[No public or meaningful name is available]

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

other: read across from analogue substance

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

yes

Remarks:

limited given data

GLP compliance:

yes

Remarks:

HAZLETON LABORATORIES AMERICA, INC.

Type of assay:

unscheduled DNA synthesis

Test material

Test material information

1

Reference substance name:

Monosodium aqua-[5-[[2,4-dihydroxy-5-[(2-hydroxy-3,5-dinitrophenyl)azo]phenyl]azo]-2-naphthalensulfonate], iron complex

EC Number:

400-720-9

EC Name:

Monosodium aqua-[5-[[2,4-dihydroxy-5-[(2-hydroxy-3,5-dinitrophenyl)azo]phenyl]azo]-2-naphthalensulfonate], iron complex

Cas Number:

126851-40-9

Molecular formula:

C22H11FeN6NaO10S.H2O

IUPAC Name:

Monosodium aqua-[(5-((E)-(5-((Z)-(3,5-dinitro-2-oxidophenyl)diazenyl)-2-hydroxy-4-oxidophenyl)diazenyl)naphthalene-2-sulfonate)ferrate(-)]

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc.
- Age at study initiation: adult
- Weight at study initiation: 150 - 300 g
- Assigned to test groups randomly: yes
- Diet (e.g. ad libitum): Purina Certified Rodent Chow (Formula 5002); ad libitum
- Water (e.g. ad libitum): water; ad libitum
- Acclimation period: minimum of 5 days prior to use

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Amount of vehicle (if gavage or dermal): 9 ml/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Fresh preparations of test article in vehicle were used for any testing purpose.

Duration of treatment / exposure:

4 h

Frequency of treatment:

single application

Post exposure period:

4 h

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000, 2000, 4000 mg/kg bw
Basis:
nominal in water

No. of animals per sex per dose:

3 animals/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Dimethylnitrosamine (DMN)
- Justification for choice of positive control(s): known to induce UDS in vivo in rat hepatocytes
- Route of administration: intraperitoneal
- Doses / concentrations: ca. 10 mg/kg bw

Examinations

Tissues and cell types examined:

hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In the preliminary study to determine dose and perfusion time, an attempt was made to gavage two animals with 5000 mg/kg, but the test material formed a sludge in CMC that could not be forced through a syringe. The highest dose that could be administered was between approximately 3200 mg/kg and 3400 mg/kg although one animal died due to gavage back up. The amount administered was not exact because the sludge was difficult to measure. One rat was sacrificed (liver perfusion) approximately 4.5 hours later and the other approximately 15 hours later and slides were prepared for UDS. Microscopic examination of the slides prepared at the two time points indicated that they were similar. It was decided that the UDS assay would be performed approximately 4 hours after administration of a single dose of the test material.


DETAILS OF SLIDE PREPARATION:
UDS based on the procedures in rats described by Williams (1980) and Mirsalis, Tyson and Butterworth (1982): Briefly, isolated hepatocytes were cultured for 1.5 to 2 hours at 37°C in a humidified atmosphere containing 5 % C02. Three of the replicate cultures from each animal were used for the UDS assay, thus labeld and fixed with acetic acid : ethanol (1:3) and dried for at least 24 hours.

METHOD OF ANALYSIS:
The cells were examined microscopically at approximately 1500x magnification under oil immersion and the field was displayed on the video screen of an automatic counter. UDS was measured by counting nuclear grains and subtracting the average number of grains in three nuclear-sized areas adjacent to each nucleus (background count). This value is referred to as the net nuclear grain count. The coverslips were coded to prevent bias in grain counting. The net nuclear grain count was determined for 50 randomly selected cells on each coverslip. Only nuclei with normal morphologies were scored, and any occasional nuclei blackened by grains too numerous to count were excluded as cells in which replicative DNA synthesis occurred rather than repair synthesis. The mean net nuclear grain count was determined from the triplicate coverslips (150 total nuclei) for each treatment condition. Occasionally, a coverslip is recounted at a later date or by a different technician. Since a different cell population will generally be scored, the average count for 50 cells was used in the calculation of the mean for the triplicate treatment.

Evaluation criteria:

The test material is considered active in the UDS assay at applied concentrations that cause:
- an increase in the mean net nuclear grain count to at least six grains per nucleus after subtraction of the concurrent negative control value, and/or
- an increase in the percent of nuclei having 6 or more net grains to at least 10% of the analyzed population after subtraction of the concurrent negative control value, and/or
- the percent of nuclei with 20 or more grains to reach or exceed 2% of the analyzed population

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 3200 - 5000 mg/kg bw
- Solubility: material formed a sludge in CMC that could not be forced through a syringe at 5000 mg/kg bw

Any other information on results incl. tables

Results:

Test condition	Dose Level	Animal	UDS grains/nucleus	Average* % nuclei with
				>= 6 grains	>= 20 grains
water	-	1	0.45 ± 0.18	0.7	0.0
		2	-0.43 ± 0.10	0.7	0.0
		3	-0.163 ± 1.59	0.7	0.0
DMN	10 mg/kg bw	1	25.22 ± 8.79	94.7	64.7
		2	15.47 ± 2.65	83.3	41.3
		3	17.2 ± 7.8	84.0	41.3
Saeurebraun 6229	4000 mg/kg bw	1	0.36 ± 0.52	2.0	0.0
		2	-0.08 ± 0.73	0.0	0.0
		3	0.08 ± 0.66	0.7	0.0
	2000 mg/kg bw	1	-1.32 ± 0.50	0.7	0.0
		2	-1.00 ± 0.76	0.0	0.0
		3	-1.03 ± 1.02	0.7	0.0
	1000 mg/kg bw	1	-1.49 ± 0.57	0.0	0.0
		2	-1.11 ± 0.88	5.3	0.0
		3	-0.57 ± 0.60	0.0	0.0
	5000 mg/kg bw	1	-0.20 ± 0.20	1.3	0.0
		2	-1.18 ± 0.39	0.0	0.0
		3	-0.92 ± 0.75	1.3	0.0

UDS: Average of net nuclear grain counts on triplicate coverslips (150 total cells), ± standard deviation between coverslips

*: Average values for triplicate coverslips

Applicant's summary and conclusion

Conclusions:

The substance was tested for genotoxicity following OECD 486. The tets substance was inactive in the in-vitro/in-vivo Rat Epatocyte UDS Assay over a dosing range of about 500 to 4000 mg/kg.