Tetrahydrothiopyran-3-carboxaldehyde

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:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test substance: Tetrahydrothiopyran-3-aldehyd
- Batch No.: "aus Fass 72"
- Test substance No.: 89/713
- Appearance, consistency: Colorless liquid
- Degree of purity: about 93%
- Storage: + 4° C to + 6° C

Test animals

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH, WIGA, O-W8741 Sulzfeld, FRG
- Age at study initiation: no data
- Weight at study initiation: mean weight of about 27 g
- Assigned to test groups randomly: yes
- Fasting period before study: none
- Housing: groups of 5 separately according to sex
- Diet: Standardized pelleted feed, Kliba Haltungsdiaet, Klingentalmuehle AG, CH-4303 Kaiseraugst, Switzerland, ad libitum
- Water: tap water ad libitum
- Acclimation period: no data


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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: olive oil

Details on exposure:

The aim of the present study was to test the substance Tetrahydrothiopyran-3-aldehyd for clastogenic and for spindle poison effects in NMRI mice after a single oral administration using the micronucleus test method. The test procedure and the preparation of the bone marrow were based on the method of SCHMID, W. and SALAMONE, M. et al.
The micronucleus test is a method independent of the karyotype of the test animal for an indirect detection of a chromosome-damaging (clastogenic) effect or a damage of the mitotic apparatus (spindle poison effect). Micronuclei (Howell-Jolly bodies) are scored primarily in young, polychromatic erythrocytes which contain one or sometimes more than one micronucleus occurring as a round or seldom as a crescent-shaped or bizarre structure. The size of micronuclei may indicate the mode of action of the test substance. Thus, chromosome-breaking agents primarily induce small micronuclei which are formed from acentric chromosome fragments and the diameter of which is about 1/20 to 1/5 < 1/4) of the cell diameter. Substances which induce impairments of distribution in the course of mitosis, however, lead to an increase in large micronuclei which have a diameter >1/4 of the cell diameter and which may represent entire chromosomes.

The study was carried out in October 1990 in accordance with the OECD guideline for testing of chemicals - "Genetic Toxicology: Micronucleus Test", No. 474 and in accordance with the EEC Directive 84/449, B 12. Mutagenicity (Micronucleus Test).

Test groups and doses
Male and female animals were assigned to the test groups according to a randomization plan prepared with an appropriate computer program. The dose levels were set as follows:
In a pretest for the determination of the acute oral toxicity deaths were observed down to a dose of 3830 mg/kg body weight. 3160 mg/kg body weight was survived by all animals but led to signs of toxicity such as irregular respiration, abdominal and lateral position and constricted eyelids; the general state of the animals was poor. Therefore, a dose of 3000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1000 mg/kg and 333 mg/kg body weight were administered as further doses. The substance to be administered per kg body weight was dissolved in olive oil.
- Test groups 2 were given 3000 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of 30 g/100 ml.
- Test group 3 was given 1000 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of 10 g/100 ml.
- Test group 4 was given 333 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of' 3,33 g/100 ml.

Analysis
Test substance preparation analysis:
For the determination of the test substance concentration in the solvent 3 samples of the highest dose and lowest 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 by means of gas chromatography.
The stability of the test substance in the solvent olive oil over a period of 4 hours was verified analytically. The analytical investigations were carried out in the analytical laboratory of BASF AG.

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 AG as well as for the presence of germs by a contract laboratory.

Test procedure and administration
Male and female animals per sacrifice interval were given Tetrahydrothiopyran-3-aldehyd dissolved in olive oil at dose levels of 3000 mg/kg, 1000 mg/kg and 333 mg/kg body weight. Treatment consisted of a single oral administration. The volume of administration was 10 ml/kg body weight.

All test substance formulations were prepared immediately before administration. The amount of substance or volume to be administered was related to the specific weight of the individual animals on the day of the experiment. For control purposes, male and female animals were given merely the solvent olive oil by the same route.

Duration of treatment / exposure:

16, 24, and 48 hours, respectively

Frequency of treatment:

single treatment

Post exposure period:

none

Doses / concentrations

Remarks:

Doses / Concentrations:
3000 mg/kg, 1000 mg/kg and 333 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

5 females per dose

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:

Clinical examinations
After the administration of the test substance the animals were examined for any evident clinical signs of toxicity.

Tissues examined:
- bone marrow

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, 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.

Evaluation criteria:

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 micronuclei
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) (d =diameter of micronucleus, D = cell diameter)
The size of micronuclei may give an indication an 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:

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

Results and discussion

Test results

Sex:

female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Analysis
Test substance preparation analysis:
Depending on the dose, about 100 % of the theoretical values could be determined analytically.
The homogeneity of the test substance in the solvent olive oil was guaranteed by constant stirring during the removal and administration of the test substance formulation and by analytical determination of 3 individual samples of the highest and lowest 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 3000 mg/kg body weight led to irregular respiration, squatting posture and constricted eyelids about 30 minutes after test substance administration. After about 1 hour in few cases abdominal and lateral position was additionally observed and the general state of the animals was poor. The day after treatment the animals did not show clinical signs any longer. In the 1000 mg/kg and 333 mg/kg groups only irregular respiration was found about 30 minutes after treatment which lasted for about further 30 minutes. 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 olive oil in a volume of 10 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 3000 mg/kg body weight, 1.4 ‰ polychromatic erythrocytes containing micronuclei were found after 16 hours, 0.9 ‰ after 24 hours and 1.1 ‰ after 48 hours. In the two lower dose groups rates of micronuclei of about 0.7 ‰ (1000 mg/kg group) and 0.8 ‰ (333 mg/kg group) were detected after a sacrifice interval of 24 hours in each case.

With 7.2 ‰ the positive control substance cyclophosphamide for clastogenicity, as expected, led to a clear increase in the number of polychromatic erythrocytes containing exclusively small micronuclei at a dose level of 20 mg/kg body weight. With 72.6 ‰ 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. 24.2 ‰. 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 Tetrahydrothiopyran-3-aldehyd did not lead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d > D/4) did not deviate from the solvent control value at any of the sacrifice intervals. No inhibition of erythropoiesis induced by the treatment of mice with Tetrahydrothiopyran-3-aldehyd was detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups.

CONCLUSIONS
According to the results of the present study, there are thus no biologically relevant, significant differences in the frequency of erythrocytes containing micronuclei either between the solvent control and the 3 dose groups (3000 mg/kg, 1000 mg/kg and 333 mg/kg) or between the various sacrifice intervals (16, 24, and 48 hours). Thus, under the experimental conditions chosen here, the test substance Tetrahydrothiopyran-3-aldehyd has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis.

Any other information on results incl. tables

SUMMARY

The substance Tetrahydrothiopyran-3-aldehyd was tested for clastogenicity and for the ability to have spindle poison effects in NMRI mice using the micronucleus test method. For this purpose, Tetrahydrothiopyran-3-aldehyd, dissolved in olive oil, was administered once orally to male and female animals at dose levels of 3000 mg/kg, 1000 mg/kg and 333 mg/kg body weight in a volume of 10 ml/kg body weight in each case. As negative control, male and female mice were administered merely the solvent olive oil by the same route. As positive control for clastogenicity, 20 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. As positive control for spindle poison effects, 0.15 mg of vincristine/kg body weight, dissolved in aqua dest., was administered once intraperitoneally to male and female animals in a volume of 10 ml/kg body weight.

 

Animals which were administered the solvent or the positive control substances cyclophosphamide or vincristine did not show any clinical signs of toxicity. A dose of 3000 mg/kg body weight led to evident signs of toxicity. In the 1000 mg/kg and 333 mg/kg groups only irregular respiration was observed about 30 minutes after treatment.

 

The animals were sacrificed and the bone marrow of the two femora was prepared 16, 24 and 48 hours after administration in the highest dose group of 3000 mg/kg body weight. In the test groups of 1000 mg/kg and 333 mg/kg body weight, in the negative control group and in the

positive control groups the 24-hour sacrifice intervals were 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 Tetrahydrothiopyran-3-aldehyd in doses of 3000 mg/kg, 1000 mg/kg and 333 mg/kg body weight did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always in the same range as that of the negative control in all dose groups and at all sacrifice intervals. No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

 

Thus, under the experimental conditions chosen here, the test substance Tetrahydrothiopyran-3-aldehyd does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of' chromosome distribution in the course of mitosis.