pentasodium 4-hydroxy-7-[(sulfonatomethyl)amino]-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]-8-[(2-sulfonato-4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-2-sulfonate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Jun. 24, 2002 to Aug. 21, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted according to OECD Guideline 474, EPA OPPTS 870.5395 and EU method B.12. in compliance with GLP.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): Reaktiv-Scharlach F01-0467

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: HARLAN WINKELMANN Gartenstr. 27, D-33178 Borchen, SPF breeding colony
- Age at study initiation: Approximately 6 wk
- Weight at study initiation (mean): 187.9 g (males) ; 146.5 g (females)
- Housing: Macrolon cages (type 4) on soft wood granulate in groups of 5 animals
- Diet: ssniff R/M (V1534), ad libitum, except for the period in which the animals were kept in diuresis cages
- Water: Tap water in plastic bottles, ad libitum, except for the period in which the animals were kept In diuresis cages
- Acclimation period: At least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C
- Humidity (%): 50 °C
- Photoperiod (hrs dark / hrs light): 12 h dark/12 h light

IN-LIFE DATES: From Jun. 24, 2002 to Jun. 26, 2002

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Sesame oil
- Concentration of test material in vehicle: 0, 20 %
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: On the days of administration the test substance was dissolved in sesame oil at a appropriate concentration (0 and 20 %). A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.

Duration of treatment / exposure:

Not applicable

Frequency of treatment:

Twice separate doses separated by an interval of 24 h

Post exposure period:

24 h after last dosing

Doses / concentrations

Remarks:

Doses / Concentrations:
2000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: Oral
- Doses / concentrations: 40 mg/kg bw and 0.4 % (w/v) concentration

Examinations

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: No preliminary experiments were performed, as corresponding toxicological information was available. Since 2000 mg/kg bw resulted in no lethality in acute oral toxicity testing a limit test with this dose was performed.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Twice at an interval of 24 h and animals were killed by carbon dioxide asphyxiation 24 h after dosing.

DETAILS OF SLIDE PREPARATION: For each animal, about 3 mL fetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 min at approx, 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 h.The specimens were fixed in methanol and stained with May-Grunwald/Giemsa.



METHOD OF ANALYSIS: Prior to microscopic assessment, all slides were furnished with code numbers, so that the counting was blind. The following counts were made:
Number of polychromatic erythrocytes (PCE) with micronuclei per 2000 erythrocytes
Number of micronuclei (MN) in 2000 erythrocytes.
Ratio of polychromatic erythrocytes to 200 normochromatic erythrocytes

Evaluation criteria:

A substance is considered positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group

Statistics:

Monotone-dose-relationship, One-sided Wilcoxon tests were performed starting with the highest dose group.
These tests were performed with a multiple level of significance of 5%

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: LD50>2000 mg/kg bw


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No significant difference as compared to controls.
- Ratio of PCE/NCE (for Micronucleus assay): The ratio of PCE to total erythrocytes remained essentially unaffected by the test substance and was not less than 20 % of the control values
- Appropriateness of dose levels and route: Yes
- Statistical evaluation: Yes

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the test conditions, the test substance is considered not clastogenic in the micronucleus test in SD rats.

Executive summary:

A study was conducted to assess the potential of the test substance to cause chromosomal damage (clastogenicity) in a rat bone marrow micronucleus test according to OECD Guideline 474, EPA OPPTS 870.5395 and EU method B.2. in compliance with GLP.

 

The test substance was dissolved in sesame oil and was given twice at an interval of 24 h as an oral dose of 2000 mg/kg bw to male and female rats (Hsd:Sprague Dawley). At study start the animals were 6 wk of age and had mean body weights of 187.9 g (M) and 146.5 g (F). According to the test procedure the animals were killed 24 h after administration.

Cyclophosphamide was used as positive control substance and was administered once orally at a dose of 40 mg/kg bw.

The number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with test substance and differed less than 20 % of the control value.

Cyclophosphamide induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

 

Under the test conditions, the test substance is considered not clastogenic in the micronucleus test in SD rats.