Benzoic acid, 3,5-diamino-4-[2-[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-2-[2-[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-, sodium salt (1:?)

Genetic toxicity: in vitro

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

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

480-890-9

EC Name:

-

Cas Number:

906532-68-1

Molecular formula:

C23H24N6O17S5 · xNa

IUPAC Name:

sodium 3,5-diamino-2-[(E)-2-{2-sulfo-4-[2-(sulfooxy)ethanesulfonyl]phenyl}diazen-1-yl]-4-[(E)-2-{4-[2-(sulfooxy)ethanesulfonyl]phenyl}diazen-1-yl]benzoate

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 results

Test results 1

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

Test results 2

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	1
50	0.5
150	1
500	1.5
1500	0.5
5000	0.5
Positive control (MMC) 0.33	25
Scheme II (+S9, 3h)
Negative control, 0	0
50	1.5
150	8
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	3
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.