Reaction mass of trisodium 4-amino-3-[[4-[[4-[(2-amino-4-hydroxyphenyl)azo]phenyl]amino]-3-sulphonatophenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate and trisodium 4-amino-3-[[4-[[4-[(4-amino-2-hydroxyphenyl)azo]phenyl]amino]-3-sulphonatophenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

October 22nd 1990 to April 5th, 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

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)

Version / remarks:

The UDS test is an assay for the detection of chemically induced effects on DNA.

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Test material

Test material information

Constituent 1

Reference substance name:

Direct Black 168:1 - Similar Substance 01

IUPAC Name:

Direct Black 168:1 - Similar Substance 01

Test animals

Species:

rat

Strain:

Wistar

Remarks:

WU

Details on species / strain selection:

The rat is an animals used as suitable experimental animal in genotoxicity investigations (and physiological, pharmacological and toxicological studies) for many years. Many data are available from such investigations that could be helpful in the interpretattion of results and for the design and performance of the UDS test.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SAVO-Ivanovas, med. Versuchstierzuchten GmbH.
- Weight at study initiation: appr. 200 g.
- Assigned to test groups randomly: yes.
- Fasting period before receiving test material: starved overnight (4 hours treatment) or appr. 6 hours (16 hours treatment).
- Housing: individually, in Makrolon Type I cages, with wire mesh top and granulated soft wood bedding.
- Diet: pelleted standard diet.
- Water: tap water, ad libitum.
- Acclimation period: minimum 5 days.
- Other: the animals underwent quarantine in the animals house of the laboratory for at least one week after their arrival. During this period the animals did not show signs of illness or altered behaviour.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C.
- Humidity: 30 - 70 %.
- Photoperiod: 12 hrs dark / 12 hrs artificial light (6.00 a.m - 6.00 p.m).

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

- Vehicle: bedistilled water.
- Justification for choice of solvent/vehicle: it was chosen according to its relative nontoxicity for the animals.

Details on exposure:

- Preparation of dose solution: the test article was formulated in aqua bidestilled, on the day of the experiment immediately before treatment.
- Volume administered: 10 ml/kg bw

Duration of treatment / exposure:

4 hours and 16 hours treatment.

Frequency of treatment:

Single standard dose.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

4 - hours treatment period.

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

16-hours treatment period.

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

16-hours treatment period.

No. of animals per sex per dose:

5 males per test group.

Control animals:

yes, concurrent vehicle

Positive control(s):

2-acetylaminofluorene
- Preparation: dissolved in DMSO/polyethylene glycol (1+9). The solution was prepared on day of administration.
- Route and frequency of administration: orally, single.
- Dose: 100 mg/kg b.w.
- Volume administered: 10 ml/kg b.w.

Examinations

Details of tissue and slide preparation:

ISOLATION OF THE PRIMARY HEPATOCYTES
The animals were sacrificied by liver perfusion. After anesthetizing the rats with 1.5 ml/kg b.w. Hypnodil i.p. the liver was perfused through the vena portae with Hank's balanced salt solution (HBSS) supplemented with collagenase (0.05 % w/v) adjusted to pH 7.4 and maintained at 37 °C. The hepatocytes were isolated from the liver and washed twice with HBSS. The crude cell suspension was filtered through a 94 μm stainless steel mesh to yield a single cell suspension. The quality of the actual performed perfusion was determined by the tryptan blue dye exclusion method. The number of the isolated cells was also determined.

CULTURE CONDITIONS
The washed hepatocytes were centrifuged and transferred into Williams medium E (WME) supplemented with 2.38 mg/ml hepes, 100 units/ml penicillin, 0.10 mg/ml streptomycin, 0.29 mg/ml glutamin, 0.50 μg/ml insulin and 100 μl/ml fetal calf serum. The medium without the cells was adjusted to pH 7.6. At least 5 cultures were established for each animal. Aliquotes of 2.5 ml with freshly isolated hepatocytes in complete culture medium (1.0 x 10^5 cells/ml) were added to 35 mm six-well cluster dishes containing one gelatinized 25 mm round plastic coverslip per well. After an attachment period of appr. 1.5 h in a 95 % air / 5 % CO2 humidified incubator at 37 °C the culture medium was discarded. Then the cell layer was rinsed once with PBS to remove non-adherent cells.

OTHER
Three animals per group were evaluated for UDS. The two remaining animals per test group would be evaluated if an animal dies spontaneously or in case of technical problems concerning the isolation of hepatocytes.

DETERMINATION OF UDS
3HTdR (5 μCi/ml, specific activity 20 Ci/mol) in 2.0 ml culture medium (WME, 1 % FCS) was added to the cultures. After a labelling time of 4 hours the cells were washed twice with WME supplemented with 1 % FCS and 0.25 mM unlabelled thymidine. Cultures were incubated overnight using the same medium. The medium was then replaced by a hypotonic solution of 1 % sodium citrate for 10 minutes to swell the nuclei for better grain quantification. The cells on the coverslips were then fixed by three changes of methanol:acetic acid (3+1 v/v) for 15 mins each, rinsed with 96 % ethanol and air dried. Two of the cultures were used for determination of cytotoxicity and attachment efficiency with the neutral red assay whereas three cultures were used for the UDS assay.

ANALYSIS-AUTORADIOGRAPHIC PROCESSING
The cover slips were mounted the side carrying the cells up on a glass slides and coated with ILFORD K-2 photographic emulsion in the dark. The coated slides were stored in light-proof boxes in the presence of a drying agent for 12-14 days at 4 °C. The photographic emulsion was then developed with KODAK D-19 at room temperature, fixed in TETENAL and stained with 0.4 % aceto orcein.

QUANTIFICATION OF UDS
Evaluation was performed microscopically on coded slided using NIKON microscopes with 100 x oil immersion objectives. The number of siver grains above the nucleus was counted automatically using the ARTEK 880 counter. The number of grain counts of one nuclear-sized cytoplasm adjacent to the nucleus was estimated. At least two slides per animal and 50 cells per slide were evaluated. Heavily labelled S-phase cells were excluded from counting.

ASSESSMENT OF ATTACHMENT OF CELLS
Two cultures of each animals were examined by using the neutral red absorption assay. After an attachement period of 1.5 hours the cells were washed and refed with medium containing neutral red (50 μg/ml). After an incubation period of 3 hours the cultures were rinsed and the incorporated dye eluted with 50 % ethanol supplemented with 1 % acetic acid.

CRITERIA FOR DOSE SELECTION
The highest recommended dose level to be used in the assay is 1000 mg/kg b.w. The toxicity data of the test article available indicated that 1000 mg/kg b.w. is the maximum tolerated dose causing toxic reactions without having major effects on the survival of the animals, therefore no pre-experiment was performed.

Evaluation criteria:

A test article is classified as positive if it induces either a statistically significant dose-related increase in radiolabel incorporation expressed as grains per nucleus or a reproducible and statistically significant positive response for at least one of the test points. A test article producing neither a statistically significant dose related increase in radiolabel incorporation expressed as grain per nucleus nor a statistically significant and reproducible positive response at any one of the test points is considered non-effective in this system.

Statistics:

Statistical significance can be evaluated by means of the non-parametric Mann-Whitney test. A statistical evaluation of the results of biometry was not necessary to perform as the number of nuclear an net grain counts of the groups treated with the test article were in the range of the corresponding controls.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No toxic reactions of the animals occurred at any of the tratement periods or dose groups. In addition, neither the viability nor the in vitro attachment of the hepatocytes was dramatically affected due to the in vivo pre-treatment with the test article.
The interindividual variations obtained for the numbers of isolated hepatocytes as well as for the attachment-efficiency (NR-assay) are in the range of the testing laboratory historical laboratory control. In case of hepatotoxicity the NR-vales expected would be approximately zero.

No dose level of the test article revealed UDS induction in the hepatocytes of the trated animals as compared to the current negative controls. Neither the nuclear grains nor the resulting net grains were enhanced due to the in vivo treatment of the animals with the test article for 4 hours or 16 hours, respectively.

POSITIVE CONTROL
An appropriate reference mutagen (2-AAF, 100 mg/kg bw) was used as positive control. In vivo treatment with 2-AAF revealed disctinct increases in the number of nuclear and net grain counts.

PRE-EXPERIMENT
The highest recommended dose level to be used in the assay resulted to be 1000 mg/kg bw. since the toxicity data of the test article given by the sponsor indicated that 1000 mg/kg bw should have no makor effects on the survival of the animals a pre-experiment was not necessary to perform.

Any other information on results incl. tables

The viability and attachment of the hepatocytes.

Viability and attachment of hepatocytes.

Dose  (mg/kg b.w.)	Tretament period	animal no.	Viability *	Number of isolated cells (x 106)	Attachement  (O.D 540 mm)**
1000	4h	1	71	102	0.367
	4h	2	74	367	0.495
	4h	4	65	152	1.030
Vehicle (aqua bidest.)	16h	6	81	392	0.428
	16h	7	67	326	0.321
	16h	8	81	321	0.554
100	16h	11	82	270	0.226
	16h	12	76	629	0.198
	16h	13	73	424	0.536
1000	16h	18	69#	304	0.270
	16h	19	79#	373	0.355
	16h	20	72#	272	0.366
100 (2AAF)	16h	21	81	376	0.410
	16h	22	77	343	0.449
	16h	23	71	169	0.438

* = viability determined by means of trypan blue dye exclusion assay.

** = mean value of two independent cultures.

# = some of the dead cells elicited black pigmentation indicating an accumulation of the test article in hepatocytes.

The counts of grains per nucleus, grains per cytoplasm area and the net grains per nucleus are summarised for each animal.

Summary of results for individual animals.

Treatment	Animal no.	Grains per nucleus		Grains per cytoplasm area		Net grains per nucleus
		Mean *	± SD	Mean *	± SD	Mean *	± SD
Solvent control (a.dest/16h)	1	4.95	3.42	7.01	3.10	-2.06	2.41
	2	3.57	2.18	5.69	2.28	-2.12	2.61
	3	3.79	2.32	7.86	3.27	-4.07	3.48
Positive control -  (2AAF 100 mg/kg /16h)	1	37.01	19.15	9.68	4.94	27.33	19.10
	2	22.44 #	9.27	11.50	4.61	10.94	8.74
	3	17.93	8.10	12.74	4.56	5.19	8.76
Dose level 1 (100 mg/kg / 16h)	1	5.88	3.50	9.15	4.13	-3.27	3.66
	2	4.93	2.59	7.27	2.19	-2.34	2.89
	3	6.35	3.15	7.51	3.26	-1.16	3.45
Dose level 2 (1000 mg/kg / 4h)	1	4.10	2.27	6.18	2.32	-2.08	2.51
	2	3.92	2.01	6.59	2.63	-2.67	2.22
	3	4.48	2.26	5.73	2.47	-1.25	2.54
Dose level 3 (1000 mg/kg / 16h)	1	4.92	2.66	7.40	3.00	-2.48	2.53
	2	4.08	2.40	6.49	2.88	-2.41	2.73
	3	8.80	6.00	13.69	8.27	-4.89	5.88

* = mean of 100 cells

# = mean of 50 cells; due to technical reasons only one slide was scorable.

The counts of grains per nucleus, grains per cytoplasm area and the net grains per nucleus are summarised for each dose group.

Summary of results for dose groups.

Treatment	Dose level	Grains per nucleus		Grains per cytoplasm area		Net grains per nucleus
		Mean *	± SD	Mean *	± SD	Mean *	± SD
Solvent control	a.dest/16h	4.10	2.75	6.85	3.04	-2.75	3.01
Positive control -  2AAF	100 mg/kg /16h	26.46 #	16.31	11.27	4.90	15.20	17.13
Dose level 1	100 mg/kg / 16h	5.72	3.15	7.98	3.38	-2.26	3.45
Dose level 2	1000 mg/kg / 4h	4.17	2.19	6.17	2.50	-2.00	2.49
Dose level 3	1000 mg/kg / 16h	5.93	4.52	9.19	6.22	-3.26	4.17

* = mean of 3 animals, 100 cells each.

# = mean of 3 animals, two animals 100 cells each, one animal 50 cells.

Applicant's summary and conclusion

Conclusions:

The test article did not induce DNA-damage leading to repair synthesis in the hepatocytes of the treated rats.

Executive summary:

The substance was assessed in the in vivo UDS assay for its potential to induce DNA repair (UDS) in the hepatocytes of rats. The substance was orally administered the substance in concentrations of 1000 mg/kg b.w for the 4 -hours treatment period and in concentrations of 100 and 1000 mg/kg b.w for the 16 -hours treatment period. Bedistilled water was used as vehicle and served as the negative control while an appropriate reference mutagen (2 -AAF) was used as the positive control. Five male rats were used in each dose group. After the treatment period the animals were narcotized and sacrificied by liver perfusion. Primary hepatocyte cultures were established and exposed to 3HTdR which is incorporated if UDS occurs. For each dose level hepatpcytes from three treated animals were assessed for occurence of UDS.

No toxic reactions of the animals occured at any of the treatment periods or dose group. The viability and the in vitro attachment of the hepatocytes was not affected due to the in vivo pre-treatment with the test article. The interindividual varriations obtained for the numbers of isolated hepatocytes as well as for the attachment efficiency were in the range of the historical laboratory control. No dose level of the test article revealed UDS induction in the hepatocytes of the treated animals as compared to the concurrent negative controls. Neither the nuclear grains nor the resulting net grains were enhanced due to the in vivo treatment ofthe animals with the test article for 4 or 16 hours.

Conclusion

The test article did not induce DNA-damage leading to repair synthesis in the hepatocytes of the treated rats.