2,4,6-tri-n-propyl-2,4,6-trioxo-1,3,5,2,4,6-trioxatriphosphorinane

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995-10-16 to 1995-12-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Directive 92/69, L 383 A, Annex B. 12., p. 154 -156

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PPA 4/94
- Expiration date of the lot/batch: June 1996
- Purity test date: March 6th, 1995

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: darkness at approximately 20°C in a fume cupboard
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stability in the vehicle is guaranteed for 4 hours

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment the test substance was dissolved in DMSO (dried) at an appropriate concentration. A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Tierzucht Schonwalde GmbH i.G., Hauptstrafte 62, 16352 Schonwalde, Germany
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: males: mean 40.2 g+/- 2.73 g, females: mean 31.9 g +/- 2.93 g
- Assigned to test groups randomly: yes
- Housing: in fully air-conditioned rooms in makrolon cages type Type 3 (five animals per cage) on soft wood granulate
- Diet: rat/mice diet ssniff® R/M-H (V 1534), ad libitum ssniff® GmbH, Postbox 2039, 59480 Soest, Germany
- Water: tap water in plastic bottles, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±3°C
- Humidity (%): 50 ± 20 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: DMSO, dried
- Concentration of test material in vehicle: 0 (vehicle control) and 12.5 % (w/v)
- Amount of vehicle: total volume 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment the test substance was dissolved in DMSO (dried) at an appropriate concentration. A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed. DMSO (dried) was administered in the same way to the negative control groups. The study included a simultaneous positive control using Endoxan®, which was administered once orally at a dose of 50 mg per kg body weight.

Duration of treatment / exposure:

In conformity with the test procedure the animals were killed by carbon dioxide asphyxiation 12, 24 or 48 hours after administration.

Frequency of treatment:

single administration

Post exposure period:

12, 24 or 48 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

vehicle control, killing time 24 h p.a.

Doses / concentrations 2

Dose / conc.:

1 250 mg/kg bw/day (actual dose received)

Remarks:

test group, killing time 24 h p.a.

Doses / concentrations 3

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

vehicle control, killing time 48 h

Doses / concentrations 4

Dose / conc.:

1 250 mg/kg bw/day (actual dose received)

Remarks:

test group, killing time 48 h

Doses / concentrations 5

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

vehicle control, killing time 12 h

Doses / concentrations 6

Dose / conc.:

1 250 mg/kg bw/day (actual dose received)

Remarks:

test group, killing time 12 h

No. of animals per sex per dose:

5 male anf 5 female animals per group per killing time

Control animals:

yes, concurrent vehicle

Positive control(s):

Endoxan containing cyclophosphamide
- Route of administration: oral by gavage
- Doses / concentrations: 50 mg/kg bw, killing time 24 h p.a.

Examinations

Tissues and cell types examined:

bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Oral administration of 1500 mg per kg body weight resulted in mortality in male mice. The highest sub lethal dose of 1250 mg per kg body weight was selected for the main study.

TREATMENT AND SAMPLING TIMES
In conformity with the test procedure the animals were killed by carbon dioxide asphyxiation 12, 24 or 48 hours after administration.

DETAILS OF SLIDE PREPARATION:
For each animal, about 3 mL foetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at approx. 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 hours.
Staining was performed as follows:
- 5 minutes in methanol
- 5 minutes in May-Grunwald's solution
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Entellan

METHOD OF ANALYSIS:
1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. All bone marrow smears for evaluation were coded to ensure that the group from which they were taken remained unknown to the investigator. The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes, and the number of normocytes with micronuclei occurring in the 1000 normocytes, were evaluated statistically.

Evaluation criteria:

Both biological and statistical significances were considered together for evaluation purposes.
A substance is considered positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes for at least one of the time points compared with the concurrent negative control group. A test substance producing no significant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
For the assay to be valid, individual and/or group mean values for the positiv control should exeed the laboratory's historical control range.

Statistics:

A Wilcoxon-Test (one-sided) was evaluated to check the validity of the study. This is done by comparing the number of polychromatic erythrocytes with micronuclei in the positive control with the negative control.
A Wiicoxon-Test (one-sided) was performed for each treatment interval (12h, 24h, 48h) and for polychromatic and normochromatic erythrocytes. These tests are performed sequentially with a multiple level of significance of 5%.
The following comparisons are performed only if there is a difference between the positive control and the negative control (24 h). This was performed with a Wilcoxon-Test (two-sided) with a 5 %-level of significance. Wilcoxon-Tests (two-sided) are performed sequentially for the ratio of polychromatic erythrocytes for each treatment interval (12h, 24h, 48h) at a multiple level of significance of 5 %. The data obtained were also compared with historical controls.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Doses producing toxicity: 1250 mg/kg, 48 hours after application all animals were free of clinical signs of toxicity.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: Oral administration of 2000 and 1500 mg per kg body weight resulted in mortality in 1 out of 3 males and 1 out of 3 females (2000 mg/kg bw) and 2 out of 3 males (1500 mg/kg bw), respectively. No mortality occured in the 1000 mg/kg bw dose group.
- Clinical signs of toxicity in test animals:
2000 mg/kg bw dose group:
stilted gait, squatting posture, palpebreal fissure narrow, coat bristling, spontaneous activity decreased, cyanosis
1250 mg/kg bw doese group:
spontaneous activity decreased, palpebreal fissure narrow, palpebreal fissure very narrow, squatting posture, stilted gait and coat bristling
1500 mg/kg bw dose group:
stilted gait, squatting posture, palpebreal fissure narrow, palpebreal fissure closed, coat bristling, spontaneous activity decreased, eyelids adhering and sonoures rales
1000 mg/kg bw dose group:
no clinical signs were observed

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: The incidence of micronucleated polychromatic and normochromatic erythrocytes in the test group was within the normal range of the negative control groups.
- Ratio of PCE/NCE: The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by the test compound.

Any other information on results incl. tables

For details on results, please refer to the attached document.

Applicant's summary and conclusion

Conclusions:

The test substance did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not mutagenic in the micronucleus test.

Executive summary:

 

The micronucleus test was carried out with the test substance to assess its potential to cause

chromosomal damage (clastogenicity). The test compound was dissolved in DMSO (dried) and was given once as an orally dose of 1250 mg per kg body weight to male and female mice, based on the results of a previous dose range finding assay (see preliminary study). According to the test procedure the animals were killed 12, 24 or 48 hours after administration.

Endoxan® was used as positive control substance and was administered once orally at a dose of 50 mg per kg body weight.

The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with the test substanceand was statistically not different from the control values.

Endoxan®induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to normocytes was not changed to a significant extent.

Under the conditions of the present study the results indicate that the test substanceis not mutagenic in the micronucleus test.