4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-06-17 - 2015-07-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well-documented GLP OECD 487 guideline study without deviations on the registered substance itself.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

OECD Guideline for the Testing of Chemicals No. 487 “In vitro Mammalian Cell Micronucleus Test”, adopted 26 September 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D-65189 Wiesbaden

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin

EC Number:

277-633-6

EC Name:

4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin

Cas Number:

73912-21-7

Molecular formula:

C27H35O3P

IUPAC Name:

7,13-dicyclohexyl-5,15-dimethyl-9,11-dioxa-10-phosphatricyclo[10.4.0.0³,⁸]hexadeca-1(12),3(8),4,6,13,15-hexaen-10-ol

Test material form:

solid: pellets

Details on test material:

- Substance type: pure substance
- Storage condition of test material: In the refrigerator at 2-8°C, moisture protected

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Human lymphocytes
Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a male donor (31 years old) for Experiment I and from a male donor (25 years old) for Experiment II. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-Test: 1.2 to 1200.0 μg/mL (with regard to the solubility of the test item)
Experiment I, ±S9, 4h exposure: 1.2, 2.3, 4.7, 9.4, 18.8, 37.5, 75.0, 150.0, 300.0, 600.0, 1200.0 µg/ml
Experiment II, -S9, 20h exposure: 29.1, 34.9, 41.9, 50.2, 60.3, 72.3, 86.8, 104.2, 125.0, 150.0 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

0.5% DMSO (99.99% purity)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: demecolcin

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h (pulse exposure) or 20h (continuous exposure)
- Expression time (cells in growth medium): 16h or 0h in medium + 20h with CytB
- Fixation time (start of exposure up to fixation or harvest of cells): 40h

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B (4 μg/mL)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 parallel cultures

NUMBER OF CELLS EVALUATED: at least 1000 binucleate cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-block proliferation index as indication of cytostasis

Evaluation criteria:

Evaluation of cytotoxicity and cytogenetic damage:
Evaluation of the slides was performed using NIKON microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the CBPI (Cytokinesis-block proliferation index) was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

Statistics:

Statistical analysis
Statistical significance will be confirmed by using the Chi-squared test (α < 0.05) using the validated R Script CHI2.Rnw for those values that indicate an increase in the number of cells with micronuclei compared to the concurrent solvent control. Other statistical methods may be used if appropriate.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH / osmolality: No relevant influence on osmolarity or pH was observed.
- Precipitation: Precipitation of the test item in the culture medium was observed in Experiment I at 37.5 μg/mL and above in the absence of S9 mix and at 75.0 μg/mL and above in the presence of S9 mix and in Experiment II in the absence of S9 mix at 86.8 μg/mL and above at the end of treatment.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In both cytogenetic experiments, in the absence and presence of S9 mix, no cytotoxicity was observed.

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

The study was conducted under GLP according to OECD guideline 487 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation. Positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin to induce micronuclei in mammalian cells.
Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, of 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to precipitating or the highest evaluable concentrations.

Executive summary:

In a mammalian cell cytogenetics assay, Micronucleus test (OECD 487), primary human lymphocyte cultures were exposed to 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin in DMSO at concentrations of 1.2, 2.3, 4.7, 9.4, 18.8, 37.5, 75.0, 150.0, 300.0, 600.0, 1200.0 µg/ml for 4h with and without metabolic activation and 29.1, 34.9, 41.9, 50.2, 60.3, 72.3, 86.8, 104.2, 125.0, 150.0 µg/ml for 20h without metabolic activation (Phenobarbital/β-naphthoflavone induced rat liver S9).

4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin was tested up to precipitating concentrations. Positive controls induced the appropriate response.  There was no evidence or a concentration related positive response of Micronuclei induced over background.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 487 for in vitro cytogenetic mutagenicity data.