Tris(methylphenyl) phosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is not considered to be genotoxic on the basis of available data, as well as data on analogous substances.

Link to relevant study records

Reference

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

July - October 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Study, conducted to recognised study guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Structural chromosome aberrations in somatic and/or germ cells

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79: Chinese hamster lung, male
ECACC Cat. No.: 86041102
Lot No.: 05F013
Supplier: ECACC (European Collection of Cells Cultures)
Morphology: Fibroblast

The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for genetic toxicity assays with low background aberrations. These cells are chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12 14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male). This cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks were kept in a freezer at -80 ± 10 deg C. The stock was checked for mycoplasma infection before freezing. No infection of mycoplasma was noted.

Trypsin-EDTA (0.25% Trypsin, 1mM EDTA) solution was used for cell detachment to subculture. The laboratory cultures were maintained in 75 cm2 plastic flasks at 37 deg 0.5 °C in a humidified atmosphere containing approximately 5 % CO2 in air. The V79 cells for this study were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 1% Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 ug/mL amphoptericin-B) and 10 (v/v) % heat-inactivated foetal bovine serum (DMEM-10, culture medium). During the treatments, the serum content of the medium was reduced to 5 (v/v) % (DMEM-5).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital (PB) and b-naphthoflavone (BNF) rat liver, S9

Test concentrations with justification for top dose:

Assay 1
3-hr treatment without S9 Mix, harvest 20 hours from the beginning of treatment
1.37, 4.12, 12.35, 37.04, 111.1, 333.3, 1000 µg/ml

3-hr treatment withS9 Mix, harvest 20 hours from the beginning of treatment
1.37, 4.12, 12.35, 37.04, 111.1, 333.3, 1000 µg/ml+S9 mix 50 µL/ml

Assay 2

3-hr treatment without S9 Mix, harvest 20 hours from the beginning of treatment
0.0169 , 0.0508 , 0.152, 0.457, 1.37, 4.12, 12.35, 37.04, 111.1, 333.3, 1000 µg/ml

3-hr treatment withS9 Mix, harvest 20 hours from the beginning of treatment
1.37, 4.12, 12.35, 37.04, 111.1, 333.3, 1000 µg/ml+S9 mix 50 µL/ml

Vehicle / solvent:

Name: Dimethyl sulfoxide
Abbreviation: DMSO
Lot No.: 1400666*
Appearance: Clear liquid
Supplier: Sigma-Aldrich Co.
Expiry date: June 2014
Storage condition: Room temperature, under N2

Name: Dimethyl sulfoxide
Abbreviation: DMSO
Lot No.: BCBB7249**
Appearance: Clear liquid
Supplier: Sigma-Aldrich Co.
Expiry date: January 2016
Storage condition: Room temperature, under N2

*: used in the preliminary experiments, Assay 1 and Assay 2
**: used in the repeated Assay 1

Untreated negative controls:

yes

Remarks:

DMSO - Negative (solvent) control was run concurrently with treatment groups

Negative solvent / vehicle controls:

yes

Remarks:

Negative (solvent) control was run concurrently with treatment groups

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Positive control without metabolic activation: Ethyl methanesulfonate Positive Control with metabolic activation: Cyclophosphamide monohydrate

Migrated to IUCLID6: and Cyclophosphamide monohydrate

Details on test system and experimental conditions:

Formulation

Stock solutions of the test items were prepared at 500 mg/mL (preliminary experiments) or 100 mg/ml (main experiments) concentrations as described. The necessary amount of Kronitex TCP was weighed into a calibrated volumetric flask. A partial volume of solvent was added and the solution was stirred until homogeneity is reached, then the volume was adjusted to the required level. From the stock solution serial dilutions were prepared to obtain the dosing solutions for lower doses. The stock solution and the solvent were filtered sterile using a 0.22 µm syringe filter (Millipore). The solutions were prepared right before the treatment of the cells.

NEGATIVE AND POSITIVE CONTROLS

Negative (solvent) and positive controls were included in the experiments. Furthermore, untreated controls were also included in the preliminary experiments. Routine safety precautions (lab coat, gloves, safety glasses and face mask) were applied to assure personnel health and safety.

EXTERNAL METABOLIC ACTIVATION SYSTEM

In the experiments with metabolic activation in this study, a cofactor-supplemented post-mitochondrial S9 fraction prepared from activated rat liver was used as an appropriate metabolic activation system.

The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of LAB Research Ltd. according to Ames et al. [1] and Maron and Ames [7]. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.

The supplier, batch number and expiry date of the used chemicals described in this section are summarized in Table 6 (Section 5.8. Chemicals Used in the Experiments). The composition of solution refers to 1000 mL.


Induction of Rat Liver Enzymes

Male Wistar rats (197-238 g, animals were 43-47 days old at the initiation) were treated with Phenobarbital (PB) and -naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 hour before sacrifice when food was removed.

Sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels. Initiation of the induction of liver enzymes used for preparation S9 used in this study was 08 March 2010.


Preparation of Rat Liver Homogenate S9 Fraction

On Day 4, the rats were euthanized and the livers were removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized. Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-5 mL portions, frozen quickly and stored at -80  10ºC.

The sterility of the preparation was confirmed.

The protein concentration of the preparation was determined by a chemical analyzer at 540 nm in the Clinical Chemistry Laboratory of LAB Research Ltd. The protein concentration of the S9 fraction used was determined to be 34.0 g/L. The date of preparation of S9 fraction for this study was 11 March 2010 (LAB code: E10716).

The biological activity in the Salmonella assay of S9 was characterized using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batch of S9 used in this study functioned appropriately.

Preparation of S9 Mix

The metabolic activation system (S9 mix) was prepared in the Cytogenetic Laboratory of LAB Research Ltd, according to Natarajan et al. (1976) and was formulated on the day of use as follows:

S9 fraction 3 mL
HEPES 20 mM 2 mL
KCl 330 mM 1 mL
MgCl2 50 mM 1 mL
NADP 40 mM 1 mL
Glucose-6-phosphate 50 mM 1 mL
DME medium 1 mL

Prior to addition to the culture medium the S9 Mix was kept in an ice bath.


TEST PROCEDURE

Toxicity and Concentration Selection

Treatment concentrations for the mutation assay were selected based on the results of a short preliminary test. In this Concentration Selection Cytotoxicity Assay, two separate assays were performed. In Assay A, cells were treated for 3-hours in the presence and absence of S9-mix followed by harvesting at 20 hours. In Assay 2, cells were treated for 3 hours in the presence of S9-mix and for 20 hours in the absence of S9-mix with a 28-hour harvesting time. The assays were performed with a range of test item concentrations to determine cytotoxicity. Treatment was performed as described for the main test. However, single cultures were used and positive controls were not included. Visual examination of the final culture medium was conducted at the beginning and end of the treatments.

At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (solvent) control as % relative survival.

Treatment of the Cells

For the cytogenetic experiments, 1-3 day old cultures (more than 50 % confluent) were used. Cells were seeded into 92 x 17 mm tissue culture dishes at 5 x 105 cells/dish concentration and incubated for approximately 24 hours at 37C in 10 mL of culture medium (DMEM-10). Duplicate cultures were used for each test item concentration or controls. After the seeding period, the medium will be replaced with 9.9 mL treatment medium (DMEM-5). Cells were treated with different concentration test item solutions (treatment volume: 100 µL/dish), negative (solvent) or positive control solution for the given period of time at 37C in the absence or presence of S9-mix. After the exposure period, the cultures were washed with culture medium. Then, 10 mL of fresh culture medium were added into the dishes and cells were incubated further until the harvesting time.

Harvesting was performed after 20 hours (approximately 1.5 normal cell cycles) or 28 hours (approximately 2 normal cell cycles) from the beginning of treatment.

Solubility of the test item in the final treatment medium was visually examined at the beginning and end of the treatment.

For concurrent measurement of cytotoxicity an extra dish was plated for each sample and treated in the same manner. At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (solvent) control as % relative survival.


Preparation of Chromosomes

2 to 2.5 hours prior to harvesting, cell cultures were treated with Colchicine (0.2 µg/mL). The cells were swollen with 0.075 M KCl hypotonic solution, then were washed in ice-cold fixative (Methanol : Acetic-acid 3 : 1 (v : v) mixture) until the preparation became plasma free (3-4 washes). Then, a suspension of the fixed cells was dropped onto clean microscope slides and air-dried. The slides were stained with 5 % Giemsa solution, air-dried and coverslips were mounted.

Examination of Slides

The slides where given random unique code numbers at the Test Facility by a person who will not be involved in the metaphase analysis. The code labels will cover all unique identification markings on the slides to ensure that they will be scored without bias.

The coded slides will be sent to Test Site and scored for chromosomal aberrations under the control of the Principal Investigator, Natalie Danford, BSc, MPH, PhD. When the metaphase analysis was completed for each test, the slide codes were broken and the number of metaphases with aberrations (excluding gaps) and the types of aberrations for each culture were presented in tables.

One hundred metaphases with 222 chromosomes (dicentric chromosomes were counted as two chromosomes) from each culture were examined for the presence or absence of chromosomal aberrations (approximately 1000x magnification), where possible. Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.

The aberrations were defined in the following way:

Gap: small unstained lesion smaller than the width of a chromatid and with minimal misalignment of the chromatid(s)
Break: unstained lesion larger than the width of a chromatid, or with clear misalignment
Exchange: breakage and reunion of chromatids within a chromosome, or between chromosomes
Chromatid-type: structural chromosome damage expressed as breakage of single chromatids or breakage and reunion between chromatids
Chromosome-type: structural chromosome damage expressed as breakage, or breakage and reunion, of both chromatids at an identical site.
Fragments could arise from breakage and exchange events. When the origin of a fragment was clear, it was recorded under that category (e.g. a dicentric chromosome with a fragment was recorded as one chromosome exchange event). When the origin of the fragment was not clear, it was recorded as a chromatid break. Metaphases with more than five aberrations (excluding gaps) were recorded as showing multiple damage. The examination of slides from a culture was halted when 15 or more metaphases with aberrations (excluding gaps) have been recorded for that culture.

Additionally, the number of polyploid and endoreduplicated cells was scored. Polyploid metaphases are defined as metaphases with approximate multiples of the haploid chromosome number (n), other than the diploid number (i.e. ca. 3n, 4n etc). Endoreduplicated metaphases have chromosomes with 4, 8, etc. chromatids. Marked reductions in the numbers of cells on the slides were recorded if needed.

The Vernier co-ordinates of at least five metaphases (with aberrations, where possible) were recorded for each culture.

Evaluation criteria:

EVALUATION OF THE RESULTS

The assay is considered valid, if the following criteria are met:
-The solvent control data were within the laboratory’s normal range for the spontaneous aberration frequency.
-The positive controls induced increases in the aberration frequency, which are significant.

The test item is considered to have shown clastogenic activity in this study if all of the following criteria are met:
-Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps were considered).
-The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
-The increases are statistically significant.
-The increases are not associated with large changes in pH or osmolarity of the treated cultures.
The historical control data for this laboratory were also considered in the evaluation. Evidence of a dose-response relationship was considered to support the conclusion.

The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed.

Statistics:

For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis will be the number of cells with one or more chromosomal aberrations (with and without gaps).

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Weak cytotoxicity of the test item was observed in the 1000 - 111.1 µg/mL concentration range (relative survival values of 52, 42 and 50 % at 1000, 333.3 and 111.1 µg/mL concentrations, respectively)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

SOLUBILITY AND CONCENTRATION SELECTION

Based on the results of a preliminary solubility test, the test item was insoluble in Distilled water; however it was well-soluble in Dimethyl sulfoxide (DMSO). A clear solution was obtained at the concentration of 500 mg/mL using this solvent. As DMSO is compatible with the test system, it was chosen as solvent for the study.

Two Concentration Selection Cytotoxicity Assays (Assay A using 20-hour harvesting time, and Assay B using 28-hour harvesting time) were performed as part of the study to establish an appropriate concentration range for the Chromosome Aberration Assays, both in the absence and in the presence of a metabolic activation system.

Toxicity was determined by cell counting and results noted as % survival compared to the negative (solvent) control. Detailed results of the cytotoxicity assays are presented in Tables 2 and 3 of Appendix 1.

The highest examined concentration in the preliminary test was 5000 µg/mL (the recommended maximum concentration). A total of seven test item concentrations between 5000 and 6.86 g/mL (1:3 serial dilutions) were used to evaluate toxicity in the presence and absence of metabolic activation in each cytotoxicity assays. Cytotoxicity was observed in all of the preliminary experiments. Insolubility (oil-like drops, film and/or precipitate) was detected in the final treatment medium at the end of the treatment in the 5000-185.19 g/mL concentration range in all of the preliminary experiments.

Treatment concentrations for the chromosome aberration assays were selected on the basis of results of the performed Concentration Selection Cytotoxicity Assays according to the OECD guideline instructions regarding relatively insoluble substances and/or cytotoxic substances. Based on these results, 1000 g/mL concentration was chosen as the top concentration for Assay 1 (3-hour treatment with and without metabolic activation) and Assay 2 (3-hour treatment with metabolic activation and 20-hour treatment without metabolic activation).


CHROMOSOME ABERRATION ASSAYS


In Assay 1, Kronitex TCP was tested at concentrations of 1000; 333.3; 111.1, 37.04; 12.35; 4.12 and 1.37 µg/mL during a 3-hour treatment with and without metabolic activation. Sampling time was 20 hours in both cases.

Note: due to an unexpected reason, the slides of Assay 1 (performed 01-03 September 2010), could not be evaluated as the insufficient number of metaphases could not allow making a scientifically satisfactory evaluation. Therefore, Assay 1 was repeated in a new experimental period (21-23 September 2010) using the same experimental setup. The results of the repeated experiment will be reported, however all the raw data of the invalid experiment will be kept and archived in the raw data binder.

In Assay 1, in case of 3-hour treatment with metabolic activation, insolubility (oil-like film or minimal amount of oil-like film on the surface) was detected at 1000 and 333.3 µg/mL concentrations in the final treatment medium at the beginning and end of the treatment. Weak cytotoxicity of the test item was observed in the 1000 - 111.1 µg/mL concentration range (relative survival values of 52, 42 and 50 % at 1000, 333.3 and 111.1 µg/mL concentrations, respectively). Based on these results, 111.1; 37.04; 12.35; 4.12 and 1.37 µg/mL concentrations were selected for metaphase analysis. Results of the metaphase analysis are shown in Table 4 below. The test item did not induce an increase in the number of cells with structural chromosome aberrations without gaps at any examined concentrations. There were no statistical differences between the test item treated and solvent control groups and no dose-response relationship was noted.

In Assay 1, in case of 3-hour treatment without metabolic activation, insolubility (oil-like film on the surface) was detected at 1000 and 333.3 µg/mL concentrations in the final treatment medium at the beginning and end of the treatment. Furthermore, insolubility (minimal amount of oil-like film) was detected at 111.1 µg/mL concentration in the final treatment medium at the beginning of the treatment. Cytotoxicity of the test item was observed in the 1000 - 111.1 µg/mL concentration range (relative survival values of 33, 38 and 35 % at 1000, 333.3 and 111.1 µg/mL concentrations, respectively). Based on these results, 111.1; 37.04; 12.35; 4.12 and 1.37 µg/mL concentrations were selected for metaphase analysis. Results of the metaphase analysis are shown in Table 5 below.

The test item did not induce an increase in the number of cells with structural chromosome aberrations without gaps at any examined concentrations. There were no statistical differences between the test item treated and solvent control groups and no dose-response relationship was noted.

In Assay 2, Kronitex TCP was tested at concentrations of 1000; 333.3; 111.1, 37.04; 12.35; 4.12 and 1.37 µg/mL during a 3-hour treatment with metabolic activation and at concentrations of 1000; 333.3; 111.1, 37.04; 12.35; 4.12; 1.37; 0.457; 0.152; 0.0508 and 0.0169 µg/mL during a 20-hour treatment without metabolic activation. Sampling time was 28 hours in both cases.

In Assay 2, in case of 3-hour treatment with metabolic activation, insolubility (precipitate) or minimal amount of insolubility (minimal amount of precipitate and slight opalescence) was detected at 1000 and 333.3 µg/mL concentrations in the final treatment medium at the beginning and end of the treatment. Strong cytotoxicity of the test item was observed in the 1000 - 111.1 µg/mL concentration range (relative survival values of 14, 13, and 16 % at 1000, 333.3 and 111.1 µg/mL concentrations, respectively). Based on these results, 111.1; 37.04 and 12.35 µg/mL concentrations were selected for metaphase analysis. Results of the metaphase analysis are shown in Table 6 below. The test item did not induce an increase in the number of cells with structural chromosome aberrations without gaps at any examined concentrations. There were no statistical differences between the test item treated and solvent control groups and no dose-response relationship was noted.

In Assay 2, in case of 20-hour treatment without metabolic activation, insolubility (precipitate) or minimal amount of insolubility (slight opalescence) was detected at 1000 and 333.3 µg/mL concentrations in the final treatment medium at the beginning and end of the treatment. Strong cytotoxicity of the test item was observed in the 1000 – 12.35 µg/mL concentration range (relative survival values of 18, 26, 25, 29, and 29 % at 1000, 333.3, 111.1, 37.04 and 12.35 µg/mL concentrations, respectively). Based on these results, 12.35, 4.12 and 1.37 µg/mL concentrations were selected for metaphase analysis. Results of the metaphase analysis are shown in Table 7 below. The test item did not induce an increase in the number of cells with structural chromosome aberrations without gaps at any examined concentrations. There were no statistical differences between the test item treated and solvent control groups and no dose-response relationship was noted.

Note: Statistically significant difference compared to the solvent control was noted for the 4.12 µg/mL concentration without metabolic activation in the number of structural chromosome aberrations including gaps. However, the number of aberration was in the historical control range, furthermore, no difference was detected at higher tested concentration.

The occurrence of polyploid and endoreduplicated metaphases is presented in Tables 10 and 11. In some cases, polyploid metaphases were found after treatment with the different concentrations of the test item. In Assay 2, in case of 3-hour treatment with metabolic activation at 111.1µg/mL concentration, unexpectedly high number (39) of polyploid metaphases was observed (the parallel duplicate culture resulted in 4 polyploid metaphases). No endoreduplicated metaphases were found after treatment with the different concentrations of the test item.


VALIDITY OF THE STUDY

The solvent control data were within the laboratory’s normal range for the spontaneous aberration frequency.

The positive controls of Ethyl methanesulfonate (EMS) and Cyclophosphamide (CP) caused the expected biologically relevant and statistically significant increases of cells with structural chromosome aberrations (Tables 4-5 and 6-7) with one exception.

In Assay 1, in case the 3-hour treatment without metabolic activation, the resulted numbers of structural chromosome aberration without gaps for the EMS positive control item were 4 and 3 for the duplicate cultures, respectively. This increase compared to the solvent control was statistically significant; however, it was out of the historical control range. Therefore, this part of Assay 1 will be repeated in an additional experiment to provide more adequate positive control data.

Note: In Assay 1, in case of the 3-hour treatment with metabolic activation, one of the duplicate cultures of the CP positive control item did not contain any analyzable metaphases on the slide. However, the replicate culture showed the expected statistically significant increase in the number of structural chromosome aberration without gaps, so it was considered to prove the proper performance of the test. New slides of that specific culture are under evaluation to provide additional data.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Tabulated data is presented below within the attachments.

Conclusions:

Interpretation of results (migrated information):
negative

The test item Kronitex TCP was tested for potential clastogenic activity using the Chromosome Aberration Assay. The study included two Concentration Selection Cytotoxicity Assays and two a Chromosome Aberration Assays.

The performed experiments (with the exception of the experiment without metabolic activation of Assay 1) were considered to be valid and to reflect the real potential of the test item to cause structural chromosomal aberrations in the cultured V79 Chinese hamster cells used in this study.



Treatment with the test item did not result in a statistically or biologically significant dose-dependent increase in the frequency of the cells with structural chromosome aberrations without gaps either in the presence or absence of a rat metabolic activation system which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/-naphthoflavone-induced rats.

In conclusion, Kronitex TCP tested up to the cytotoxic concentrations in the presence and absence of metabolic activation, did not induce structural chromosome aberrations in this study in V79 Chinese Hamster lung cells. Therefore, Kronitex TCP is considered non-clastogenic in this test system.

Executive summary:

The test substance is considered to be non-clastogenic in this test system. No classification is applicable.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Tricresylphosphate (TCP) is a member of a category of substances known collectively as the “Phosphinated Flame Retardants”. This selection of chemicals is managed by the “Phosphates Flame Retardant Consortium” (PFRC).This group of Phosphates displays many similar properties across the group as a whole. These have been subject to recent assessment and review.

 

ANNEX XI (General rules for adaptation of the standard testing regime set out in annexes VII to X) of the REACH Regulation states the following regarding the use of read-across:

 

‘Substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or "category" of substances. Application of the group concept requires that physicochemical properties, human health effects and environmental effects or environmental fate may be predicted from data for reference substance(s) within the group by interpolation to other substances in the group (read-across approach). This avoids the need to test every substance for every endpoint.

The similarities may be based on:

 

1) a common functional group;

2) the common precursors and/or the likelihood of common breakdown products via physical and biological processes, which result in structurally similar chemicals; or

3) a constant pattern in the changing of the potency of the properties across the category.

 

If the group concept is applied, substances shall be classified and labelled on this basis.’

 

ECHA Guidance on information requirements and chemical safety assessment Chapter R.6: QSARs and grouping of chemicals (May 2008) provides more detailed information on using the read-across approach:

 

‘Chemical categories accomplish the goal of obtaining hazard information through the evaluation of all available experimental data for the individual chemicals in the category, so that reliable estimates that are adequate for classification and labelling and/or risk assessment can be made without further testing of the individual members of the category. If there is sufficient experimental data to support the category evaluation that the chemicals in the category behave in a similar or predictable manner, thenthis approachcan be used to assess the chemicals instead of conducting additional testing. Therefore, it is proposed that to address the Genetic Toxicity endpoint, a weight of evidence approach is utilised. A full justification for the substance similarity is appended below for information.

 

There exists many mutagenicity studies on the members of the phosphate esters group. The following data endpoints are utilised in this read across assessment:

 

CAS 1330-78-5 EC 215-548-8 Tris(methylphenyl) phosphate [tricresyl phosphate]

 

In vitro

 

Bacterial reverse mutation assay; negative (with and without metabolic activation.); Cascieri T Jr., Weiner, M. L. (1978)

 

Bacterial reverse mutation assay ; negative (with and without metabolic activation.); MacKellar D G (1976d)

 

The above two tests were conducted using only 4 strains; S. typhimurium TA 1538was not assessed. The study is considered to be acceptable, particularly when taken in the scope of the weight of evidence approach utilised.

 

Sister chromatid exchange assay in mammalian cells (cytogenetic assay); negative ; GREAT LAKES CHEMICAL CORPORATION (1979)

 

In vitro mammalian chromosome aberration test (chromosome aberration); negative with and without metabolic activation ; Hargitai, J (2010)

 

CAS 68937-41-7 EC 273-066-3 Phenol, isopropylated, phosphate (3:1)

 

In vitro

 

Bacterial reverse mutation assay; negative (with and without metabolic activation.); MacKellar D G (1977a)

 

Bacterial reverse mutation assay; negative (with and without metabolic activation.); MacKellar D G (1977b)

 

Mammalian cell gene mutation assay; negative for BALB/c 3T3 cells; EUROPEAN COMMISSION - European Chemicals Bureau (2000)

 

Mammalian cell gene mutation assay; negative for BALB/c 3T3 cells; EUROPEAN COMMISSION - European Chemicals Bureau (2000)

 

Mammalian cell gene mutation assay; negative for mammalian cell line, other: mouse embryo fibroblasts (BALB/3T3); EUROPEAN COMMISSION - European Chemicals Bureau (2000)

 

In vitro mammalian chromosome aberration test (chromosome aberration); negative with and without metabolic activation ; Gudi R, Rao M. (2005)

 

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro (DNA damage and/or repair); Negative for lymphocytes: rat(all strains/cell types tested); met. act.: without; EUROPEAN COMMISSION - European Chemicals Bureau (2000)

 

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro (DNA damage and/or repair); Negative for lymphocytes: rat(all strains/cell types tested); met. act.: without; EUROPEAN COMMISSION - European Chemicals Bureau (2000)

 

in vitro mammalian cell transformation assay (genome mutation); ambiguous for mouse lymphoma L5178Y cells(all strains/cell types tested); met. act.: with;

negative for mouse lymphoma L5178Y cells(all strains/cell types tested); met. act.: without; European Chemicals Bureau (2000)

 

In vivo

 

Somatic mutation assay (Chinese hamster Cricetulus grinseus) (chromosome aberration); negative; European Chemicals Bureau (2000)

 

Somatic mutation assay (Chinese hamster) (chromosome aberration); negative; European Chemicals Bureau (2000)

 

Somatic mutation assay (Chinese hamster) (chromosome aberration); positive; European Chemicals Bureau (2000)

 

Somatic mutation assay (Chinese hamster) (chromosome aberration); positive (exerted a slight mutagenic action on the somatic cells tested); European Chemicals Bureau (2000)

 

Micronucleus assay (DNA damage and/or repair); negative; European Chemicals Bureau (2000)

 

Sister chromatid exchange assay (DNA damage and/or repair); negative; European Chemicals Bureau (2000)

 

Sister chromatid exchange assay (DNA damage and/or repair); negative; European Chemicals Bureau (2000)

 

Dominant lethal assay (genome mutation) [Drosophila melanogaster male]; negative; European Chemicals Bureau (2000)

 

EC 907-387-3 Diphenyl tolyl phosphate [Reaction mass of bis(methylphenyl) phenyl phosphate and diphenyl tolyl phosphate and triphenyl phosphate and tris(methylphenyl) phosphate]

 

In vitro

 

Bacterial reverse mutation assay; negative (with and without metabolic activation.); Herbold B.A. (1988)

 

Mammalian cell gene mutation assay (gene mutation)(Chinese hamster); negative; Wollny H.-E. (2010)

 

Bacterial reverse mutation assay; negative (with and without metabolic activation.); JETOC (1997b) / Shibuya, T. et al. (1995)

 

In vitro mammalian chromosome aberration test (chromosome aberration) (Chinese hamster); negative ; JETOC (1997b)

 

In vitro mammalian chromosome aberration test (chromosome aberration) (Chinese hamster); positive ; Tanaka (1995)

 

In vivo

 

Micronucleus assay (chromosome aberration); negative; Chemicals Investigation Promoting Council, Japan. (1996)

 

Micronucleus assay (chromosome aberration); negative; BG Chemie (1993)

 

CONCLUSION

On the basis of the weight of evidence approach, it is demonstrated that genetic toxicity is not associated with the phosphates as a group. This conclusion is supported by the fact that there is no evidence of mutagenic or carcinogenic activity of tricresyl phosphate in male or female F344/N rats that received 75, 150, or 300 ppm. There was no evidence of carcinogenic activity of tricresyl phosphate in male or female B6C3F, mice that received 60, 125 or 250 ppm. Furthermore, the UK Member State Authority also concluded as follows:

“There is no indication from the studies available that tricresyl phosphate is mutagenic”.

This data is summarised as follows:

	EC 215-548-8	EC 273-066-3	EC 907-387-3	Category Summary	Weight of Evidence Conclusion
Bacterial Gene Mutation	Two studies, both negative with and without activation	Two studies, both negative with and without activation	Two studies, both negative with and without activation	Six studies, all negative with and without activation	Not genotoxic
In vitromammalian chromosomal abberations	One study, negative with and without activation	One study, negative with and without activation	One study, negative without activation and positive with activation	Three studies, all negative with and without activation	Not genotoxic
In vitromammalian gene mutation	One study, negative with and without activation	Four studies, three negative with and without activation, one study with ambigous result following activation and negative without activation	One study, negative with and without activation	Six studies, five negative with and without activation, one with negative without activation, and one ambiguous without activation	Not genotoxic
In vivochromosomal abberations	Read across to category	Eight studies, six negative and two positive	Two studies, both negative	Ten studies, eight negative and two positive	Not genotoxic

 

Justification for selection of genetic toxicity endpoint

GLP study conducted on the substance itself.  Read across data on structural analogues is also presented.

Justification for classification or non-classification

The above results triggered no classification under the CLP Regulation (EC No 1272/2008). No classification for mutagenic effects is therefore required.