Hydrogen [29H,31H-phthalocyaninesulphonato(3-)-N29,N30,N31,N32]cuprate(1-), compound with dodecylamine (1:1)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD Guideline 474. A default reliability of 2 is used because of read-across.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

no

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

108300-90-9

Cas Number:

108300-90-9

IUPAC Name:

108300-90-9

Details on test material:

- Batch No: Partie 1 + 2, 1985
- Analytical purity: 95 %
- Storage: + 4 °C

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH WIGA, Sulzfeld, Germany
- Assigned to test groups randomly: Male and female animals were assigned to the test groups according to a randomization plan compiled in the Department of Toxicology of BASF AG
- Housing: 5 animals per cage for one week separately according to sex; individual housing before the beginning of the experiment
- Diet: Standardized pelleted feed (Kliba Haltungsdiät, Klingentalmühle Kaiseraugst, Switzerland), ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

DMSO

Details on exposure:

Details on test groups, number of animals and dose levels are listed in table 1 in "any other information on materials and methods".
All test substance formulations were prepared immediately before administration.
After the administration of the test substance the animals were examined for any evident clinical signs of toxicity .

Duration of treatment / exposure:

single oral administration

Frequency of treatment:

single oral administration

Post exposure period:

16, 24 or 48 h

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 250, 500, 1000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5 animals per sex per dose.

Positive control(s):

Cyclophosphamide
- Route of administration: orally by gavage

Examinations

Details of tissue and slide preparation:

PREPARATION OF THE BONE MARROW:
The bone marrow was prepared according to the method described by Schmid and Staiger (Mut Res 7: 99 - 108, 1969).
The two femora were prepared from the animals and all soft tissues were removed After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube in reciprocal directions using a cannula filled with Hanks solution which was at 37 °C (about 2 ml/femur. Subsequently the suspension was mixed thoroughly with a pipette, centrifuged at 1500 rpm for 5 minutes, the supernatant was pipetted off except for a few drops, and the precipitate was resuspended.
1 drop of this suspension was dropped onto clean microscopicslides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.

STAINING:
The slides were stained in eosin and methylene blue solution for 5 minutes, rinsed in aqua dest. and then placed in fresh aqua dest. for 2 or 3 minutes. They were finally stained in Giemsa solution for 12 minutes. After being rinsed twice in aqua dest. and clarified in xylene, the preparations were embedded in Entellan.

MICROSCOPIC EVALUATION:
As a rule, 1000 polychromatic erythrocytes from each of the male and female animals of every test group are evaluated and investigated for micronuclei. The normochromatic erythrocytes (= normocytes), which occur, are also scored. The following parameters are recorded:

- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
The increase in the micronucleus rate in polychromatic erythrocytes of treated animals as compared with thesolvent control group provides an index of a chromosomebreaking (clastogenic) effect or of a spindle activity of the substance tested.

- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
The rate of micronuclei in normochromatic erythrocytes at the early sacrifice intervals represents the situation before test substance administration and may serve as a control value. A substance-induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice intervals.

- Ratio of polychromatic to normochromatic erythrocytes
This ratio indicates an influence of the test substance specifically on the bone marrow.

- Number of small micronuclei (d < D/4) and of large micronuclei (d >= D/4); (d = diameter of micronucleus, D= cell diameter )
The size of micronuclei may give an interpretation on the mode of action of tha test substanca i.e. a clastogenic or a spindle poison effect.


The preparations were coded for analysis.

Statistics:

Two statistical tests were used to answer the questions of whether there are significant differences between control group and dose groups concerning the rate of micronuclei in polychromatic erythrocytes: first, the exact test according to FISHER, which was applied to register significant differences between the relative frequencies of a characteristic of two groups and second, the asymptotic U test according to MANN-WHITNEY (rank test modified according to WILCOXON. The relative frequencies of cells with micronuclei were used as a criterion of the rank determination for the U test. The two tests were calculated at the levels of 95 % and 99 %. Significances at the 95 % level were marked with * (Fisher Yates Test) and with + (U Test), significances at the 99 % level were marked with ** (Fisher Yates Test) and with ++ (U Test).

Results and discussion

Additional information on results:

Clinical examinations:
Tha single oral administration of the solvent/carrier in a volume of 4 ml/kg body weight was tolerated by all animals without any signs or symptoms. The doses of 1000 mg/kg, 500 mg/kg and 250 mg/kg body weight led to irregular respiration about 30 minutes after test substance administration. In the 1000 mg/kg group in addition piloerection was observed. After 2- 3 hours signs of toxicity were no longer found. The single oral administration of the positive control substance cyclophosphamide in a dose of 40 mg/kg body weight did not cause any evident signs of toxicity.

Microscopic evaluation:
The single oral administration of DMSO in a volume of 4 ml/kg body weight led to 1.4 % polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval. After the single administration of the highest dose of 1000 mg/kg body weight, 2.2 % polychromatic erythrocytes containing micronuclei were found after 16 hours, 1.9 % after 24 hours and 1.2 % after 48 hours. In the two lower dose groups rates of micronuclei of about
2.0 % (500 mg/kg group) and 1.4 % (250 mg/kg group) were detected after a sacrifice interval of 24 hours in each case. With 22.6 % however, the positive control substance cyclophosphamide, as axpected, led to a clear increase in the rate of polychromatic erythrocytes containing micronuclei at a dose evel of 40 mg/kg body weight .
The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals. Thus the test substance Pigmentblau 1755 did not lead to any increase in the rate of micronuclei . The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) did not deviate from the solvent control value at any of the sacrifice intervals . Nor were large micronuclei (d >= D/4) observed either in the solvent control group or in the three dose groupa of Pigmentblau 1755.
No inhibition of erythropoiesis induced by the treatment of mice with Pigmentblau 1755 was detected; the ratio of polychromatic to normochromatic erytrhocytes was always in the same range as that of the control values in all dose groups .

Analysis:
Depending on the dose, about 98 - 105 % of the theoretical values could be determined analytically.

Any other information on results incl. tables

Table 1: Summary of the results from all test groups

Group	Interval 16 hours
	Polychromatic erythrocytes investigated	Normocytes/10000 polychromatic erythrocytes	cells with micronuclei
			per 1000 polychromatic erythrocytes	per 1000 normochromatic erythrocytes
Solvent control DMSO	-
1000 mg/kg bw	10000	3298	2.2	1.52
500 mg/kg bw	-
250 mg/kg bw	-
positive control cyclophosphamide 40 mg/kg bw	-
	Interval 24 hours
	Polychromatic erythrocytes investigated	Normocytes/10000 polychromatic erythrocytes	cells with micronuclei
			per 1000 polychromatic erythrocytes	per 1000 normochromatic erythrocytes
Solvent control DMSO	10000	3153	1.4	0.95
1000 mg/kg bw	10000	3180	1.9	0.94
500 mg/kg bw	10000	3481	2.0	0.57
250 mg/kg bw	10000	3049	1.4	1.64
positive control cyclophosphamide 40 mg/kg bw	10000	4091	22.6	1.71
	Interval 48 hours
	Polychromatic erythrocytes investigated	Normocytes/10000 polychromatic erythrocytes	cells with micronuclei
			per 1000 polychromatic erythrocytes	per 1000 normochromatic erythrocytes
Solvent control DMSO	-
1000 mg/kg bw	10000	3604	1.2	0.55
500 mg/kg bw	-
250 mg/kg bw
positive control cyclophosphamide 40 mg/kg bw	-

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative