Dibutyl hydrogen phosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Dibutyl hydrogen phosphate was investigated in the Ames test. No evidence of mutagenic activity of dibutyl hydrogen phosphate was seen (with and without mutagenic activation). Additionally, dibutyl hydrogen phosphate was evaluated negative in a cytogenetic assay and also in a CHO/HGPRT test, also with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Principles of method if other than guideline:

Ames test with S.typhimurium TA 100, TA 1535, TA 98, TA 1537, E.coli WP2 uvrA; plate incorporation assay

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat liver S-9 mix, induced with phenobarbital and 5.6-benzoflavone

Test concentrations with justification for top dose:

0, 4.882, 9.755, 19.53, 39.06, 78.12, 156.2 µg/plate

Vehicle / solvent:

acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: -S9, AF-2 (TA100, WP2, TA98), sodium azide (TA 1535) and9-aminoacridine (TA1537); +S9, 9-aminoanthracen (all strains)

Details on test system and experimental conditions:

Ames test

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxicity was observed at 156.2 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

see result Tables 1 and 2 - attached

Dibutyl hydrogen phosphate was negative in Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Echerichia coli WP2 uvrA with and without metabolic activation.

Conclusions:

Interpretation of results: negative

Executive summary:

An Ames test with S.typhimurium strains TA 100, TA 1535, TA 98, TA 1537, and E.coli WP2 uvrA was conducted with dibutyl hydrogen phosphate according the guidelines for screening mutagenicity testing of chemicals (Japan).

Dibutyl hydrogen phosphate was tested in concentrations of 4.882 to 156.2 µg/plate. Toxicity was observed at 156.2 µg/plate, with and without metabolic activation. The test results were negative in all strains, with and without metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: scientifically acceptable and sufficiently documented

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Principles of method if other than guideline:

Mutagenicity of dibutyl hydrogen phosphate was investigated in a chromosomal aberration test with Chinese hamster CHL/IU cells.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: Chinese hamster CHL/IU cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix from rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

without S9 mix (continuous treatment for 24 and 48 hours): 0, 0.06, 0.12, 0.24 mg/mL
without S9 mix (short-term treatment for 6 hours): 0, 0.10, 0.21, 0.41 mg/mL
plus S9 mix (short-term treatment for 6 hours): 0, 0.14, 0.27, 0.54 mg/mL

Justification of dose based on cell growth experiments.

Vehicle / solvent:

acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

plates per test: 2
No. of cells analysed per culture: 200

Evaluation criteria:

Number of structural aberrations and number of cells with aberrations; polyploidy

Statistics:

yes

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

-S9 (continuous treatment): > 0.24 mg/ml; -S9 (short-term treatment): > 0.41 mg/ml; +S9 (short-term treatment): > 0.54 mg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

see tables 1 and 2 attached

Neither structural nor numerical chromosomal aberrations were induced in CHL/IU cells up to the concentration inducing 50% cell growth, in the presence or absence of an exogenous metabolic activation system.

Conclusions:

Interpretation of results: negative

Executive summary:

The mutagenicity of dibutyl hydrogen phosphate was investigated in a chromosomal aberration test with Chinese hamster CHL/IU cells, following the Japanese Guidelines for Mutagenicity Testing of Chemicals. The cell cultures were treated for 24 and 48 hours without S9 mix (continuous treatment) and for 6 hours with and without S9 mix. Concentrations were based on cytotoxicity, including test item concentrations that induced 50% cell growth. Neither clastogenic effects (structural and numerical chromosomal aberrations) nor polyploidy became obvious in these experiments. Dibutyl hydrogen phosphate can thus be considered as negative in the chromosomal aberration test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

other: the cells have a stable karyothype with a modal chromosome number of 22

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

exposure S9
period mix
concentrations in µg/mL
Experiment I
4 hours - 137.5 275.0 550.0 1100.0 2200.0
4 hours + 137.5 275.0 550.0 1100.0 2200.0
Experiment II
24 hours - 137.5 275.0 550.0 1100.0 2200.0
4 hours + 137.5 275.0 550.0 1100.0 2200.0

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Vehicle used: Concurrent solvent controls (deionised water (local tap water, deionised at Harlan CCR)) were performed.

Osmolarity and pH value:
The osmolarity and pH-value were determined in the solvent control and in the maximum concentration of the pre-experiment without metabolic activation without effects.


Culture Medium
For seeding and treatment of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 µg/mL) and amphotericin B (1 %). For the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

Metabolic activation system
Phenobarbital/beta-naphthoflavone induced rat liver S9

Seeding
Two to three days after sub-cultivation stock cultures were trypsinized at 37 °C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10 % FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2 % in PBS.

The PBS is composed as follows (per litre): NaCl 8000 mg, KCl 200 mg, KH2PO4 200 mg, Na2HPO4 150 mg.

Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution (PBS) containing 200 mg/l EDTA (ethylene diamine tetraacetic acid). Approximately 1.5×10E6 (single culture) and 5×10E2 cells (in duplicate) were seeded in plastic culture flasks. The cells were grown for 24 hours prior to treatment.

Pre-test on toxicity
A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. The general culture conditions and experimental conditions in this pre-test were the same as described for the mutagenicity experiment below. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

Treatment
After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 µl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to the test item for 24 hours in complete medium, supplemented with 10 % FBS, in the absence of metabolic activation.

The "saline G" solution had the following constituents (per litre): NaCl 8000 mg, KCl 400 mg, Glucose 1100 mg, Na2HPO4×2H2O 192 mg, KH2PO4 150 mg. The pH was adjusted to 7.2.

The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment as described below.

Three or four days after treatment 1.5×10E6 cells per experimental point were subcultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5×10E5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.

The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.

The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

Data Recording

The data generated were recorded in the raw data.

Acceptability of the Assay

The gene mutation assay is considered acceptable if it meets the following criteria:

- the numbers of mutant colonies per 10E6 cells found in the solvent controls fall within the laboratory historical control data range

- the positive control substances should produce a significant increase in mutant colony frequencies.

- the cloning efficiency II (absolute value) of the solvent controls must exceed 50 %

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.

A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

A positive response is described as follows:

A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.

The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.

However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of

the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Relevant cytotoxic effects were noted at the maximum concentration of the first experiment without metabolic activation. No relevant cytotoxic effects occurred in any other experimental part of this study

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation of the test item was observed up to the maximum concentration of all experiments.
Relevant cytotoxic effects indicated by a relative cloning efficiency I and/or relative cell density below 50% in both parallel cultures were noted at the maximum concentration of the first experiment without metabolic activation. No relevant cytotoxic effects occurred in any other experimental part of this study.
No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The mutant frequency did not exceed the historical range of solvent controls. The induction factor exceeded the threshold of three times the corresponding solvent control in the first culture of the first experiment without metabolic activation at 137.5 μg/mL. This increase however, was judged as biologically irrelevant since all absolute values of the mutation frequency remained within the range of historical solvent controls. Furthermore, the increase was not reproduced in the parallel culture performed under identical experimental conditions and was not dose dependent as indicated by the lacking statistical significance.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A single dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the second culture of the first experiment without metabolic activation. This trend however, was judged as irrelevant as it actually was reciprocal, going down versus increasing concentrations.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 8.9 up to 32.9 mutants per 10 E6 cells; the range of the groups treated with the test item was from 0.0 up to 31.3 mutants per 10 E6 cells.
EMS (150 μg/mL) and DMBA (1.1 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.
see attached result tables

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments (see attached result tables).

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Conclusions:

Interpretation of results: negative

Executive summary:

A HPRT test was performed according to OECD TG 476 to investigate the potential of dibutyl hydrogen phosphate to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration of 2200 µg/mL was equal to approximately 10 mM. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, dibutylphosphate was negative in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In an in-vivo micronucleus assay in mice no indication for a mutagenic effect was found.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

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

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

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

Dose / conc.:

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

Dose / conc.:

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

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

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%.

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

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

An Ames test with S.typhimurium strains TA 100, TA 1535, TA 98, TA 1537, and E.coli WP2 uvrA was conducted with dibutyl hydrogen phosphate according the guidelines for screening mutagenicity testing of chemicals (Japan). Dibutyl hydrogen phosphate was tested in concentrations of 4.882 to 156.2 µg/plate. Toxicity was observed at 156.2 µg/plate, with and without metabolic activation. The test results were negative in all strains, with and without metabolic activation.

The mutagenicity of dibutyl hydrogen phosphate was investigated in a chromosomal aberration test with Chinese hamster CHL/IU cells, following the Japanese Guidelines for Mutagenicity Testing of Chemicals. The cell cultures were treated for 24 and 48 hours without S9 mix (continuous treatment) and for 6 hours with and without S9 mix. Concentrations were based on cytotoxicity, including test item concentrations that induced 50% cell growth. Neither clastogenic effects (structural and numerical chromosomal aberrations) nor polyploidy became obvious in these experiments. Dibutyl hydrogen phosphate can thus be considered as negative in the chromosomal aberration test.

A HPRT test was performed according to OECD TG 476 to investigate the potential of dibutyl hydrogen phosphate to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration of 2200 µg/mL was equal to approximately 10 mM. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, dibutyl hydrogen phosphate was negative in this HPRT assay.

 

The mouse 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 dibutyl hydrogen phosphate 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 dibutyl hydrogen phosphate is negative in the micronucleus test.

In all in vitro genetic toxicity assays (Ames test, cytogenetic test, CHO/HGPRT test) and the in vivo micronucleus test dibutyl hydrogen phosphate was negative.

 

Justification for classification or non-classification

Dibutyl hydrogen phosphate was negative in vitro and in vivo. According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.