Reaction products of 7-amino-4-hydroxynaphthalene-2-sulfonic acid reacted with 2-((1,3,5-triazin-2-yl)amino)ethan-1-ol, coupled with diazotised 3-amino-4-hydroxybenzenesulfonic acid, subsequently coupled with diazotised 2-amino-5-methoxybenzenesulfonic acid, chelated with copper, sodium salt

Genetic toxicity: in vivo

Some information in this page has been claimed confidential.

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-04-05 to 1991-06-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

endogenous gene animal assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

Direct Red 83:1

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Mice albino CD1 strain
to assess the potential of Kayarus Supra Rubine
BIN to produce damage to chromosomes or aneuploidy when administered
orally to mice. Groups of 10 mice
5 males (23-28g)
and 5 females (20-25g)
Approx. 5-8 weeks old
Minimum of acclimatisation period of 5 days
Gavage of a single oral dose of Kayarus Supra Rubine BLN at
5000 mg/kg.
Animals were killed 24, 48 or 72 hours later, the bone marrows extracted and smear preparations made and stained. Polychromatic
and normochromatic erythrocytes were scored for the presence of micronuclei.
Further groups of mice were dosed with arachis oil B.P. or cyclophosphamide, to serve as vehicle and positive controls respectively

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Oil Arachis B.P.
Identification and stability of the vehicle control were not determined.

Details on exposure:

- Storage temperature of food: Room temperature

Duration of treatment / exposure:

Animals were observed 1 hour after dosing and subsequently once daily for 3 days. Any deaths and evidence of overt toxicity were recorded at each observation.

Frequency of treatment:

Once daily for 3 days

Doses / concentrations

Dose / conc.:

5 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Range finding Toxicity Study:
Dose level: 5000 mg/kg
Concentration: 500 mg/mL
Dose Volume: 10 ml/kg
No. of male 5
No. of female 5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide Monohydrate was freshly prepared as required as a solution at the appropriate concentration in distilled water. Identification and stability of control material and the preparations were not determined.

Examinations

Tissues and cell types examined:

Erythrocytes (micronucleaded polychromatic and normochromatic)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Range finding study



DETAILS OF SLIDE PREPARATION:
Immediately following sacrifice (i.e. 24, 48 or 72 hours), one femur was dissected and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixe in absolute methanol, and stained in May-Grünwald/Giemsa.

EVALUATION OF SLIDES
Stained bone marrow smears were examined at random using light microscopy at x 1000 magnification. The incidence of micronucleated cells per 1000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored.
Micronuclei are normally circular in shape, although occasionally they may be oval or half moon shaped, and have a sharp contour with even staining. In additional the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 polychromatic erythrocytes was counted; these cells were also scored for incidence of micronuclei.
The ratio of normochromatic to polychromatic erythrocytes was calculated together with appropriate group mean values for males and females separately and combined.

Evaluation criteria:

There are several criteria for determining a positive result, such as:
- a dose-related increase in the number of micronucleated cells or
-a clear increase in the number of micronucleated cells in a single dose group at a single sampling time.
A test substance for which the results do not meet the above criteria is considered nonmutagenic in this test.
-Biological relevance of the results.
-Statistical significance
A positive response for bone marrow toxicity is demonstrated when the dose group mean normochromatic to polychromatic ratio is shown to be statistically significant from the concurrent vehicle control group.

Statistics:

If necessary, and where possible, all data were statistically analysed using appropriate statistical methods as recommended by the UKENS sub-committee and guidelines for mutagenicity testing Report part III (1989)

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

No significant change in the NCE/PCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE FINDING TOXICITY STUDY:
A range-finding study was performed to determine a suitable dose level for the micronucleus study. The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of cytotoxicity up to a maximum recommended dose of 5000 mg/kg.
Groups of mice were dosed as follows:
DOSE LEVEL CONCENTRATION DOSE VOLUME NUMBER OF MICE
mg/kg mg/mL mL/kg Male Female

5000 500 10 7 7

All animals were dosed at once only by the appropriate dose level by gavage. The volume administered to each animal was calculated according to its fasted bodyweight at the time of dosing.
Animals were observed 1 hour after dosing and subsequently once daily for 3 days. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed.
The mortality data are summarised as follows:
DOSE LEVEL SEX NUMBER OF ANIMALS TREATED DEATH ON DAY TOTAL DEATH
0 1 2 3 1/14
5000 mg/kg Male 7 0 0 0 0
Female 7 1 0 0 0
No clinical signs were observed in any of the animals dosed with KAYARUS SUPRA RUBINE BLN.
There was only a premature death after one hour.
5000 mg/kg was selected as the maximum recommended dose for use in the micronucleus study.
RESULTS OF DEFINITIVE STUDY: see below.
- Statistical evaluation:If necessary, and where possible, all data were statistically analysed using appropriate statistical methods as recommended by the UKENS sub-committee and guidelines for mutagenicity testing Report part III (1989)
- Ratio of PCE/NCE (for Micronucleus assay):
There was no significant change in the NCE/PCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups.

Any other information on results incl. tables

Test design:

Dose Group	Dose Level mg/kg	Concentration mg/mL	Dose Volume ml/kg	Kill time (hours after dosing)	Animal     Male	Numbers   female
Vehicle control (Arachis oil B.P.)	0	0	10	72	1-5	6-10
Vehicle control (Arachis oil B.P.)	0	0	10	48	11-15	16-20
Vehicle control (Arachis oil B.P.)	0	0	10	24	21-25	26-30
Positive Control (Cyclophosphamide)	50	5	10	24	31-35	36-40
Kayarus Supra Rubine BLN	5000	500	10	72	41-45	46-50
Kayarus Supra Rubine BLN	5000	500	10	48	51-55	56-60
Kayarus Supra Rubine BLN	5000	500	10	24	61-65	66-70

 

Comparison was made between no. of micronucleated polychromatic erythrocytes occurring in each of the three test material groups and the no occurring in the corresponding vehicle control groups.

A positive mutagenic response is demonstrated when a statistically significant increasing the number of micronucleated polychromatic erythrocytes is observed for ether of the kill times.

A positive response of bone marrow toxicity is demonstrated when the dose group mean normochromatic to polychromatic ratio is shown to be statistically significant from the concurrent vehicle control group.

 Statistical methods, if necessary: which are recommended by the UKEMS sub-committee on guidelines for mutagenicity testing, Report, part III (1989)

 

Results:

Mortality Data and Clinical observations:

There were no premature deaths in any of the dose groups.

Clinical signs in animals dosed with Kayarus Supra Rubine BLN: diarrhoea and coat stained with test substance.

Evaluation of Bone Marrow Slides:

There was no significant increase in the frequency of micronucleated PCE's in any of the test material dose groups when compared to their concurrent vehicle control groups, the increases
seen in the 72 and 24-hour sampling time groups were within accepted ranges.

 There was no significant increase in the frequency of micronucleated NCE's in any of the test material dose groups when compared to their concurrent vehicle control groups.

There was no significant change in the NCE/PCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence
confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test. The test material, Kayarus Supra Rubine BLN, was found not to produce micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

 

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The test material, Kayarus Supra Rubine BLN, was considered to be non-genotoxic under the conditions of the test.

Executive summary:

In order to assess the potential of the test substance to produce damage to chromosomes or anopleudias when administered oral to mice, a study was performed according to the OECD guidelines No 474 "Genetic toxicity: Micronucleus Test" and method B12 of Commission Directive 84/449/EEC.

Following a preliminary range finding study to confirm the oral toxicity of Kayarus Supra Rubine BLN, the study was conducted using the test item at the maximum recommended dose level (5000 mg/kg)

In the micronucleus study a group of ten animals (5 male and 5 female) were given a single dose of the test substance.Animals were killed 24, 48 or 72 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei.

Further groups of mice were dosed with arachis oil B.P. or cyclophosphamide, to serve as vehicle and positive controls, respectively.

Results:

Mortality Data and Clinical observations:

There were no premature deaths in any of the dose groups.

Clinical signs in animals dosed with Kayarus Supra Rubine BLN: diarrhoea and coat stained with test substance.

Evaluation of Bone Marrow Slides:

There was no significant increase in the frequency of micronucleated PCE's in any of the test material dose groups when compared to their concurrent vehicle control groups, the increases
seen in the 72 and 24-hour sampling time groups were within accepted ranges.

 There was no significant increase in the frequency of micronucleated NCE's in any of the test material dose groups when compared to their concurrent vehicle control groups.

There was no significant change in the NCE/PCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups.

 Based on the above findings and under the test conditions the test substance, was considered to be non-genotoxic under the conditions of the test.