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Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
Mutagenic activity of Phosphoric acid, butyl ester was investigated in three bacterial reverse mutation assays (Ames test; test strains used: S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538, TA 2638 and E. coli WP2 uvr A) and in an in vitro chromosome aberration study in Chinese Hamster CHL cells. Furthermore, a read across was performed to gene mutations studies (HPRT test) with the structural analogues reaction mass of “Methyl dihydrogen phosphate and orthophosphoric acid and dimethyl hydrogen phosphate”, “Phosphoric acid, mixed esters with butyl alcohol and ethylene glycol” and “Hexadecyl dihydrogen phosphate” (for read across justification please refer to attached document, IUCLID Chapter 7.6.1). Negative results were obtained in the Ames tests, the gene mutation tests and the chromosome aberration study in Chinese hamster cells with and without metabolic activation. Phosphoric acid, butyl ester was also proved to be non-clastogenic in an in vivo Micronucleus assay in mice.
Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Mar - Aug 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline conform GLP study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 7 weeks
- Weight at study initiation: males - 33.3 g, females - 27.5 g
- Assigned to test groups randomly: yes
- Housing: in makrolon cages type 3 (5 per cage) on softwood granulate
- Diet (e.g. ad libitum): rat/mice diet ssniff R/M-H (V 1534), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 5 days

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

IN-LIFE DATES: From: 18.5. To: 22.6.1999
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: deionized water
- Concentration of test material in vehicle: 1%, 3% or 10%
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On days of administration the test substance was emulsified in deionized water at the appropriate concentrations. A magentic stirrer was used to keep the preparations homogeneous until dosing had been completed.
Duration of treatment / exposure:
twice at an interval of 24 h
Frequency of treatment:
twice
Post exposure period:
24 h after dosing
Remarks:
Doses / Concentrations:
100 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
50 mg/kg bw Endoxan containing Cyclophosphamide
Tissues and cell types examined:
erythrocytes from femor bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a preliminary dose range findingstudy, oral administration of 1200 kg/kg bw and higher doses caused strong clinical signs of toxicity and resulted in mortality in male and female mice. Oral administration of 1000 mg/kg bw resulted in mortality in one male mouse.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The test substance was administered twice at an interval of 24 h orally by gavage to the test animals at doses of 100, 300 and 1000 mg/kg bw. The vehicle and positive control was administered in the same way.
Following dosing, the animals were examined regularly for mortality and clinical signs of toxicity.

DETAILS OF SLIDE PREPARATION:
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 min. 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 h. The cells were stained with Giemsa.

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 erythrcytes 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 erythrocytey with micronuclei out of the 2000 counted erythrocytes. All bone marrow smears for evaluation were coded to ensure that the group from which they were taken remained unknown to the investigator.

Criteria for a positive response:
Both biological and statstical significances were considered together for evaluation puproses.
A substance is considered as positive if there is a significant dose-related increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group. A test substance producing no significant dose-related 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 reythrocytes with micronclei 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-relationshi one-sided Wilcoxon tests were performed starting with the highest dose group. These tests were performed with a multiple level of significance of 5%.
Sex:
female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at 1000 mg/kg bw
Vehicle controls validity:
valid
Positive controls validity:
valid
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Oral administration of 1000 mg/kg bw resulted in the death of 2 female out of 5 animals treated. These animals wer replaced and survived after treatment. The following clinical sign was observed: motor activity decreased.
The dissection of the 2 female animlas which died within 24 h 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 inicidence of micronucleated polychromatic erythrocytes in the dose groups of Phosphoric acid, butyl ester was within the normal range of the negative control group. No statisitcally significant increase of micronucleated polychromatic erythrocytes was observed. The ratio of polychromatic erythrocytes to total erythrocytes remained essentailly unaffected by the test compound and was not less than 20% of the control values.

Cyclophosphamide induced a marked and statistically significant increae in the number of polychromatic erythrocytes with micronuclei, thus indicating the sensitivty of the test system.
Conclusions:
Interpretation of results (migrated information): negative
The results lead to the conclusion that Phosphoric acid, butyl ester did not cause a substantial increase of microncleated polychromatic erythrocytes and is not mutagenic in the micronucleus test under the conditions described.
Executive summary:

The test substance was administered twice at an interval of 24 h orally by gavage to the test animals at doses of 100, 300 and 1000 mg/kg bw. The vehicle and positive control was administered in the same way. Following dosing, the animals were examined regularly for mortality and clinical signs of toxicity.

The number of polychromatic erythrocytes was examined.

Oral administration of 1000 mg/kg bw resulted in the death of 2 female out of 5 animals treated. These animals wer replaced and survived after treatment. The following clinical sign was observed: motor activity decreased. The dissection of the 2 female animlas which died within 24 h 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 inicidence of micronucleated polychromatic erythrocytes in the dose groups of Phosphoric acid, butyl ester was within the normal range of the negative control group. No statisitcally significant increase of micronucleated polychromatic erythrocytes was observed. The ratio of polychromatic erythrocytes to total erythrocytes remained essentailly unaffected by the test compound and was not less than 20% of the control values. Cyclophosphamide induced a marked and statistically significant increae in the number of polychromatic erythrocytes with micronuclei, thus indicating the sensitivty of the test system.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Phosphoric acid, butyl ester showed negative results in the study for the induction of gene mutations (bacterial reverse mutation assay) by frameshift or base-pair substitutions with and without metabolic activation. The study was performed with the test strains S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538, TA 2638 and E. coli WP2 uvr A. Test concentrations up to the limit concentration of 10 µL/plate were tested in the experiment. The test compound proved to be not mutagenic to the bacterial strains.

Structural analogues of Phosphoric acid, butyl esteralso yielded negative results in an in vitro gene mutation study in mammalian cells in concentration up to 5000 µg/ml.

Phosphoric acid, butyl esterwas assessed for its potential to induce chromosome aberrations in Chinese hamster cells (CHL) in vitro. The test item did not induce chromosome aberrations.

The substance failed to induce clastogenicity in an in vivo Micronucleus test in mice.

Justification for selection of genetic toxicity endpoint
This study is selected as key study representing the toxicological endpoint "Genetic toxicity" since it was an in vivo study and examines the most sensitive genotoxic mechanism. The study was performed according to the current OECD Guideline 474 and und GLP.

Justification for classification or non-classification

In conclusion, Phosphoric acid, butyl ester (or its structural analogues) is not mutagenic in the bacterial reverse mutation assay, the in vitro gene mutation study, the in vitro chromosome aberration in the presence and absence of metabolic activation and in an in vivo Micronucleus assay in mice up to the tested concentrations. 

Phosphoric acid, butyl ester does not have to be not classified for mutagenicity since this substance (or its structural analogues) did not reveal any mutagenic effect in the bacterial reverse mutation assay in the presence or absence of metabolic activation in concentrations up to 5000 µg/plate, in the in vitro gene mutation assay (up to 5000 µg/mL), in the in vitro chromosome aberration study or in an in vivo Micronucleus assay in mice.

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