |
2 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||||||||||||||||
2 |
100 |
|
|
|
0 |
0 |
1 |
0 |
0 |
1 |
0 |
1 |
0 |
0 |
0 |
0 |
|||||||||||||||||
1 + 2 |
200 |
2.5 |
2.5 |
0.5 |
0 |
0 |
3 |
0 |
0 |
1 |
0 |
1 |
0 |
0 |
0 |
0 |
|||||||||||||||||
Test item: 252.7 µg / mL |
|
||||||||||||||||||||||||||||||||
1 |
100 |
|
|
|
1 |
0 |
3 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||||||||||||||||
2 |
100 |
|
|
|
1 |
0 |
2 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||||||||||||||||
1 + 2 |
200 |
3.5 |
2.5 |
0.0 |
2 |
0 |
5 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||||||||||||||||
Test item: 1354.3 µg / mL |
|
||||||||||||||||||||||||||||||||
1 |
100 |
|
|
|
0 |
0 |
10 |
0 |
0 |
3 |
0 |
0 |
0 |
0 |
1 |
0 |
|||||||||||||||||
2 |
100 |
|
|
|
0 |
0 |
7 |
0 |
0 |
2 |
0 |
2 |
0 |
0 |
0 |
0 |
|||||||||||||||||
1 + 2 |
200 |
10.0 |
10.0 |
2.0 |
0 |
0 |
17 |
0 |
0 |
5 |
0 |
2 |
0 |
0 |
1 |
0 |
* Including cells carrying exchanges
Abbreviations
g = gap, ig = iso-gap (gaps are achromatic lesions of chromatid or chromosome type where no or only a minimal misalignment of chromosomal material is visible), b = break, ib = iso-break, f = fragment, if = iso-fragment, d = deletion, id = iso-deletion, ma = multiple aberration (= more than 4 events in one cell [excluding gaps]), ex = chromatid type exchange, cx = chromosome type exchange, cd = chromosomal disintegration (= pulverization)
TABLE 6 Structural Chromosome Aberrations Main
Experiment;
(Preparation Interval 22 hrs with S9 Mix:
Exposure Period 4 hrs)
Slide |
Cells |
% Aberrant cells |
Aberrations |
|
|||||||||||||||||||||||||||||
no. |
scored |
incl. |
excl. |
carrying ex-changes |
Gaps |
Chromatid type |
Chromosome type |
Other |
|
||||||||||||||||||||||||
|
|
gaps* |
gaps* |
|
g |
ig |
b |
f |
d |
ex |
ib |
if |
id |
cx |
ma |
cd |
|||||||||||||||||
|
|
||||||||||||||||||||||||||||||||
Solvent control: DMSO 0.5 % |
Without S9 mix |
||||||||||||||||||||||||||||||||
1 |
100 |
|
|
|
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||||||||||||||||
2 |
100 |
|
|
|
0 |
0 |
2 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||||||||||||||||
1 + 2 |
200 |
1.0 |
1.0 |
0.0 |
0 |
0 |
2 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||||||||||||||||
Positive control: CPA 7.5 µg / mL |
|
||||||||||||||||||||||||||||||||
1 |
100 |
|
|
|
0 |
0 |
12 |
0 |
0 |
1 |
1 |
0 |
0 |
0 |
0 |
0 |
|||||||||||||||||
2 |
100 |
|
|
|
0 |
0 |
15 |
0 |
0 |
3 |
1 |
0 |
0 |
0 |
0 |
0 |
|||||||||||||||||
1 + 2 |
200 |
12.5 |
12.5 |
2.0 |
0 |
0 |
27 |
0 |
0 |
4 |
2 |
0 |
0 |
0 |
0 |
0 |
|||||||||||||||||
Test item: 15.4 µg / mL |
|
||||||||||||||||||||||||||||||||
1 |
100 |
|
|
|
1 |
0 |
11 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
|||||||||||||||||
2 |
100 |
|
|
|
0 |
0 |
9 |
0 |
0 |
1 |
0 |
2 |
0 |
0 |
1 |
0 |
|||||||||||||||||
1 + 2 |
200 |
11.5 |
11.0 |
0.5 |
1 |
0 |
20 |
0 |
0 |
1 |
1 |
2 |
0 |
0 |
1 |
0 |
|||||||||||||||||
Test item: 26.9 µg / mL** |
|
||||||||||||||||||||||||||||||||
1 |
50 |
|
|
|
1 |
0 |
21 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||||||||||||||||
2 |
50 |
|
|
|
0 |
0 |
38 |
4 |
0 |
0 |
2 |
0 |
0 |
0 |
7 |
0 |
|||||||||||||||||
1 + 2 |
100 |
45.0 |
44.0 |
0.0 |
1 |
0 |
59 |
5 |
0 |
0 |
2 |
0 |
0 |
0 |
7 |
0 |
|||||||||||||||||
Test item: 47.2 µg / mL |
|
||||||||||||||||||||||||||||||||
1 |
100 |
|
|
|
1 |
0 |
14 |
1 |
0 |
1 |
0 |
1 |
0 |
0 |
0 |
0 |
|||||||||||||||||
2 |
100 |
|
|
|
2 |
0 |
13 |
0 |
0 |
3 |
0 |
0 |
0 |
0 |
0 |
0 |
|||||||||||||||||
1 + 2 |
200 |
16.0 |
16.0 |
2.0 |
3 |
0 |
27 |
1 |
0 |
4 |
0 |
1 |
0 |
0 |
0 |
0 |
|||||||||||||||||
Test item: 82.5 µg / mL** |
|
||||||||||||||||||||||||||||||||
1 |
50 |
|
|
|
2 |
0 |
41 |
0 |
0 |
3 |
0 |
0 |
0 |
0 |
4 |
0 |
|||||||||||||||||
2 |
50 |
|
|
|
2 |
0 |
31 |
0 |
0 |
3 |
1 |
0 |
0 |
0 |
3 |
0 |
|||||||||||||||||
1 + 2 |
100 |
52.0 |
52.0 |
5.0 |
4 |
0 |
72 |
0 |
0 |
6 |
1 |
0 |
0 |
0 |
7 |
0 |
|||||||||||||||||
Test item: 144.4 µg / mL** |
|
||||||||||||||||||||||||||||||||
1 |
50 |
|
|
|
0 |
0 |
53 |
3 |
0 |
2 |
2 |
2 |
0 |
0 |
10 |
0 |
|||||||||||||||||
2 |
50 |
|
|
|
0 |
0 |
30 |
1 |
0 |
2 |
0 |
0 |
0 |
0 |
6 |
0 |
|||||||||||||||||
1 + 2 |
100 |
61.0 |
61.0 |
4.0 |
0 |
0 |
83 |
4 |
0 |
4 |
2 |
2 |
0 |
0 |
16 |
0 |
* Including cells carrying exchanges
** Evaluation of 50 metaphases per culture
Abbreviations
g = gap, ig = iso-gap (gaps are achromatic lesions of chromatid or chromosome type where no or only a minimal misalignment of chromosomal material is visible), b = break, ib = iso-break, f = fragment, if = iso-fragment, d = deletion, id = iso-deletion, ma = multiple aberration (= more than 4 events in one cell [excluding gaps]), ex = chromatid type exchange, cx = chromosome type exchange, cd = chromosomal disintegration (= pulverization)
TABLE 7 Biometry
Statistical significance at the five per cent level (p < 0.05) for aberration frequency was evaluated by means of the Fisher’s exact test. Evaluation was performed only for cells carrying aberrations excluding gaps.
Biometry of the Main Experiment
Test group versus |
Preparation |
Exposure |
S9 mix |
p-value |
|
Test group |
144.4µg/mL |
22 hrs |
4 hrs |
- |
0.376 |
" |
252.7µg/mL |
22 hrs |
4 hrs |
- |
0.376 |
" |
1354.3µg/mL |
22 hrs |
4 hrs |
- |
0.003S |
" |
15.4µg/mL |
22 hrs |
4 hrs |
+ |
< 0.001S |
" |
26.9 µg/mL |
22 hrs |
4 hrs |
+ |
< 0.001S |
" |
47.2 µg/mL |
22 hrs |
4 hrs |
+ |
< 0.001S |
" |
82.5 µg/mL |
22 hrs |
4 hrs |
+ |
< 0.001S |
" |
144.4 µg/mL |
22 hrs |
4 hrs |
+ |
< 0.001S |
Positive control versus |
|
|
|
|
|
EMS |
825.0 µg/mL |
22 hrs |
4 hrs |
- |
0.003S |
CPA |
7.5 µg/mL |
22 hrs |
4 hrs |
+ |
< 0.001S |
n.c. Not
calculated as the aberration rate is equal or lower than the control rate
S Aberration
rate is statistically significant higher than the control rate
Applicant's summary and conclusion
- Conclusions:
- In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation, when tested up to cytotoxic and/or precipitating or the highest evaluable concentration.
The study is considered to be relevant, reliable, adequate for risk assessment, and adequate for classification purposes. - Executive summary:
Study Design:
This in vitro assay was performed to assess the potential of the test item to induce structural chromosomal aberrations in the absence and presence of an exogenous metabolic activation system (liver S9 mix from phenobarbital/beta-naphthoflavone treated male rats). In each experimental group two parallel cultures were analysed. Per culture 100 metaphases were evaluated for structural chromosomal aberrations, except for three values in the presence of S9 mix, where 50 metaphases were scored. The highest applied concentration in the main study (2370.0 Ng/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight and the purity (92.9 %) of the test item and with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiments was performed considering the toxicity data and test item precipitation and in accordance with OECD Guideline 473.
Results:
In the absence of S9 mix, cytotoxicity was observed at the highest evaluated concentration.
In the presence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage.
Clastogenicity was observed in the absence of S9 mix after treatment with 1354.3 Ng/mL (10.0 % aberrant cells, excluding gaps) and in the presence of S9 mix after treatment with all evaluated concentrations. All values for percent aberrant cells (excluding gaps) are statistically significant and above the range of the laboratory historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps).
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.
Conclusion:
Under the experimental conditions reported, the test item induced structural chromosomal aberrations in human lymphocytes in vitro. Therefore, the test item is considered to be clastogenic in this chromosome aberration test in the absence and presence of metabolic activation, when tested up to cytotoxic and/or precipitating or the highest evaluable concentration
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