Phosphoric acid, decyl octyl ester

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 29, 2011 to January 04, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosome aberration test (migrated information)

Test material

Test material information

Constituent 1

Reference substance name:

Phosphoric acid, mono- and di-C6-10-alkyl esters

EC Number:

269-616-7

EC Name:

Phosphoric acid, mono- and di-C6-10-alkyl esters

Cas Number:

68307-94-8

Molecular formula:

No information available

IUPAC Name:

Esterification Products of Phosphorus Pentoxide and Alcohols C6-C10 (even numbered)

Test material form:

liquid

Method

Target gene:

Not applicable.

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 16 h under typical experimental exposure conditions. Cell Culture: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at 37°C with 5% CO2 in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test (Cell Growth Inhibition Test): The dose range of test substance used was 19.53 to 5000 µg/mL
Experiment 1: 4 (20) h without S9: 0*,6.25, 12.5, 25, 50*, 75*, 100*, 150, 200 µg/mL, 4 (20) h with S9: 0*, 6.25, 12.5, 25*, 50*, 75*, 100*, 150, 200, µg/mL
Experiment 2: 24 h without S9: 0*,12.5, 25, 50*, 75*, 100*, 200 µg/mL, 4 (20) h with S9: 0*, 12.5, 25, 50*, 75*, 100*, 200, µg/mL
*Dose levels selected for metaphase analysis

Vehicle / solvent:

Dimethyl sulphoxide

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

Methods of application: In medium
Duration- Pre-incubation period: 48 h Exposure duration:
Experiment 1 – 4 h with and without S9.
Experiment 2 – 24 h without S9, 4 h with S9.
Expression time (cells in growth medium): 20 h for 4 h exposure
Selection time (in incubation with a selective agent): Not applicable
Fixation time (start of exposure up to fixation or harvest of cells): 24 h
Spindle inhibitor (Cytogenetic assays): Demecolcine
Stain (for cytogenetic assays): When slides were dry they were stained in 5% giemsa for 5 minutes, rinsed, dried and cover slipped using mounting medium.
Number of replications: Duplicate cultures
Number of cells evaluated: 100/culture
Determination of cytotoxicity - Method
Mitotic index – A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index as a percentage of the vehicle control value.

Scoring of Chromosome Damage
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

Other examinations
Determination of polyploidy: In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported.

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's exact test.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Refer to information on results and attached tables.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Results
Preliminary toxicity test (cell growth inhibition test): The mitotic index data are presented in Appendix 1 (5) and (6) (see attached background material - Appendix 1). The test substance exhibited clear evidence of dose-related toxicity in all three exposure groups. A precipitate was observed in the parallel blood-free cultures at the end of the exposure period initially at 156.25 µg/mL and 312.5 µg/mL without and with S9-mix, respectively. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to 78.13 µg/mL in the continuous exposure and 4(20) h with S9, whereas metaphase cells were present at 156.25 µg/mL in the 4(20) h without S9 exposure group. Therefore, the dose selection for Experiments 1 and 2 was based on toxicity in accordance with the OECD 473 test guideline.

Chromosome aberration test - Experiment 1
The dose levels of the controls and the test substance are given in the table below:
Group Final concentration of Esterification products of Phosphorus Pentoxide and Alcohols C6-C10 (Even numbered) (µg/mL)
4 (20) h without S9: 0*, 6.25, 12.5, 25, 50*, 75*, 100*, 150, 200, MMC 0.4*
4 (20) h with S9: 0*, 6.25, 12.5, 25*, 50*, 75*, 100*, 150, 200, CP 5**

Dose levels selected for metaphase analysis
MMC: Mitomycin CCP: Cyclophosphamide

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to the test substance dose level of 100 µg/mL in the absence and presence of metabolic activation (S9). The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in Form 1, Appendix 2 (see attached background material - Appendix 2). These data show an approximate 50% growth inhibition was achieved at 100 µg/mL in the presence of S9. However, in the absence of S9, an 18% growth inhibition was observed at 100 µg/mL but there were no metaphases present above this dose level. The selection of the maximum dose level for metaphase analysis was based on toxicity, and was 100 µg/mL both in the absence and presence of S9, because there were no metaphases present above this dose level. The chromosome aberration data are given in Form 1, Appendix 2 (see attached background material - Appendix 2). All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control substances induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test substance did not induce any statistically significant increases in the frequency of cells with aberrations in either the absence or presence of metabolic activation (S9). The polyploid cell frequency data are given in Form 1, Appendix 2. The test substance did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Chromosome aberration test - Experiment 2:
The dose levels of the controls and the test substance are given in the table below:
Group Final concentration of Esterification products of Phosphorus Pentoxide and Alcohols C6-C10 (Even numbered) (µg/mL)
24 h without S9: 0*, 12.5, 25, 50*, 75*, 100*, 200, MMC 0.2*
4 (20) h with S9: 0*, 12.5, 25, 50*, 75*, 100*, 200, CP 5**

Dose levels selected for metaphase analysis
MMC: Mitomycin CCP: Cyclophosphamide

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test substance dose level of 100 µg/mL in the presence and absence of S9. The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in Form 2, Appendix 2 (see attached background material - Appendix 2). These data show 29% and 21% growth inhibition was achieved at 100 µg/mL in the absence and presence of S9, respectively. The selection of the maximum dose level for metaphase analysis was similar to Experiment 1, and was based on toxicity. The chromosome aberration data are given in Form 2, Appendix 2 (see attached background material - Appendix 2). All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control substances induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test substance did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of metabolic activation. The polyploid cell frequency data are given in Form 2, Appendix 2 (see attached background material - Appendix 2). The test substance did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Applicant's summary and conclusion

Conclusions:

Under study conditions, the test substance was considered to be non-clastogenic to human lymphocytes in the chromosomal aberration assay, with and without metabolic activation

Executive summary:

An in vitro study was conducted to determine the clastogenic potential of the test substance, mono-, di- and tri- C6-10 PSE, in cultured human lymphocytes, according to the OECD Guideline 473, EU Method B10, EPA OPPTS 870.5375, in compliance with GLP. Duplicate cultures of human lymphocytes, treated with the test substance, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, a 4 h exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20 h expression period (tested at 0, 6.25, 12.5, 25, 50, 75, 100, 150, 200 µg/mL test concentrations) and a 4 h exposure in the absence of metabolic activation (S9) with a 20 h expression period (tested at: 0, 6.25, 12.5, 25, 50, 75, 100, 150, 200 µg/mL test concentrations). In Experiment 2, the 4 h exposure with addition of S9 was repeated (using a 1% final S9 concentration) (tested at: 0, 12.5, 25, 50, 75, 100, 200 µg/mL test concentrations, mitomycin C 0.2 µg/mL); whilst in the absence of metabolic activation the exposure time was increased to 24 h (tested at: 0, 12.5, 25, 50, 75, 100, 200 µg/mL test concentrations, cyclophosphamide 5 µg/mL). The dose levels used in the main experiments were selected using data from the preliminary toxicity test. All vehicle (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control substances induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated. The test substance did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced or exceeded approximately 50% mitotic inhibition. Under study conditions, the test substance was considered to be non-clastogenic to human lymphocytes in the chromosomal aberration assay, with and without metabolic activation (XXXX, 2012).