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EC number: 700-427-9 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Link to relevant study records
- 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
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: this study was planned and executed in accordance with relevant guidelines as well as the requirements of the GLP
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction
- Test concentrations with justification for top dose:
- test without metabolic activation and 3 h exposure period - 0.5, 1, 2, 4, 8 and 16 μg/ml
test with metabolic activation and 3 h exposure period - 4, 8, 16, 32, 64 and 128 μg/ml
test without metabolic activation and 24 h exposure period - 0.25, 0.5, 1, 2, 4, 8 and 16 μg/ml - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO is commonly used in Mammalian Chromosome Aberration Tests of water insoluble substances. It does not react with Atlen SK and is compatible with the survival of the cells and the S9 activity - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- tests without metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- tests with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 h (tests with and without metabolic activation) and 24 h (additional test without metabolic activation)
- Expression time (cells in growth medium): 2 d
- Fixation time (start of exposure up to fixation or harvest of cells): 1 d
NUMBER OF REPLICATIONS: experiments were conducted in duplicate culture
SPINDLE INHIBITOR (cytogenetic assays): demecolcine
STAIN (for cytogenetic assays): Giemsa stain
NUMBER OF CELLS EVALUATED: for the analysis 4x10^5 cells were seeded in duplicate; 100 metaphases per experiment were evaluated for aberrations
DETERMINATION OF CYTOTOXICITY
- Method: cytotoxicity of Atlen SK was determined in earlier Mammalian Cell Gene Mutation Test on the basis of plating efficiency - Evaluation criteria:
- The metaphases were analysed for the following structural aberrations: chromatid gaps and breaks, isochromatid gaps and breaks and exchange (dicentrics, double minutes, quadriradials, triradials and rings).
Gaps, as their genetic significance is not clearly understood, were not included in the assessment of chromosomal damage and were not evaluated statistically.
Unequivocal increases of aberrations above control levels which occurred at one or more concentrations were indication that substance gave positive results in the tests. - Statistics:
- The results were statistically evaluated using the test of difference of two relative values.
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- cytotoxicity was observed at concentration of 128 μg/ml in the 3 h experiment with metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- In the 3 h experiments without and with metabolic activation the percentages of aberrant metaphases (excluding gaps) in Alten SK treated groups were less than 3 % and less than 2 % respectively (Tables 1-4). No evidence of reduction of mitotic index was found in both tests. In the additional experiment with 24 h exposure (without metabolic activation) the percentage of aberrant metaphases (excluding gaps) was not different at any concentration level from that of the solvent control (Table 5). The percentages of aberrant metaphases (excluding gaps) in solvent controls ranged from 1 - 2 % (Tables 1 - 5). Positive control susbtances induced statistically significant increases in the incidence of aberrant metaphases (Tables 1 -6). For tables see section Any other information on results incl. tables.
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
Atlen SK was found to be negative in the In Vitro Mammalian Chromosome Aberration Test (both in experiment without metabolic activaton as well as in experiment with metabolic activation). - Executive summary:
Presented results originate from a guideline study conducted in accordance with the requirements of the GLP. Hence, this information can be considered reliable and suitable for use as the key study for this endpoint.
Reference
Table 1. Results of in vitro chromosome aberration analysis of Atlen SK effect without metabolic activation a,b (treatment 3-h) / A
Sample |
conc. (μg/ml) |
MI (%) |
Aberrant metaphases (%) |
Number of chromosomal aberrations per 100 metaphases |
|||||||||
Chromatid |
Isochromatid |
Exchange |
Total number of CA |
||||||||||
g |
b/f |
g |
b/f |
dic |
r |
dmin |
qr |
tr |
|||||
SC |
|
14.8 |
1 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
Tested Substance |
0.5 |
15.0 |
2 |
2 |
1 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
2 |
1.0 |
16.9 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
2.0 |
15.9 |
1 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
|
4.0 |
15.5 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
8.0 |
15.9 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
PC |
0.4 |
7.8 |
28*** |
3 |
30 |
1 |
0 |
0 |
0 |
0 |
24 |
8 |
62*** |
Table 2.Results of in vitro chromosome aberration analysis of Atlen SK effect without metabolic activation a,b (treatment 3-h) / B
Sample |
conc. (μg/ml) |
MI (%) |
Aberrant metaphases (%) |
Number of chromosomal aberrations per 100 metaphases |
|||||||||
Chromatid |
Isochromatid |
Exchange |
Total number of CA |
||||||||||
g |
b/f |
g |
b/f |
dic |
r |
dmin |
qr |
tr |
|||||
SC |
14.0 |
14.0 |
2 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
2 |
Tested Substance |
0.5 |
13.5 |
2 |
2 |
1 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
2 |
1.0 |
14.9 |
3 |
0 |
3 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
3 |
|
2.0 |
12.6 |
1 |
1 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
|
4.0 |
13.4 |
1 |
3 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
|
8.0 |
14.9 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
PC |
0.4 |
7.0 |
27*** |
2 |
23 |
0 |
0 |
1 |
1 |
0 |
21 |
3 |
49*** |
Table 3. Results of in vitro chromosome aberration analysis of Atlen SK effect with metabolic activation a,b (treatment 3-h) / A
Sample |
conc. (μg/ml) |
MI (%) |
Aberrant metaphases (%) |
Number of chromosomal aberrations per 100 metaphases |
|||||||||
Chromatid |
Isochromatid |
Exchange |
Total number of CA |
||||||||||
g |
b/f |
g |
b/f |
dic |
r |
dmin |
qr |
tr |
|||||
SC |
|
17.5 |
1 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
Tested Substance |
4 |
21.4 |
2 |
1 |
3 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
3 |
8 |
16.6 |
2 |
0 |
2 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
2 |
|
16 |
13.0 |
1 |
1 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
|
32 |
17.5 |
2 |
1 |
0 |
0 |
1 |
0 |
0 |
0 |
1 |
0 |
2 |
|
64 |
15.0 |
1 |
1 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
|
PC |
40 |
6.3 |
33*** |
3 |
32 |
0 |
0 |
0 |
0 |
0 |
23 |
13 |
68*** |
Table 4. Results of in vitro chromosome aberration analysis of Atlen SK effect with metabolic activation a,b (treatment 3-h) / B
Sample |
conc. (μg/ml) |
MI (%) |
Aberrant metaphases (%) |
Number of chromosomal aberrations per 100 metaphases |
|||||||||
Chromatid |
Isochromatid |
Exchange |
Total number of CA |
||||||||||
g |
b/f |
g |
b/f |
dic |
r |
dmin |
qr |
tr |
|||||
SC |
|
16.6 |
1 |
2 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
Tested Substance |
4 |
22.0 |
1 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
8 |
17.0 |
2 |
2 |
2 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
2 |
|
16 |
14.3 |
1 |
1 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
|
32 |
19.2 |
1 |
2 |
1 |
0 |
0 |
0 |
0 |
0 |
3 |
0 |
4 |
|
64 |
14.0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
PC |
40 |
6.8 |
33*** |
1 |
37 |
1 |
0 |
1 |
1 |
0 |
25 |
12 |
76*** |
Table 5. Results of in vitro chromosome aberration analysis of Atlen SK effect without metabolic activation a,b (treatment 24-h) / experiment A/experiment B
Sample |
conc. (μg/ml) |
MI (%) |
Aberrant metaphases (%) |
Number of chromosomal aberrations per 100 metaphases |
|||||||||
Chromatid |
Isochromatid |
Exchange |
Total number of CA |
||||||||||
g |
b/f |
g |
b/f |
dic |
r |
dmin |
qr |
tr |
|||||
SC |
|
10.0 /8.3 |
1/1 |
1/1 |
0/0 |
0/0 |
0/0 |
1/1 |
0/0 |
0/0 |
0/0 |
0/0 |
1/1 |
Tested Substance |
0.25 |
12.5 /13.5 |
1/1 |
0/0 |
1/1 |
0/0 |
0/0 |
0/0 |
0/0 |
0/0 |
0/0 |
0/0 |
1/1 |
0.50 |
7.5 /10.7 |
1/0 |
1/1 |
0/0 |
0/0 |
0/0 |
1/0 |
0/0 |
0/0 |
0/0 |
0/0 |
1/0 |
|
1.00 |
9.9 /9.7 |
1/1 |
0/2 |
0/1 |
0/0 |
0/0 |
1/0 |
0/0 |
0/0 |
0/0 |
0/0 |
1/1 |
|
2.00 |
8.8 /9.2 |
0/1 |
0/0 |
0/1 |
0/0 |
0/0 |
0/0 |
0/0 |
0/0 |
0/0 |
0/0 |
0/1 |
|
4.00 |
7.0 /8.3 |
1/0 |
0/1 |
1/0 |
0/1 |
0/0 |
0/0 |
0/0 |
0/0 |
0/0 |
0/0 |
1/0 |
|
8.00 |
3.0 /3.2 |
0/1 |
1/2 |
0/0 |
0/0 |
0/0 |
0/1 |
0/0 |
0/0 |
0/0 |
0/0 |
0/1 |
|
PC |
0.04 |
7.3 /10.1 |
32***/28*** |
5/1 |
20/18 |
0/0 |
1/1 |
0/0 |
0/0 |
2/0 |
14/15 |
6/4 |
43***/38*** |
a A number of 100 metaphases were scored in each sample. The numbers of chromatid and isochromatid gaps were recorded for each treatment group; however, since their genetic significance is not clearly understood, they are not included in the assessment of chromosomal damage.
b SC, solvent control, (DMEM containing 0.5 % DMSO); Tested substance, ATLEN SK (0.5 – 8.0 or 4 – 64 or 0.25 – 8 μg/ml; PC, positive control (0.4 or 0.04 μg/ml of mitomycin C or 40 μg/ml of cyclophosphamide was used as the positive control); CA, chromosomal aberrations; g, gap; b/f, break and/or fragment; dic, dicentric; dmin, double minute; r, ring; qr, quadriradial; tr, triradial;
MI, mitotic index of each culture was calculated by counting the number of metaphases per 1000 cells
*** p<0.001 significantly different from the solvent control value
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Atlen SK was evaluated for mutagenic potential in three in vitro tests.
In Ames test it was tested at concentrations 5, 2.5, 1.0, 0.5, 0.1, 0.01, 0.001, 0.0001 and 0.00001 mg/plate on S. typhimurium (strains TA 100, TA 98, TA 97, TA 1535 and TA 102) with and without metabolic activation. In this test Atlen SK produced neither a statistically significant dose-related increase in the number of revertants nor a statistically significant and reproducible positive response at any one of the test points and according to these results is considered non mutagenic in this system.
In mammalian cell gene mutation test Atlen SK was tested at concentrations 1, 2, 4, 6, 8, 16 and 32 μg/ml on Chinese hamster lung fibroblasts (V79) with and without metabolic activation. In this test Atlen SK induced neither dose dependent nor reproducible increase in the mutation frequency.
In mammalian chromosome aberration test Atlen SK was tested at concentrations 0.25, 0.5, 1, 2, 4, 8, 16, 32 64 and 128 μg/ml on Chinese hamster lung fibroblasts (V79) with and without metabolic activation. In this test Atlen SK did not induce statistically significant increases in the incidence of aberrant metaphases.
Justification for selection of genetic toxicity endpoint
One of the studies carried out on mammal.
Justification for classification or non-classification
Data available for Alten SK is conclusive but it is not sufficient for the classification of Atlen SK with regard to mutagenicity/genetic toxicity.
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