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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian bone marrow chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
421-880-6
EC Name:
-
Cas Number:
201792-73-6
Molecular formula:
C34H25N11Na2O11S3
IUPAC Name:
disodium 4-amino-6-{2-[4-({4-[2-(2,4-diaminophenyl)diazen-1-yl]phenyl}sulfamoyl)phenyl]diazen-1-yl}-5-hydroxy-3-[2-(4-nitrophenyl)diazen-1-yl]naphthalene-2,7-disulfonate

Test animals

Species:
mouse
Strain:
CD-1
Details on species / strain selection:
Male and female CD-1 mice in the age range of 7-1 O weeks were used for Phase I and mice in the age range 5-6 weeks were used for Phase II ofthe study. The animals were supplied by Charles River Breeding Laboratories, Margate, UK.
Sex:
male/female
Details on test animals or test system and environmental conditions:
On arrival the mice were housed on mobile mouse cage racks and given food, CT 1 ( supplied by Special Diets Services, Stepfield, Witham, Essex, UK) and water ad libitum.
The animai rooms used for Phases I and II are designed to be maintained within a temperature range of l 9-23°C, and within a relative humidity range of 40-70%. Although small excursions in temperature were observed, these were considered not to affect the integrity of the study.
Lighting was controlled to prThe animal room was under positive pressure with respect to the access corridor and had at least 15 air changes per hourovide 12 hours artificial light followed by 12 hours darkness.

The animals were killed by asphyxiation in halothane Ph. Eur. (FLUOTHANE, Zeneca Pharmaceuticals) followed by cervical dislocation 24 and 48 hours after receiving a single oral dose of the test substance.
Femurs were removed and stripped clean ofmuscle. The iliac end ofthe femur was removed and a fine paint brush was rinsed in saline, wiped to remove the excess and wetted with a solution of albumin (6% w/v in physiological saline). This was then dipped into the marrow canal and two smears were painted on an appropriately labelled clean, dry microscope slide.
This procedure was repeated to give four smears of marrow per slide.
The slides were allowed to air dry and were stained with polychrome methylene blue and eosin using an automatic staining machine

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
corn oil
Details on exposure:
Phase I involved the determination of a maximum tolerated dose (MTD), based on patterns of lethalities or severe toxicity observed over a four-day observation period following a single oral dose.
After acclimatisation, the mice for Phase II were randomly distributed on to racks and the animals were identified by cage cards and by ear punching.
In Phase II, male and female animals were weighed and given a single oral dose of com oil (20 ml/kg), cyclophosphamide (65mg/kg) or Tested Substance (5000 mg/kg)
Duration of treatment / exposure:
An individuai stock suspension ofthe test substance was prepared in com oil far each group of animals.
The positive controI substance was prepared as a solution in physiological saline
All test and positive contrai substance dosing preparations were prepared as close to the time of dosing as possible. The test substance, vehicle and positive contro! substance were dosed at a volume of 20ml/kg bodyweight.
Frequency of treatment:
one single oral dose
Post exposure period:
4 days
Doses / concentrations
Dose / conc.:
5 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males and 5 females per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
The animals were killed by asphyxiation in halothane Ph. Eur. (FLUOTHANE, Zeneca Pharmaceuticals) followed by cervical dislocation 24 and 48 hours after receiving a single oral dose of the test substance.
Femurs were removed and stripped clean ofmuscle. The iliac end ofthe femur was removed and a fine paint brush was rinsed in saline, wiped to remove the excess and wetted with a solution of albumin (6% w/v in physiological saline). This was then dipped into the marrow canal and two smears were painted on an appropriately labelled clean, dry microscope slide.
This procedure was repeated to give four smears of marrow per slide.
The slides were allowed to air dry and were stained with polychrome methylene blue and eosin using an automatic staining machine.

Evaluation criteria:
Slides were coded and scored blind. Two thousand (2 x 1000) polychromatic erythrocytes were examined far the presence of micronuclei far each animal. The slides were also examined for evidence of cytotoxicity, which may be manifest by alterations in the ratio of different cell types in the bone marrow. This was assessed by counting the ratio of polychromatic to normochromatic erythrocytes in a sample of 1000 erythrocytes
Statistics:
The incidence of micronucleated polychromatic erythrocytes and percentage polychromatic erythrocytes in the erythrocyte sample, were considered by analysis of variance at 24 and 48 hours, separately for males and females.
The data for the incidence of micronucleated polychromatic erythrocytes were transformed using a square root transformation, prior to analysis. The data for the percentages of polychromatic erythrocytes were transformed using the double arcsine transformation of Freeman and Tukey (1950), prior to analysis.
Analyses were carried out using the GLM procedure in SAS (1989). Each treatment group mean was compared with the contrai group mean at the corresponding sampling time using a one-sided Student' s t-test, based on the errar mean square in the analysis.
The data have been interpreted as follows:-
a) No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes above concurrent vehicle contro! incidences - NEGATIVE.
b) A statistically significant increase in the incidence of micronucleated polychromatic erythrocytes above the concurrent vehicle contro! incidences but whichfalls within the laboratory historical vehicle control range - NEGATIVE.
e) A statistically and biological significant increase in the incidence of micronucleated polychromatic erythrocytes which is in excess of a three-fold increase when compared with both historical and concurrent vehicle control incidences - POSITIVE.
d) An incidence of micronucleated polychromatic erythrocytes which is statistically significantly different from the concurrent vehicle contro! incidences, but less than 3-fold in excess ofboth historical and concurrent vehicle contro! incidences may require further evaluation.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Phase I - Determination of the maximum tolerated dose
Substance was adinistered as a single oral dose to a group of 5 male and 5 female mice at a dose levèl of 5000mg/kg. No significant adverse reactions to treatment were observed with the exception of one male which was killed in extremis on Day 4; the death of this animai is considered not to be treatment related.
The maximum tolerated dose (MTD) was selected as 5000mg/kg for both males and females, this being the limit dose for this assay. This dose level was administered in Phase II of the study

Phase Il - Micronucleus test
No significant adverse reactions to treatment were observed in either males and females dosed with tested Substance. Clinica! signs observed included staining ofthe coat, signs of diarrohea, signs ofurinary incontinence, yellow stained urine and black faeces.
No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes, over the vehicle contro! values, were observed in either males or females at either sampling time investigated.

No statistically or biologically significant differences in the percentage of polychromatic erythrocytes, between the vehicle contro! and tested Substance treated animals, were observed in either males or females at either sampling time investigated.
The test system positive contro!, cyclophosphamide, induced statistically and biologically significant increases in the frequency of micronucleated polychromatic erythrocytes in both male and female mice at the 24 hour sampling time.

Any other information on results incl. tables

Colouration of urine from the mice dosed with tested Substance indicated that the test substance was absorbed and systemically distributed following dosing via the oral route.

No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes, compared to the vehicle contrai values, were seen in any Substance treated mice at either ofthe sampling times investigated.

The sensitivity of the test system was clearly demonstrated by the marked increases in the frequencies of micronucleated polychromatic erythrocytes induced by the positive control substance, cyclophosphamide.

The data from the study do not indicate any clastogenic activity of Substance in the mouse bone marrow when tested up to the limit dose for both male and female mice.

Applicant's summary and conclusion

Conclusions:
Under the conditions of test, the Substance is not clastogenic in the mouse bone marrow micronucleus test
Executive summary:

The test substance has been evaluated for its ability to induce micronucleated polychromatic erythrocytes in the bone marrow of CD-I mice, according to OECD Guideline 474.

The substance was tested at the limit dose of 5000mg/kg in both males and females.

Colouration ofurine from the mice dosed with Substance indicated that the test substance was absorbed and systemically distributed following dosing via the oral route.

No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes, compared to the vehicle contrai values, were seen in any Substance treated mice at either ofthe sampling times investigated.

The sensitivity of the test system was clearly demonstrated by the marked increases in the frequencies of micronucleated polychromatic erythrocytes induced by the positive contro! substance, cyclophosphamide.

The data from the study do not indicate any clastogenic activity of the Substance in the mouse bone marrow when tested up to the limit dose for both male and female mice.