Bis(hydroxylammonium) sulphate

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:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Cited as Directive 84/449/EEC, B.12

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis(hydroxylammonium) sulphate

EC Number:

233-118-8

EC Name:

Bis(hydroxylammonium) sulphate

Cas Number:

10039-54-0

Molecular formula:

H3NO.1/2H2O4S

IUPAC Name:

bis(hydroxyammonium) sulfate

Details on test material:

- Name of test material (as cited in study report): Hydroxylammonium-Sulfat (Festsalz)
- Physical state: solid
- Date of manufacturing: April 4, 1991
- Appearance, consistency: white crystals
- Analytical purity: 98.6 %
- Lot/batch No.: 91/188
- Storage condition of test material: refrigerator

The stability of the test substance throughout the study period has been verified analytically by reanalysis.

The homogeneity of the test substance was guaranteed on account of the high purity.

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals:
--------
The investigations were carried out in healthy male and female NMRI mice, Charles River GmbH, WIGA, 0-W8741 Sulzfeld, Germany. Animals with a mean weight of about 27 g were used for the study .

Housing and diet:
---------------
For the duration of about one week the animals were housed in Makrolon cages, type M III, in groups of 5 separately according to sex in fully air-conditioned rooms in which central air conditioning guaranteed a range of 20 - 24°C for temperature and a range of 30 - 70 % for relative humidity. Before the start of the treatment the animals were transferred to Makrolon cages, type M I, and housed individually under the same conditions until the end of the test.

The animals were identified using cage cards .

The day/night rhythm was 12 hours (12 hours light from 6.00 - 18.00 hours and 12 hours darkness from 18.00 - 6.00 hours) .

Standardized pelleted feed (Kliba Haltungsdiaet, Klingentalmuehle AG, Kaiseraugst, Switzerland) and drinking water from bottles were available ad libitum.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Aqua dest.

Details on exposure:

Test substance preparation analysis :
------------------------------
For the determination of the test substance concentration in the solvent 3 samples per dose were taken from the test substance preparation using a stomach tube and transferred to test tubes. The samples were kept at room temperature until the treatment of the last animal (approximately 1 hour) and then deep-frozen until they were determined analytically. The determination of the concentration in the solvent was carried out .

The stability of the test substance in the solvent aqua dest. was determined analytically.

The analytical investigations were carried out in the analytical laboratory of BASF Aktiengesellschaft.

Feed analysis :
The feed used in the study was assayed for chemical and microbiological contaminants.

Water analysis :
The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and by the Technical Services of BASF Aktiengesellschaft as well as for the presence of germs by a contract laboratory.

Duration of treatment / exposure:

Sacrifice intervals were 16, 24 or 48 h after administration of the test material.

Frequency of treatment:

single treatment

Post exposure period:

Sacrifice intervals were 16, 24 or 48 h after administration of the test material.

Doses / concentrations

Remarks:

Doses / Concentrations:
single dose each of 300, 600 and 1200 mg/kg bw in 10 ml water/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

As a positive control, 20 mg of cyclophosphamide/kg body weight or 0.15 mg of vincristine/kg body weight, both dissolved in aqua dest., were administered to male and female animals once orally or intraperitoneally respectively each in a volume of 10 ml/kg body weight.

Examinations

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

Preparation of the bone marrow
---------------------------
- 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 using a cannula filled with fetal calf serum which was at 37°C (about 2 ml/ femur).
- The suspension was mixed thoroughly with a pipette, centrifuged at 1500 rpm for 5 minutes, the supernatant was removed except for a few drops, and the precipitate was resuspended.
- 1 drop of this suspension was dropped onto clean microscopic slides, 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 Corbit-Balsam.


Microscopic evaluation
-------------------
In general, 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 micronucle
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome-breaking (clastogenic) effect or of a spindle activity of the substance tested.

- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
The number 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) td = diameter of micronucleus, D = cell diameter
The size of micronuclei may give an indication on the possible mode of action of the test substance i.e. a clastogenic or a spindle poison effect.

Slides were coded before microscopic analysis.

Statistics:

Statistical evaluation
-----------------
A statistical evaluation was not necessary to perform. The number of polychromatic micronucleated erythrocytes after test substance treatment was nearly the range of the actual control value and within the historical values .

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Depending on the dose, about 103 - 108 % of the theoretical values could be determined analytically. The stability of the test substance in the solvent over a period of 4 hours was verified analytically. The homogeneity of the test substance in the solvent aqua dest. was guaranteed by constant stirring during the removal and administration of the test substance formulation and by analytical determination of 3 individual samples of each concentration.

Feed analysis :
In view of the aim and duration of the study the contaminants occurring in commercial feed might not influence the results.
Water analysis :
In view of the aim and duration of the study there are no special requirements exceeding the specification of drinking water.

Clinical examinations:
The single oral administration of the solvent in a volume of 10 ml/kg body weight was tolerated by all animals without any signs or symptoms. A dose of 1200 mg/kg body weight led to evident signs of toxicity such as irregular respiration, piloerection, apathy, abdominal position, closed eyelids and blueish skin within 30 minutes after test substance administration. In few cases staggering and squatting posture were additionally observed and the general state of the animals was poor. Some of these signs were still observed two days after treatment of the animals.
600 mg/kg and 300 mg/kg body weight led to irregular respiration and piloerection within 1 hour after administration. In the 600 mg/kg group in few cases squatting posture was also observed. After about 2 hours these signs were not found any longer. Neither the single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg body weight nor that of vincristine in a dose of 0.15 mg/kg body weight caused any evident signs of toxicity.

Microscopic evaluation:
The single oral administration of aqua dest. in a volume of 10 ml/kg body weight led to 1.69 ‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval. After the single administration of the highest dose of 1200 mg/kg body weight, 1.2 ‰ polychromatic erythrocytes containing micronuclei were found after 16 hours, 1.0 ‰ after 24 hours and 2.7 ‰ after 48 hours. In the two lower dose groups rates of micronuclei of about 1.8 ‰ (600 mg/kg bw group) and 1.3 ‰ (300 mg/kg bw group) were detected after a sacrifice interval of 24 hours in each case.
With 8 .8 ‰ the positive control substance cyclophosphamide for clastogenicity led to the expected increase in the number of polychromatic erythrocytes containing exclusively small micronuclei at a dose level of 20 mg/kg body weight.
With 103.8 ‰ the positive control vincristine for spindle poison effects also led to a clearly enhanced number of micronuclei containing polychromatic erythrocytes with the expected amount of large micronuclei, i.e. 14.4 ‰.
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 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 negative control group or in the three dose groups. A slight inhibition of erythropoiesis induced by the treatment of mice withthe test material was detected a a dose of 1200 mg/kg body weight at sacrifice intervals of 16 and 24 hours.

Any other information on results incl. tables

Table 2: Summary of all test groups: Results of all groups for polychromatic and normochromatic erythrocytes

Group	Interval 16 hours
	Polychromatic erythrocytes investigated	Normocytes/total amount polychromatic erythrocytes	cells with micronuclei (‰)
			polychromatic erythrocytes	normochromatic erythrocytes
Solvent control aqua dest.	-
1200 mg/kg bw	10000	6428	1.2	0.16
600 mg/kg bw	-
300 mg/kg bw	-
positive control cyclophosphamide 20 mg/kg bw	-
positive control vincristine 0.15 mg/kg bw	-
Group	Interval 24 hours
	Polychromatic erythrocytes investigated	Normocytes/total amount polychromatic erythrocytes	cells with micronuclei (‰)
			polychromatic erythrocytes	normochromatic erythrocytes
Solvent control aqua dest.	10000	4271	1.6	1.17
1200 mg/kg bw	10000	6561	1.0	0.46
600 mg/kg bw	10000	4223	1.8	0.47
300 mg/kg bw	10000	4906	1.3	1.02
positive control cyclophosphamide 20 mg/kg bw	5000	2997	8.8	1.33
positive control vincristine 0.15 mg/kg bw	5000	5789	103.8	2.07
Group	Interval 48 hours
	Polychromatic erythrocytes investigated	Normocytes/total amount polychromatic erythrocytes	cells with micronuclei (‰)
			polychromatic erythrocytes	normochromatic erythrocytes
Solvent control aqua dest.	-
1200 mg/kg bw	10000	4560	2.7	0.22
600 mg/kg bw	-
300 mg/kg bw	-
positive control cyclophosphamide 20 mg/kg bw	-
positive control vincristine 0.15 mg/kg bw	-