Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1982
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report Date:
1983

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
The study was performed in year 1982. The mammalian micronucleus test was adopted as OECD guideline 474 in 1983.
Principles of method if other than guideline:
The test procedure and the preparation of the bone marrow were based on the data given by Schmid W. and Salamone, M. et al.: Schmid, W., The micronucleus test for cytogenic analysis. In: Hollaender, A. (eds), Chemical Mutagens Principles and Methods for their Detection, Volume 4, Plenum Press, New York (1976). Schmid, W., The micronucleus test. In: Kilbey et al., Handbook of Mutagenicity Test Procedures, Elsevier Scientific Publishing Company, Amsterdam-New York-Oxford (1977). Salamone, M. et al., Towards an improved micronucleus test. Studies on 3 model agents, mitomycin C, cyclophosphamide, and dimethylbenzanthracene, Mut. Res., 74, 347-356 (1980).
GLP compliance:
no
Remarks:
The study was performed before GLP was mandatory. However the conditions of good laboratory practice have alwyas been observed. The study is scientifically valid.
Type of assay:
other: indirect detection of a chromosome-damaging (clastogenic) effect or a damage of the mitotic apparatus (spindle poison effect)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Test item: (N-Cyclohexyl-diazeniumdioxy)-potassium
IUPAC name: Cyclohexylhydroxydiazene 1-oxide, potassium salt
Chemical name: Cyclohexylhydroxydiazene 1-oxide, potassium salt; synonyma: (N-Cyclohexyl-diazeniumdioxy)-potassium, K-HDO, K-NCH, Xyligen K powder, Xyligen K
Molecular formula: C6 H11 K N2 O2
Molecular mass: 182.27
Specific details on test material used for the study:
Test substance No.: 82/172
Appearance, consistency: White flakes
Degree of purity: 90.5% (water 9%)
Storage: 4 °C
Solvent: Aqua dest.

Test animals

Species:
mouse
Strain:
NMRI
Details on species / strain selection:
The investigations were carried out in male and female NMRI mice, Charles-River WIGA, D-8741 Sulzfeld, FRG.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles-River WIGA, D-8741 Sulzfeld, FRG.
- Weight at study initiation: 25 g - 40 g
- Assigned to test groups randomly: yes, under following basis: Animals weighing between 25 g and 40 g were assigned to the test groups by strict randomization.
- Housing:
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 oc
- Humidity (%): 30 - 70%
- Air changes (per hr): fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 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).

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Male and female animals per sacrifice interval were given Xyligen-K Powder dissolved in aqua dest. at dose levels of 68.1 mg/kg, 21 .5 mg/kg and 6.8 mg/kg body weight. Treatment consisted of a single oral administration. The volume of administration was 10 ml/kg body weight.
Duration of treatment / exposure:
single oral administration.
Frequency of treatment:
one single administration only
Post exposure period:
Sacrifice intervals between 16 hours and 48 hours
Doses / concentrationsopen allclose all
Dose / conc.:
68.1 mg/kg bw/day
Remarks:
Test group I, sacrifice interval: 16 hours
Dose / conc.:
68.1 mg/kg bw/day
Remarks:
Test group II; sacrifice interval: 24 hours
Dose / conc.:
21.5 mg/kg bw/day
Remarks:
Test group III, sacrifice interval: 24 hours
Dose / conc.:
6.8 mg/kg bw/day
Remarks:
Test group IV; sacrifice interval: 24 hours
No. of animals per sex per dose:
5 animals per sex and dose
Control animals:
yes, concurrent no treatment
Positive control(s):
As a positive control, 60 mg of cyclophosphamide/kg body weight, dissolved in aqua dest., was administered once orally to male and female animals in a volume of 10 ml/kg body weight.

Examinations

Tissues and cell types examined:
bone marrow / polychromatic erythrocytes
Details of tissue and slide preparation:
PREPARATION OF THE BONE MARROW
The bone marrow was prepared according to the method described by SCHMID, W.
- The two femora were prepared from the animals sacrificed by cervical dislocation, and all soft parts 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 fetal, calf serum which was at room temperature (about 2 ml/femur).
- 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 re-suspended.
- 1 drop of this suspension was dropped onto clean microscopic slides in each case using a Pasteur pipette. Smears were prepared using slides with ground edges. The preparations were dried in the air and subsequently stained.

STAINING:
- Stain in eosin and methylene blue solution for 5 minutes.
- Rinse in aqua dest., then place in fresh aqua dest. for
- 2 or 3 minutes.
- Stain in Giemsa solution for 12 minutes.
After being rinsed twice in aqua dest. and clarified in Xylene, the preparations were embedded in Entellan.
Evaluation criteria:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
- Ratio of polychromatic to normochromatic erythrocytes
- Number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4) (d = diameter of micronucleus, D = cell diameter)
Statistics:
Two statistical tests were used to answer the questions of whether there are significant differences between control group and dose group or between the individual 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 per animal 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 were marked with* (95%) and with** (99%).
A sequential statistical test was used to answer the question of a possible change of the characteristic in terms of time within a dose group ("test for homogeneity in terms of time"). On the basis of the hypothesis saying that the relative frequencies of the characteristic are identical at all times, the relative frequencies which reject this hypothesis at the level of 95% or 99% were determined successively. Significances were again marked with* (95%) or with** (95%).

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
distinct clinical symptoms
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: no chromosome damaging (clastogenic) effect
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In the determination of the acute oral toxicity deaths were observed down to a dose of 100 mg/kg. The dose level at which all animals survived, but which still led to distinct clinical symptoms, such as excitation, twitchings and tonic and clonic convulsions, was 68.1 mg/kg body weight and was selected as the highest dose in the present cytogenetic investigations.


RESULTS OF DEFINITIVE STUDY
An inhibition of erythropoiesis induced by the treatment of mice with Xyligen-K Powder was not detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups.
According to the results of the present study, there are no biologically or statistically significant differences in the frequency of erythrocytes containing micronuclei either between the solvent control and the 3 dose groups (68.1 mg/kg, 21 .5 mg/kg and 6.8 mg/kg) or between the various sacrifice intervals (16, 24 and 48 hours).

Any other information on results incl. tables

Clinical signs:

Substance

Dose

Time after administration

Clinical signs

Duration of signs

Solvent aqua dest.

10 ml/kg body weight

 

No symptoms

-

K-HDO

68.1 mg/kg bw

15 minutes

irregular respiration and excitation in most of the animals. In some mice, symptoms, such as tremors, twitchings, tonic and clonic convulsions, ruffled fur and apathy, were observed.

The symptoms subsided on the day after treatment.

K-HDO

21.5 mg/kg bw

15 – 30 minutes

irregular respiration and slight excitation and in some animals ruffled fur

On the day after treatment no abnormalities were detected any longer in any of the animals.

K-HDO

6.8 mg/kg bw

15 – 30 minutes

irregular respiration and slight excitation and in some animals ruffled fur

On the day after treatment no abnormalities were detected any longer in any of the animals.

Cyclophosphamide

60 mg/kg bw

 

No evident signs of toxicity

 

Necropsy

The gross-pathological examination of the animals sacrificed at the end of the study did not reveal any changes of the internal organs which could be attributed to the test substance administered.

 

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions chosen here, the test substance Xyligen-K Powder (= KHDO) has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of distribution in the course of mitosis.
Executive summary:

The substance Xyligen-K Powder was tested for mutagenicity in NMRI mice using the micronucleus test method. For this purpose, Xyligen-K Powder, dissolved in aqua dest., was administered once orally to male and female animals at dose levels of 68.1 mg/kg, 21 .5 mg/kg and 6.8 mg/kg body weight in a volume of 10 ml/kg body weight. For control purposes, male and female mice were administered merely the solvent by the same route. As a positive control, 60 mg of cyclophosphamide/kg body weight, dissolved in aqua dest., was administered once orally to male and female animals in a volume of 10 ml/kg body weight.

Animals which were administered the solvent aqua dest. or the positive control substance cyclophosphamide did not show any clinical signs of toxicity. However, about 15 minutes after the administration of Xyligen-K Powder the dose of 68.1 mg/kg body weight led to irregular respiration and excitation in most of the animals. In some mice, tremors, twitchings, tonic and clonic convulsions, ruffled fur and apathy were observed. The symptoms subsided on the day after treatment. About 15 - 30 minutes after the administrations of 21 .5 mg/kg and 6.8 mg/kg body weight irregular respiration and slight excitation and, in some animals, ruffled fur were also observed in the major part of the animals. On the day after treatment no abnormalities were detected any longer in any of the animals.

The gross-pathological examination of the internal organs of the male and female animals of all test groups sacrificed at the end of the study did not reveal any pathological changes that could be attributed to the test substance administered.

The animals were sacrificed and the bone marrow of the two femora was prepared 16, 24 and 48 hours after administration in the solvent control group and in the highest dose group of 68.1 mg/kg body weight. In the test groups 2 of 21 .5 mg/kg and 6 .8 mg/kg body weight and in the positive control group the 24-hour sacrifice interval was investigated only. After staining of the preparations 1000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 1000 polychromatic erythrocytes were also registered. According to the results of the present study, the single oral administration of Xyligen-K Powder in doses of 6.8 mg/kg, 21 .5 mg/kg and 68.1 mg/kg body weight did not lead to any increase in the number of polychromatic erythrocytes containing micronuclei. The rate of micronuclei was always in the same range as that of the control in all dose groups and at all sacrifice intervals. The erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was not influenced either.

Thus, under the experimental conditions chosen here, the test substance Xyligen-K Powder does not have any chromosome-damaging (clastogenic) effect and there were no indications of any impairment of distribution in the course of mitosis.