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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
The post-mitochondrial fraction (S9) of liver from Aroclor 1254 induced Sprague-Dawley rats was purchased from MOLTOX, Molecular Toxicology Inc., USA and was used for metabolic activation.
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 0, 50, 150, 500, 1500, 5000 µg/ml
Concentration range in the main test (without metabolic activation): 0, 50, 150, 500, 1500, 5000 µg/ml
Vehicle / solvent:
Culture medium
Controls
Untreated negative controls:
yes
Remarks:
10% sterile deionized water in culture medium was used as the negative control.
Positive controls:
yes
Remarks:
Mitomycin C at 1 µM ( µg/mL) was used as the positive control for 3-hour and 20-hour treatments without S9 activation. Cyclophosphamide at 40 uM was used for 3-hr treatment with S9 activation.
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Exposure period (with metabolic activation): 3 hours
Exposure period (without metabolic activation): 3 hours
Exposure period (without metabolic activation): 20 hours

Fixation time:
With/without S9: 3h exposure, 24h fixation (experiment 1)

Without S9: 24h exposure, 24h fixation time (experiment 2)

Without S9: 48h exposure, 48h fixation time (experiment 2)

With 3h exposure, 48h fixation (experiment 2)

Results and discussion

Test resultsopen allclose all
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
3hr, at the concentraion: 150 µg/ml, 500 µg/ml
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Concurrent Cytotoxicity Test:
Measurements of cytotoxicity for all treated and negative control cultures were conducted at the time of mitotic cell harvest. The cytotoxicity at all concentrations for all three schemes did not show more than 50% cqtotoxicity. The maximum top concentration recommended was used for all three schemes.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Concurrent Cytotoxicity analysis of Everzol Orange ED-G Crude in Chinese Hamster Ovary Cells

 Concentration (μg/mL)  Cell number (X E5 cells) Survival (%)  Cytotoxicity (%) 
Scheme I (-S9, 3h)       
 Negative control 30.7 100.0 0.0 
 50 26.4  86.0  14.0 
 150 30.1 98.0  2.0 
 500 24.7  80.5  19.5 
 1500 24.7  80.5  19.5 
 5000 23.2  75.6  24.4 
Scheme II (+S9, 3h)       
 Negative contral 23.9  100.0  0.0 
50  30.7  100.0  0.0 
150  28.9 100.0  0.0 
500  13.4  56.1 43.9 
1500  13.2  55.2  44.8
5000  26.9  100.0  0.0 
Scheme III (-S9, 20h)       
Negative control 31.2  100.0  0.0 
50 31.6  100.0  0.0 
150  29.2  93.6  6.4 
500  25.5  81.7  18.3 
1500 22.6  72.4  27.6 
5000  19.0  60.9  39.1 
       

Summary of Chromosome aberrations in Chinese Hamster Ovary cells

 Concentration (μg/mL) Aberrant Cells (%)
 Scheme I (-S9, 3h)  
 Negative control, 0
 50 0.5 
 150
 500 1.5 
 1500 0.5 
 5000 0.5 
 Positive control (MMC) 0.33 25 
 Scheme II (+S9, 3h)  
 Negative control, 0
50  1.5 
 150
 500 9.5 
 1500 Too few metaphases 
 5000 Too few metaphases 
 Positive control (CPP) 22.5 
 Scheme III (-S9, 20h)  
 Negative control, 0  0.5
 50  0
 150 0.5 
 500
 1500 Too few metaphases 
5000  Too few metaphases 
   

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation
negative without metabolic activation

In conclusion, Everzol Orange ED-G Crude did not induce structural chromosome aberration in CHO cells for all concentrations in the absence of S9 but induce structural chromosome aberration at the concentrations of 150 and 500 µg/ml in the presence of S9 for 3-hour treatment.
Executive summary:

The chromosome aberration assay was conducted in three test schemes: 3-hour exposure both with and without S9 activation (schemes I and II) and 20-hour continuous exposure without S9 (scheme III). Results were conducted in duplicate cultures and with negative control and positive controls concurrently. 100 metaphases for each culture and 200 metaphases for each treatment were scored. Results showed that percents of aberrant cells induced by negative control in the scheme I, II and III were 1%, 0% and 0.5%. The positive control induced significant increases in percents of aberrant cells over the corresponding negative control. Everzol Orange ED-G did not significantly increase the frequencies of structural chromosome aberration in the schemes I and III. However, the frequencies of structural chromosome aberration in the scheme II, at concentration of 150 and 500 um/mL in the 3 hr incubation have significantly increased.

In conclusion, Everzol Orange ED-G crude induced structural chromosome aberration at the concentration of 150 and 500 μg/mL at the presence of S9 for 3-hr treatment. The concentration-response analysis and trend probability were conducted in the 3-hr treatment with S9 and results showed lacking in concentration response.