[x·sodium, y·lithium, x+y=3] 4-amino-3[[4-[[4-[(2-amino-4-hydroxyphenyl)diazenyl]phenyl]amino]-3-sulfonato phenyl]diazenyl]-5-hydroxy-6-(phenydiazenyl)naphthalene-2,7-disulfonat, mixture of isomers

Genetic toxicity: in vivo

Administrative data

Endpoint:

genetic toxicity in vivo, other

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

See the report for the analogue approach justification in section 13 "Other reports"

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

January from 02 to 26, 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted May 26th, 1983

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian germ cell cytogenetic assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Tierfarm Füllinsdorf, CH- 4414 Füllinsdorf/Basel, Switzerland.
- Age at start of acclimatization: minimum 10 weeks.
- Weight at study initiation: approx. 30 g.
- Assigned to test groups randomly: yes.
- Fasting period before study: yes, approximately 18 hours before treatment with the test article. Animals could still have water ad libitum during the fasting period.
- Housing: single in a Makrolon Type I, with wire mesh top and granulated soft wood bedding.
- Diet: pelleted standard diet.
- Water: tap water, ad libitum.
- Acclimation period: minimum 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3°C.
- Humidity: not regulated.
- Photoperiod: 12 hrs dark / 12 hrs artificial light (6.00 a.m - 6.00 p.m).

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: destilled water.
- Justification for choice of vehicle: chocen for its non-toxicity to the animals.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The test article was dissolved in distilled water and the animals received orally a single standard dose volume of 20 ml/kg b.w.

Frequency of treatment:

Once at the start of the study.

Dose / conc.:

5 000 mg/kg bw (total dose)

Remarks:

Three groups

No. of animals per sex per dose:

6 males and 6 females per dose group.

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPA)
- Preparation: the substance was dissolved in physiological saline and the solution was prepared on the day of administration.
- Route of administration: orally.
- Dosing: single dose of 40 mg/kg b.w.
- Volume administered: 10 ml/kg b.w.
- Stability: at 20 °C only 1 % of CPA is hydrolysed per day in aqueous solution.

Tissues and cell types examined:

Bone marrow cells.

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES: sampling of the bone marrow was done 24, 48 and 72 hours after treatment.

PREPARATION OF THE ANIMALS
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 ml syringe. The cell suspension was centrifuged at 1,500 rpm for 5 minutes and
the supernatant was discarded.

DETAILS OF SLIDE PREPARATION
A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample

ANALYSIS OF CELLS
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.

CRITERIA FOR DOSE SELECTION
A preliminary study on acute toxicity was performed with the same strain and under identical conditions as in the mutagenicity study. 4 animals (2 males, 2 females) received orally a single dose of 5000 mg/kg b.w of the substance dissolved in aqua dest. The volume administered was 20 ml/kg b.w.
The maximum tolerated dose or the highest dose that can be formulated and administered reproducibly sould be selected. The volume to be administered should be compatible with physiological space available. The maximum tolerated dose level was determined to be the dose that caused toxic reactions without having major effects on survival within 72 hours. Higher dosing than 5000 mg/kg b.w. was not attainable as appropriate solutions could be obtained only up to 250 mg/ml and application volumes higher than 20 ml/kg b.w. were not justifiable for the animals used.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points. A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant and reproducible positive response at anyone of the test points is considered non mutagenic in this system.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

slight toxic reactions

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with the substance were in the same range as compared to the negative control groups.

POSITIVE CONTROL
40 mg/kg b.w. cyclophosphamide showed a distinct increase of induced micronuleus frequency.

PRE-EXPERIMENT FOR TOXICITY
All treated animals expressed slight toxic reactions such as reduction of spontaneous activity. The mean number of normochromatic erythrocytes was not substantially increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating that the substance had no cytotoxic properties.

The number of PCEs (Polychromatic Erythrocytes) with micronuclei and the ratio of PCE to NCE ( Normochromatic Erythrocyte) are given for each test group in the table below.

Summary of results.

Test group	dose (mg/kg bw)	sampling time (h)	PCEs with micronuclei	range	PCE/NCE
solvent	0	24	0.09 %	0-3	1000/935
test article	5000	24	0.09 %	0-3	1000/874
cyclophosphamide	40	24	0.76 %	3-11	1000/1119
solvent	0	48	0.02 %	0-2	1000/842
test article	5000	48	0.04 %	0-1	1000/1000
solvent	0	72	0.07 %	0-2	1000/892
test article	5000	72	0.03 %	0-2	1000/769

Conclusions:

The test article did not induce micronuclei in the bone marrow cells of the mouse.

Executive summary:

The substance was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse, according to the OECD Guideline 474 and EU Method B12. Six male and six female mouses were treated orally with a single dose of 5000 mg/kg b.w. This was the maximum attainable dose as estimated by the pre-experiment. Concurrent positive (use of cyclophosphamide) and negative (use of vehicle-distilled water) control groups run. After 24 h, 48 h and 72 hours the bone marrow cells of ten animals were collected for micronuclei analysis. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with the substance were in the same range as compared to the negative control groups.

Conclusion

The test article did not induce micronuclei in the bone marrow cells of the mouse, under the test conditions.

Cross-reference 2

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From October 22 1990 to April 05, 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

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

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).

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.

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

4 - hours treatment period.

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

16-hours treatment period.

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.

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.

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.

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.

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.

Materials and methods

Test material

Test material information

1

Reference substance name:

[x·sodium, y·lithium, x+y=3] 4-amino-3[[4-[[4-[(2-amino-4-hydroxyphenyl)diazenyl]phenyl]amino]-3-sulfonato phenyl]diazenyl]-5-hydroxy-6-(phenydiazenyl)naphthalene-2,7-disulfonat, mixture of isomers

EC Number:

827-162-2

Cas Number:

2218502-12-4

Molecular formula:

C34H24N9O11S3.NaxLiy

IUPAC Name:

[x·sodium, y·lithium, x+y=3] 4-amino-3[[4-[[4-[(2-amino-4-hydroxyphenyl)diazenyl]phenyl]amino]-3-sulfonato phenyl]diazenyl]-5-hydroxy-6-(phenydiazenyl)naphthalene-2,7-disulfonat, mixture of isomers

Test material form:

solid

Results and discussion

Test results

Test results 1

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

slight toxic reactions

Test results 2

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks on result:

other: unscheduled DNA synthesis