Dibutyl hydrogen phosphate

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:

1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1998

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Principles of method if other than guideline:

A micronucleus test was carried out with dibutyl hydrogen phosphate. The test compound was emulsified in deionized water and was given twice at an interval of 24 hours as oral doses of 100, 300 and 1000 mg/kg bw to male and female mice. According to the test procedure the animals were killed 24 hours after administration.

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Dibutyl hydrogen phosphate

EC Number:

203-509-8

EC Name:

Dibutyl hydrogen phosphate

Cas Number:

107-66-4

Molecular formula:

C8H19O4P

IUPAC Name:

dibutyl hydrogen phosphate

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH
- Age at study initiation: approx. 7 weeks
- Weight at study initiation: males: 33.3 g; females: 27.5g
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

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

IN-LIFE DATES: 1999

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

deionized water; stability and homogeneity in the vehicle confirmed over 4 hours

Details on exposure:

Preliminary experiment:
first dose: 2000 mg/kg bw dibutyl phosphate (3 males and 3 females) - no clinical signs, no mortality
second dose: 1500 mg/kg bw dibutyl phosphate (3 males and 3 females) - 1 out of 3 males and 2 out of 3 females died or were sacrificed for animal welfare reasons
third dose: 1200 mg/kg bw dibutyl phosphate (3 males and 3 females) - 2 out of 3 males and 1 out of 3 females died; motor activity decreased, squatting posture, palpebral fissure narrow, coat bristling
fourth dose: 1000 mg/kg bw dibutyl phosphate (3 males and 3 females) - 0 out of 3 males and 1 out of 3 females died; motor activity decreased

Conclusion: 1000 mg/kg bw twice was used as hightest dose for the main study

Duration of treatment / exposure:

Animals were sacrificed 24 hours after the last administration of the test substance.

Frequency of treatment:

Twice at an interval of 24 hours.

Post exposure period:

24 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 males and 5 females per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide distilled in water
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:

Mouse bone marrow erythrocytes.

Details of tissue and slide preparation:

Animals were killed by carbon dioxide asphyxiation 24 hours after dosing. For each animal, about 3 ml fetal 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.

Evaluation criteria:

2000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. In addition, the ratio of polychromatic erythrocytes to 200 total erythrocy1es was determined. Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted erythrocytes.

Criteria for a positive response

Both biological and statistical significances were considered tagether for evaluation purposes. A substance is considered as positive if there is a significant dose-related increase in the number of micronucleated polychromatic erythrocy1es compared with the concurrent negative control group. A test substance producing no significant doserelated increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

A one-sided Wilcoxon-Test was evaluated to check the validity of the study. The study was considered as valid in case the proportion of polychromatic erythrocytes with micronuclei in the positive control was significantly higher than in the negative control (p=0.05).

If the validity of the study had been shown the following sequential test procedure for the examination of the mutagenicity was applied: Based on a monotone-dose relationship one-sided Wilcoxon tests were performed starting with the highest dose group. These tests were performed with a multiple level of significance of 5%.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

determined in a preliminary dose finding study

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Oral administration of 1000 mg Dibutylphosphorsäure per kg body weight resulted in the death of two female out of 5 animals treated. These animals were replaced and survived after treatment. The following clinical sign was observed: motor activity decreased.
The dissection of the two female animals which died within 24 hours after the first
application revealed the following macroscopic finding: Intestinal tract filled with orange colored spume. The dissection of all other animals revealed no test substance related macroscopic findings.
The bone marrow smears were examined for the occurrence of micronuclei in red blood cells. The summarized results and individual data of all animals in all  treatment groups are presented in the attached tables.
The incidence of micronucleated polychromatic erythrocytes in the dose groups of
Dibutylphosphorsäure was within the normal range of the negative control groups.
No statistically significant increase of micronucleated polychromatic erythrocytes was observed. The ratio of polychromatic erythrocytes to total erythrocytes remained essentially unaffected by the test compound and was not less than 20% of the control values.

Cyclophosphamide induced a marked and statistically significant increase
in the number of polychromatic erythrocytes with micronuclei, thus indicating the
sensitivity of the test system.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

Executive summary:

The micronucleus test was carried out with dibutyl hydrogen phosphate following OECD TG 474. The test compound was emulsified in deionized water and was given twice at an interval of 24 hours as oral doses of 100, 300 and 1000 mg per kg body weight to male and female mice, based on the results of previous dose range finding assays. According to the test procedure the animals were killed 24 hours after administration.

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

Oral administration of 1000 mg Dibutylphosphorsäure per kg body weight resulted in the death of 2 out of 5 females treated. These animals were replaced and survived after treatment. The following clinical sign was observed: motor activity decreased.The number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with dibutyl hydrogen phosphate and was not less than 20% of the control values.

Cyclophosphamide induced 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 total erythrocytes was not changed to a significant extent.

Under the conditions of the present study the results indicate that di-n-butylphosphate is negative in the micronucleus test.