Cuprate(6-), [2-[[[[3-[[4-chloro-6-[[4-[[4-chloro-6-[(3-sulfophenyl)amino]-1,3,5-triazin-2-yl]amino]-2,5-disulfophenyl]amino]-1,3,5-triazin-2-yl]amino]-2-hydroxy-5-sulfophenyl]azo]phenylmethyl]azo]-5-sulfobenzoato(8-)]-, pentasodium hydrogen, (SP-4-3)-

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 1994

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:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: ASTM Guideline by Butterworth et al. (1987)

Principles of method if other than guideline:

The method protocol is essentially described in Mirsalis and Butterworth (1980) as modified by Ashby et al. (1985, 1987).

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Test material

Test material information

Constituent 1

Reference substance name:

Cuprate(6-), [2-[[[[3-[[4-chloro-6-[[4-[[4-chloro-6-[(3-sulfophenyl)amino]-1,3,5-triazin-2-yl]amino]-2,5-disulfophenyl]amino]-1,3,5-triazin-2-yl]amino]-2-hydroxy-5-sulfophenyl]azo]phenylmethyl]azo]-5-sulfobenzoato(8-)]-, pentasodium hydrogen, (SP-4-3)-

EC Number:

268-655-7

EC Name:

Cuprate(6-), [2-[[[[3-[[4-chloro-6-[[4-[[4-chloro-6-[(3-sulfophenyl)amino]-1,3,5-triazin-2-yl]amino]-2,5-disulfophenyl]amino]-1,3,5-triazin-2-yl]amino]-2-hydroxy-5-sulfophenyl]azo]phenylmethyl]azo]-5-sulfobenzoato(8-)]-, pentasodium hydrogen, (SP-4-3)-

Cas Number:

68132-91-2

Molecular formula:

C38H20Cl2CuN14O18S5. H. 5Na C38H21Cl2CuN14Na5O18S5

IUPAC Name:

Cuprate(6-), [2-[[[[3-[[4-chloro-6-[[4-[[4-chloro-6-[(3-sulfophenyl)amino]-1,3,5-triazin-2-yl]amino]-2,5-disulfophenyl]amino]-1,3,5-triazin-2-yl]amino]-2-hydroxy-5-sulfophenyl]azo]phenylmethyl]azo]-5-sulfobenzoato(8-)]-, pentasodium hydrogen, (SP-4-3)-

Test material form:

other: press paste

Test animals

Species:

rat

Strain:

other: Alderley Park rat (Alpk:APfSD)

Details on species / strain selection:

Alpk:APfSD rats will be used as CTL had used this strain in toxicological studies for a long time.

Sex:

male

Details on test animals or test system and environmental conditions:

The animals used for this study will be male Alderley Park (Alpk:APfSD) rats weighing 180-280 g at the time of dosing. These animals will be suppilied by the Zeneca Barriered Animal Breeding Unit (BABU), Alderley Park, Macclesfield, Cheshire. Animals will be held in rodent rooms of animal care units on the Alderley Park site, housed under conditions appropriate to their species. Records of environmental conditions are held by the animal units at CTL.

A maximum of five rats will be housed in each cage. The animals will be supplied with food and water ad libitum. Drinking water will be supplied via an automatic watering system and the diet used will be Porton Combined Diet (PCD) supplied by Special Diets Services Ltd, Stepfieldt Witham, Essex. An analysis of each batch is supplied by Special Diets Services Ltd, and is checked by the Unit co-ordinator at BABU for acceptability of levels of possible contaminants which might influence the outcome of a study before the batch is used. Records for the receipt and storage of diet are held by the animal units at CTL. Since the animals will eventually be housed in a fume cupboard, acclimatisation is not considered appropriate.

Immediately prior to use, the animals will be transported in cages with filter lids to the testing laboratory. Whilst in this laboratory, the animals will be maintained according to SOP No CT90-023. The animals will then be weighed, tail numbered and allocated to the relevant treatment group. The animals will be allocated according to the order in which they are removed from the stock cage, ie, the first animal will be No. 1, the second, No 2 etc. The holding cage will be labelled with the Study number, the Project Licence code, the name of the licensee and the respective treatment details. Animals will be housed up to three per cage in a fume cupboard for a total period not exceeding 24 hours.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: deionised water

Details on exposure:

Dosage will be carried out according to SOP Nos CT50-137, (Dosing of Rodents by Gavage) and CT30-181, (Procedure for the In Vivo Rat Hepatocyte UDS Assay). The animals will receive a single oral dose, by gavage at a volume of 10 mL/kg bodyweight. During the course of this study, animals will have free access to PCD diet pellets ang water. A total of five animals for each test compound dose level at two separate timepoints will be treated. Clinical observation of the animals will be carried out following dosing and also immediately prior to perfusion. Any clinical signs of toxicity will be noted in the raw data. Of the two positive and two negative control animals in each experiment only one of each will normaily be scored for induction of UDS.

Details on exposure route selection: oral dosage was used since it was considered the most likely way of human exposure and related in vivo effects.

Duration of treatment / exposure:

4 d

Frequency of treatment:

Single dose

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 000 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

1 250 mg/kg bw (total dose)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Dimethylthydrazine dihydrochloride
- Route of administration: oral gavage
- Doses / concentrations: 30 mg/kg bw

Examinations

Tissues and cell types examined:

Hepatocytes (rat liver tissue)

Details of tissue and slide preparation:

The treated rats will be killed by the process of deep anaesthesia followed by perfusion and excision of the liver. Hepatocytes will be isolated from the animals by means of a two stage collagenase perfusion technique. Full details of the methodology to be used are in SOP No CT30-181.

Two or sixteen hours after dosage, animals will be anaesthetised, dissected and cannulae inserted into the hepatic portal vein and superior vena cava. Pumped Buffer 1 solution will be used to flush the liver free of blood and to remove calcium from the desmosomes. Buffer 2 will then be introduced, to which will be added calcium chloride and collagenase to cause disaggregation of the tissue into single cells. The liver will then be removed, cut open, and hepatocytes prepared by low speed centrifugation and resuspension in Williams Medium. Hepatocyte cultures, (at least three per animal), are left to attach onto tissue culture coverslips at 37°C.

The medium covering the hepatocyte cultures will be replaced with Williams Incomplete Medium containing 10 µCi/mL [3H]thymidine. After four hours incubation, the hepatocytes will be washed three times with Williams Incomplete Medium containing 0.25 mM unlabelled thymidine. Cells will be left to incubate overnight at 37°C with the same medium. This is a "cold-chase" procedure designed to remove the majority of unincorporated [3H]thymidine from the hepatocyte cultures.

Cultures will be fixed in glacial acetic acid . ethanol (1:3 v/v) and covers lips mounted onto microscope slides. Slides will be coated with photographic emulsion and left for 14 days at 4°C in the dark. The emulsion will be developed, fixed and the cell nuclei and cytoplasm stained with Meyers haemalum and eosin Y phloxine.

Slides will be examined microscopically and only the two highest dose levels not showing undue cytotoxicity, plus at least one negative and positive control animal, will be examined for UDS. Under conditions of high toxicity, cells of unusual morphology are formed, and the thymidine uptake mechanisms may consequently be affected giving false negative responses . Slides will be allocated a random code number generated by a computer. Paper labels, bearing the study number and code number, will be placed on the slides to obscure the slide identifiers by a person unconnected with slide scoring.

Coded hepatocyte cultures wi ll be examined for the induction of UDS using a microscope-mounted image analyser l inked to a computer for data analysis. The numoer of silver grains over the nucleus [N] is determined. Then an equivalent area of cytoplasm tangential to the nucleus and with the highest apparent number of silver grains is scored [C]. The difference between these two values [N-C] is the net nuclear grain count. Normally 30 cells will be scored on each of two slides for each animal . The third slide will not normally be read, unless it is considered that a total of 60 cells will not be obtained from the first two slides examined.

Evaluation criteria:

Criteria for a negative (ie non-genotoxic) response:
A negative response is obtained where the mean net nuclear grain count of each test comoound-treated animal is less than 0.

Criteria for a positive (ie genotoxic) response
An individual animal showing mean net nuclear grain counts of 0 or greater is considered to be indicative of the occurrence of a UDS response. A compound will be assigned as an unequivocal genotoxin in this assay if such a response is reproduced.

Marginal Responses
If the group mean net nuclear grain count of the treated animals is below 0 but with an individual animal exceeding 0, interpretation will consider factors as any statistically significant difference between treated and control cultures, dose response relationships, the percentages of cells in repair and the absolute values obtained for the negative control. ResuIts from the independent experiment will be examined to observe whether any effect is reproducible. In such cases, the ultimate designation will be based on experience and scientific judgement.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Hepatocytes from treated rats were exposed to [³H]-thymidine and the amount of radioactivity incorporated into the nucleus and an equal area of cytoplasm determined by autoradiography. The cytoplasmic grain count was subtracted from that of the nucleus. The value obtained, the mean net nuclear grain count [N-C], is an index of UDS activity. In this laboratory no negative control animal has shown a mean net nuclear grain count of greater than zero. An [N-C] value of greater than zero is therefore considered indicative of a UDS response. The validity criteria were fulfilled.

Each experiment was validated by concurrent control treatments of pats with sterile double deionised water, the vehicle for Procion Blue H-ERD Press Paste and with the carcinogen dimethylhydrazine dihydrochloride. Vehicle-treated rats gave rise to mean net nuclear grain counts of less than zero, whilst hepatocytes from positive control-treated animals had mean net nuclear grain counts of greater than +5. These data show that the background levels of UDS in this study were normal and that the test animals were responsive to a known carcinogen requiring metabolic activation for the demonstration of genotoxic activity.

Hepatocytes from Procion Blue H-ERD Press Paste treated animals were assessed for UDS at both dose levels tested. Treatments with Proc ion Blue H-ERD Press Paste in no case resulted in a mean net nuclear grain count greater than zero, at either time point investigated.

Applicant's summary and conclusion

Conclusions:

Based on an Unscheduled DNA Synthesis Assay, the test substance was not found to be genetically toxic when tested up to a limit dose level of 2000 mg/kg bw (oral: gavage; hepatocytes). The test substance did not induce DNA repair (as measured by unscheduled DNA synthesis) in rat liver. The test completely fulfilled the validity criteria for both positive and negative controls as defined by the standard test protocol.

Executive summary:

Reactive Blue 160 was tested for the ability to induce unscheduled DNA synthesis (UDS) in an in vivo rat hepatocyte assay. Male Alpk:APfSD rats were treated with a single oral dose of Reactive Blue 160 by gavage at dose levels of 1250 or 2000mg/kg bodyweight. The latter dose level is the limit dose level for this assay. Animals were killed and hepatocytes isolated and prepared two and sixteen hours after dosing. Two independent experiments were carried out for each time point.

Hepatocytes from treated rats were exposed to [3H]-thymidine and the amount

of radioactivity incorporated into the nucleus and an equal area of cytoplasm determined by autoradiography. The cytoplasmic grain count was

subtracted from that of the nucleus. The value obtained, the mean net nuclear grain count [N-C], is an index of UDS activity. In this laboratory no negative control animal has shown a mean net nuclear grain count of greater than zero. An [N-C] value of greater than zero is therefore considered indicative of a UDS response.

Each experiment was validated by concurrent control treatments of rats with sterile double deionised water, the vehicle for Reactive Blue 160 and with the carcinogen dimethylhydrazine dihydrochloride [DMH.2HC1]. Vehicle-treated rats gave rise to mean net nuclear grain counts of less than zero, whilst hepatocytes from DMH.2HC1-treated animals had mean net nuclear grain counts of greater than +5. These data show that the background levels of UDS in this study were normal and that the test animals were responsive to a known carcinogen requiring metabolic activation for the demonstration of genotoxic activity.

Hepatocytes from Reactive Blue 160 treated animals were assessed for UDS at both dose levels tested. Treatments with Reactive Blue 160 in no case resulted in a mean net nuclear grain count greater than zero, at either time point investigated.

It is concluded that, when tested up to a limit dose level of 2000mg/kg, the test sample of Reactive Blue 160 did not induce DNA repair (as measured by unscheduled DNA Synthesis) in rat liver.