di-p-tolyl(2,4,6-trimethylbenzoyl)phosphine oxide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study planned

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS
[Please provide information for all of the points below. The information should be specific to the endpoint for which testing is proposed. Note that for testing proposals addressing testing on vertebrate animals under the REACH Regulation this document will be published on the ECHA website along with the third party consultation on the testing proposal(s).]

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out
substance name: (di-p-tolylphosphoryl)(mesityl)methanone
CAS number: 270586-78-2
- Name of the substance for which the testing proposal will be used [if different from tested substance]
substance name: (di-p-tolylphosphoryl)(mesityl)methanone
CAS number: 270586-78-2

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:

- Available GLP studies
Genetic toxicity in vitro:
Determinationofthepotentialof
(di-p-tolylphosphoryl)(mesityl)methanone
toinduceformationofmicronuclei
inhumanlymphocytes
withthe“InVitroMammalianCellMicronucleusTest”
followingOECD487andEUB.49
Result: The result of the micronucleus test with the test item (di-p-
tolylphosphoryl)(mesityl)methanone is consideredas “positive” under the conditions of the
test.

- Available non-GLP studies
not available
- Historical human/control data
not available
- (Q)SAR
not available

- In vitro methods
Genetic toxicity in vitro:
Determinationofthepotentialof
(di-p-tolylphosphoryl)(mesityl)methanone
toinduceformationofmicronuclei
inhumanlymphocytes
withthe“InVitroMammalianCellMicronucleusTest”
followingOECD487andEUB.49
Result: The result of the micronucleus test with the test item (di-p-
tolylphosphoryl)(mesityl)methanone is consideredas “positive” under the conditions of the
test.

- Weight of evidence
1 (reliable without restriction)
GLP study following OECD487 and EUB.49

- Grouping and read-across
not available
- Substance-tailored exposure driven testing [if applicable]
not available
- Approaches in addition to above [if applicable]
not available
- Other reasons [if applicable]
not available

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- [free text] not available

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed [if relevant]
study plan:
In vivo Mammalian AlkalineComet Assay
of Liver, Glandular Stomach and Duodenum
Cells in Rats with (di-p-tolylphosphoryl)(mesityl)methanone

2.1. Aim of the Study
The in vivo mammalian alkaline Comet Assay detects DNA damage and incomplete excision repair sites in eukaryotic cells upon exposure to genotoxic compounds [10]. The term “comet” is used to identify the individual cell DNA migration patterns induced by the test item.
Ostling and Johanson developed this technique in 1984 [11]. Singh et al. (1988) later modified this technique to an alkaline Comet assay (pH > 13) to allow the detection of single strand brakes (SSB) under these conditions resulting from alkali labile sites (ALS), incomplete excision repair or oxidative base damage [12].
During the comet assay procedure individual cells are embedded in a thin agarose gel on a microscope slide. All cellular proteins are then removed from the cells by lysing with detergents and high salt concentrations. The DNA is allowed to unwind under alkaline conditions. Following the unwinding, the liberated DNA undergoes electrophoresis, allowing the broken DNA fragments or damaged DNA to migrate away from the nucleus. After staining with a DNA-specific fluorescent dye such as ethidium bromide the gel is ready for the detection of fluorescence in the head and tail of the “comet”. The relative intensity of fluorescence in the tail is a function of the frequency of DNA breaks; it can be assessed using computer-based analysis. The intensity of the comet tail relative to the total intensity (head plus tail) reflects the amount of DNA breakage [13][14].
The in vivo comet assay is most often performed in rodents, especially rats, but it can be applied to any tissue in any given in vivo model, provided that a single cell or nucleus suspension can be obtained. To fulfill animal welfare requirements, it can be combined with other toxicological and genotoxic studies (e.g. repeated dose toxicity studies [15], in vivo mammalian erythrocyte micronucleus assay [16]). The genotoxic potential of the test chemical should be assayed in the target tissue(s) of interest considering e.g. the route of human exposure, metabolism, distribution and other available data on behavior of the test substance [5]. The outcome of the assay can be dependent upon in vivo ADME (absorption, distribution, metabolism and excretion) and DNA repair processes, which vary among species, tissues and types of DNA damage.
2.2. Justification for the Selection of the Test System
OECD Guidelines for Testing of Chemicals, Section 4, No. 489 “In Vivo Mammalian Alkaline Comet Assay”, adopted July 29, 2016 [5] recommends using rats.
Rats have been used for many years as standard experimental animals in genotoxicity investigations. In addition, they have been used as experimental animal in the toxicological safety characterization of the test item. Data from such experiments are useful for the design of the alkaline Comet Assay and interpretation of its findings [15].
2.3. Justification for the Selection of the Test Method
Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 489 [5] “In Vivo Mammalian Alkaline Comet Assay“, adopted July 29, 2016 – recommends several application routes (such as intravenous, gavage). The route should be chosen in consideration of the anticipated route of human exposure and to ensure adequate exposure of the target tissue(s). Intraperitoneal injection should only be used with specific scientific justification.
2.4. Animal Welfare Statement
The principles of OECD Guidance Document No. 19: Guidance Document on the Recognition, Assessment, and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation, were followed [9].
This study will be performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorized by the Bavarian animal welfare administration.
3. Materials and Methods
3.1.1. Preparation of the Test Item Formulation
The test item will be prepared in an appropriate solvent or vehicle and diluted prior to treatment. It is
recommended that, wherever possible, the use of an aqueous solution/suspension will be considered
first, followed by consideration of a solution/emulsion in oil and the possible solution in other vehicles.
A final decision will be made based on the solubility tests. The solvent/vehicle will be chosen according
to its relative non-toxicity for the animals.
Test Item formulations will be transferred to BSL Munich together with the control formulations under
consideration of stability.

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

[bis(4-methylphenyl)phosphoroso](2,4,6-trimethylphenyl)methanone

EC Number:

884-585-5

Cas Number:

270586-78-2

Molecular formula:

C24H25O2P

IUPAC Name:

[bis(4-methylphenyl)phosphoroso](2,4,6-trimethylphenyl)methanone