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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 29 July 2011 and 04 January 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

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:

equivalent or similar to guideline

Guideline:

other: is acceptable to the Japanese New Substance Law (METI)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

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:

Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)

IUPAC Name:

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

Details on test material:

Sponsor's identification: Esterification products of Phosphorus Pentoxide and Alcohols C6-C10 (Even numbered)
Description: Amber coloured slightly viscous liquid
Batch number: CI1E0447 solvent free
CAS No.: 68307-94-8
Identifier: TIS O2891
Date received: 13 June 2011
Expiry date: 29 May 2013
Storage conditions: Room temperature in the dark

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 hours 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 item used was 19.53 to 5000 µg/ml

Experiment 1:
4(20)-hour without S9: 0*,6.25, 12.5, 25, 50*, 75*, 100*, 150, 200 µg/ml
4(20)-hour with S9: 0*, 6.25, 12.5, 25*, 50*, 75*, 100*, 150, 200, µg/ml

Experiment 2
24-hour without S9: 0*,12.5, 25, 50*, 75*, 100*, 200 µg/ml
4(20)-hour with S9: 0*, 12.5, 25, 50*, 75*, 100*, 200, µg/ml

* Dose levels selected for metaphase analysis

Vehicle / solvent:

Vehicle: 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

Remarks:

Used In the presence of S9 at 5 µg/ml

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Used In the absence of S9 at 0.4 and 0.2 µg/ml fir 4(20)-hour and 24-hour cultures respectively.

Details on test system and experimental conditions:

METHODS OF APPLICATION:
In medium

DURATION
- Pre-incubation period:
48 hours

- Exposure duration:
Experiment 1 – 4 hours with and without S9. Experiment 2 – 24 hours without S9, 4 hours with S9.

- Expression time (cells in growth medium):
20 hours for 4 hours exposure

- Selection time (in incubation with a selective agent):
Not applicable

- Fixation time (start of exposure up to fixation or harvest of cells):
24 hours

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 coverslipped 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

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

The test item did not induce any statistically significant increases in the frequency of cells with aberrations in either the absence or presence of metabolic activation (S9).

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 item 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) hour with S9, whereas metaphase cells were present at 156.25 µg/ml in the 4(20) hour 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 item 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)-hour without S9 0*,6.25, 12.5, 25, 50*, 75*, 100*, 150, 200, MMC 0.4*
4(20)-hour with S9 0*, 6.25, 12.5, 25*, 50*, 75*, 100*, 150, 200, CP 5*

*: dose levels selected for metaphase analysis
MMC: Mitomycin C
CP: 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 item 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 items 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 item 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 item 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 item are given in the table below:

Group Final concentration of Esterification products of Phosphorus Pentoxide and Alcohols C6-C10 (Even numbered) (µg/ml)
24-hour without S9 0*,12.5, 25, 50*, 75*, 100*, 200, MMC 0.2*
4(20)-hour with S9 0*, 12.5, 25, 50*, 75*, 100*, 200, CP 5*

*: dose levels selected for metaphase analysis
MMC: Mitomycin C
CP: Cyclophosphamide

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test item 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 items 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 item 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 item did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

See 'Overall Remarks, attachments'

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction.

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et ai, 1990). The method used was compatible to that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, UK, DoH Guidelines for the Testing of Chemicals for Mutagenicity as detailed in the UKEMS Recommended

Procedures for Basic Mutagenicity Tests (1990), US EPA OPTT8 870.5375 Guideline and is acceptable to the Japanese New Chemical Substance Law (METI).

Methods.

Duplicate cultures of human lymphocytes, treated with the test item, 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-hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration); whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows.

 

Group	Final concentration of Test Item (µg/ml)
4(20)-hour without S9	0, 6.25, 12.5, 25, 50, 75, 100, 150 and 200
4(20)-hour with S9 (2%)	0, 6.25, 12.5, 25, 50, 75, 100, 150 and 200
24-hour without S9	0, 12.5, 25, 50, 75, 100 and 200
4(20)-hour with S9 (1%)	0, 12.5, 25, 50, 75, 100 and 200

Results.

All vehicle (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items 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 item 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.

Conclusion.

The test item was considered to be non-clastogenic to human lymphocytes in vitro.

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