Phosphoric Acid, linear and branched Octyl Esters

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 June 2011 and 05 October 2011

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Phosphoric acid, 2-ethylhexyl ester

EC Number:

235-741-0

EC Name:

Phosphoric acid, 2-ethylhexyl ester

Cas Number:

12645-31-7

Molecular formula:

Mono-Ester: C8H19O4P Di-Ester: C16H35O4P Esters of 2-ethylhexan-1-ol with diphosphoric acid: P2O7(H)4-n(C8H17)n where n = 1-4

IUPAC Name:

Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate and esters of 2-ethylhexan-1-ol with diphosphoric acid

Details on test material:

Sponsor's identification: Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate; Identifier:TIS O2930, Nalco TIS O0543, Nalco TIS O2384, MIN PR-1137; Description: Amber coloured liquid; CAS Number:12645-31-7; Batch number:CI1C0223; Label:PR-1137 CI1C0223; Date received:04 April 2011; Expiry Date:16 March 2013; Storage conditions:Room temperature, in the dark

Method

Target gene:

To assess the potential mutagenicity of the test material on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells.

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Properly maintained: yes- Periodically checked for Mycoplasma contamination:yes- Periodically checked for karyotype stability: no- Periodically "cleansed" against high spontaneous background: yesCell Line :The Chinese hamster ovary (CHO-K1) cell line was obtained from ECACC, Salisbury, Wiltshire.Cell Culture:The stocks of cells were stored in liquid nitrogen at approximately -196°C.
Cells were routinely cultured in Hams F12 medium, supplemented with 5% foetal calf serum and antibiotics (Penicillin/Streptomycin at 100 units/100 μg per ml) at 37°C with 5% CO2 in air.Cell Cleansing:Cell stocks spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen down they were cleansed of HPRT- mutants by culturing in HAT medium for 4 days.
This is Ham's F12 growth medium supplemented with Hypoxanthine (13.6 μg/ml, 100 μM), Aminop terin (0.0178 μg/ml, 0.4 μM) and Thymidine (3.85 μg/ml, 16 μM). After 4 days in medium containing HAT, the cells were passaged into HAT-free medium and grown for 4 to 7 days. Bulk frozen stocks of HAT cleansed cells were frozen down, with fresh cultures being recovered from frozen before each experiment.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta-naphthoflavone induced rat liver, S9 mix

Test concentrations with justification for top dose:

The test item was considered to be a mixture, therefore the maximum dose level was 5000 μg/ml, the maximum recommended dose level. The dose range of test item used in the preliminary cytotoxicity test was 19.53 to 5000 μg/ml. The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows:
-Exposure Group
Final concentration of test item (μg/ml)
4-hour without S9 2.5, 5, 10, 20, 30, 40
4-hour with S9 (2%)5, 10, 20, 40, 60, 80
24-hour without S9 1.25, 2.5, 5, 10, 20, 30, 40
4-hour with S9 (1%) 5, 10, 20, 30, 40, 50, 60

Vehicle / solvent:

Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test material formed a solution with the solvent suitable for dosing at the required concentrations.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Dimethyl benzanthracene (DMBA)

Remarks:

Dimethyl benzanthracene (DMBA) at 0.5 and1.0 μg/ml was used as the positive controls in cultures with S9. All positive controls were dissolved in dimethyl sulphoxide and dosed at 1%.

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Ethyl methane sulphonate (EMS) was used at 500 and 750 μg/ml as the positive control in the 4- hour cultures without S9 and at 200 and 300 μg/ml for the 24-hour cultures without S9.

Details on test system and experimental conditions:

METHOD OF APPLICATION: Plate assay using tissue culture flasks and 6-thioguanine (6-TG) as the selective agent.
DURATION
- Exposure duration: 4 hours (with and without S9), 24 hours (without S9)
- Expression time (cells in growth medium): 7 days
SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)
NUMBER OF REPLICATIONS: Duplicate cultures
DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity flasks were incubated for 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.
ASSAY ACCEPTANCE CRITERIA
An assay will normally be considered acceptable for the evaluation of the test results only if all the following criteria are satisfied. The with and without metabolic activation portions of mutation assays are usually performed concurrently, but each portion is, in fact, an in dependent assay with its own positive and negative controls. Activation or non-activation assays will be repeated independently, as needed, to satisfy the acceptance criteria.
i) The average absolute cloning efficiency of negative controls should be between 70 and 115% with allowances being made for errors in cell counts and dilutions during cloning and assay variables. Assays in the 50 to 70% range may be accepted but this will be dependent on the scientific judgement of the Study Director. All assays below 50% cloning efficiency will be unacceptable.
ii) The background (spontaneous) mutant frequency of the vehicle controls are generally in the range of 0 to 25 x 10-6. The background values for the with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays. Assays with backgrounds greater than 35 x 10-6 will not be used for the evaluation of a test item.
iii) Assays will only be acceptable without positive control data (loss due to contamination or technical error) if the test item clearly shows mutagenic activity. Negative or equivocal mutagenic responses by the test item must have a positive control mutant frequency that is markedly elevated over the concurrent negative control.
iv) Test items with little or no mutagenic activity, should include an acceptable assay where concentrations of the test item have reduced the clonal survival to approximately 10 to 15% of the average of the negative controls, reached the maximum recommended dose (10 mM or 5 mg/ml) or twice the solubility limit of the test item in culture medium. Where a test item is excessively toxic, with a steep response curve, a concentration that is at least 75% of the toxic dose level should be used. There is no maximum toxicity requirement for test items that are clearly mutagenic.
v) Mutant frequencies are normally derived from sets of five dishes for mutant colony count and three dishes for viable colony counts. To allow for contamination losses it is acceptable to score a minimum of four mutant selection dishes and two viability dishes.
vi) Five dose levels of test item, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analysed duplicate dose levels is considered necessary in order to accept a single assay for evaluation of the test item.

Evaluation criteria:

Please see 'Assay Acceptance criteria', in details on test system and conditions section.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY CYTOTOXICITY TEST:A dose range of 19.53 to 5000 μg/ml was used in the preliminary cytotoxicity test. The results of the individual flask counts and their analysis are presented in the attached Table 1 (attached background material). It can be seen that there was very marked toxicity at and above 19.53 μg/ml in both the 4 hour exposure group in the absence of S9 and the 24 hour exposure group with no surviving cells above this dose level. In the 4 hour exposure group in the presence of S9 the toxicity was slightly less severe with approximately 70% survival at 39.06 μg/ml when compared to the negative control and a few cells surviving up to 78.13 μg/ml. A precipitate of the test item was noted in all three exposure groups at the end of exposure at and
above 1250 μg/ml.
MUTAGENICITY TEST - EXPERIMENT 1: The dose levels of the controls and the test item are given in the table below:Group Final concentration of test item (μg/ml)4-
hour without S90*, 2.5*, 5*, 10*, 20*, 30*, 40, EMS 500* and 750*4-hour with S9 (2%)0*, 5*, 10*, 20*, 40*, 60*, 80, DMBA 0.5* and 1** Dose levels plated for mutant frequency.No precipitate of the test item was seen at the end of exposure in either exposure group. The Day 0 and Day 7 cloning efficiencies are presented in the attached Table 2 and Table 3 (attached background material). The Day 0 and Day 7 cloning efficiencies for the vehicle control groups in both the with and without S9 exposure groups did not achieve 70% cloning efficiency but all achieved at least 50% cloning efficiency and were therefore considered to be acceptable. The test item demonstrated a steep toxicity curve in both exposure groups consistent with that seen in the preliminary toxicity test. In the absence of S9 the test item achieved 58% toxicity at 30 μg/ml at Day 0 when compared to the vehicle control group.
The dose level of 40 μg/ml had no surviving cells and was too toxic for plating. In the 4 hour exposure group in the presence of S9 the toxicity was too great for plating at 80 μg/ml at Day 0 with no surviving cells at this dose level. The test item achieved a 21% increase in toxicity when compared to the vehicle control group at 40 μg/ml. The dose level of 60 μg/ml was plated although it exceeded 90% toxicity as it provided an intermediate dose in a steep toxicity curve. The mutation frequency counts and mean mutation frequency per survivor values are presented in the attached Table 2 and Table 3 (attached background material). In the 4 hour exposure group in the absence of S9 there was an increase in the mutation frequency per survivor which exceeded the vehicle control value by 20 x 10-6 at 2.5 μg/ml, however since this increase was not dose related and the mutant frequency for this exposure group was generally high this was considered to be a random fluctuation. There were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10-6 in the presence of S9 with the exception of the 60 μg/ml dose level which can be excluded on the basis of excessive toxicity.
MUTAGENICITY TEST - EXPERIMENT 2: The dose levels of the controls and the test item are given in the table below:
Group Final concentration of test item (μg/ml)
24-hour without S90*, 1.25*, 2.5*, 5*, 10*, 20*, 30*, 40, EMS 200* and 300*
4-hour with S9 (1%) 0* ,5*, 10*, 20*, 30*, 40*, 50*, 60
DMBA 0.5* and 1** Dose levels plated for mutant frequency. No precipitate of the test item was seen at the end of exposure in either exposure group
The Day 0 and Day 7 cloning efficiencies for the without and with metabolic activation are present ed in the attached Tables 4 and 5 attached background material). The Day 0 cloning efficiencies for the vehicle control groups in both exposure groups did not achieve 70% but were considered acceptable as they did achieve the 50% minimum. It can be seen that the toxicity is similar to that seen in Experiment 1. The maximum dose plated for mutation frequency in the 4 hour exposure group in the presence of S9 was 50 μg/ml with an increase in toxicity of 66% when compared to the vehicle control group. The dose level of 60 μg/ml was not plated due to toxicity greater than 90%. The 24 hour exposure group demonstrated an increase in toxicity of 51% when compared to the vehicle control at 30 μg/ml. The dose level of 40 μg/ml was too toxic for plating with no surviving cells. The mutation frequency counts and mean mutation frequency per survivor per 10E6 cells values are presented in the attached Table 4 and 5 (attached background material). In the absence and presence of metabolic activation there were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10-6.It can be seen that the vehicle control values were all within the maximum upper limit of 25 x 10-6 mutants per viable cell, and that the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected. In the positive control groups dosed with DMBA at 1 μg/ml and EMS at 300 μg/ml there were insufficient cells for plating 5 mutant flasks due to the toxicity of these positive control items and the mutant frequency was therefore calculated from the counts of the available flasks and adjusted according to the number of flasks. The positive response was clearly demonstrated and therefore the reduction in mutant flasks was considered to be acceptable.

Applicant's summary and conclusion

Conclusions:

Non-mutagenic.
The test item did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in either of the two experiments. The test item was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.

Executive summary:

Introduction

The study was conducted to assess the potential mutagenicity of the test item on the hypoxanthineguanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells. The test method used was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 476' In Vitro Mammalian Cell Gene Mutation Tests', Method B17 of Commission Regulation (EC) No 440/2008, the United Kingdom Environmental Mutagen Society (Cole et al, 1990) and the US EPA OPPTS 870.5300 Guideline. The technique used is a plate assay using tissue culture flasks and 6 -thioguanine (6TG) as the selective agent.

Methods

Chinese hamster ovary (CHO) cells were treated with the test item at a minimum of six dose levels, in duplicate, together with vehicle (solvent) and positive controls. Four treatment conditions were used for the test, 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 and a 4-hour exposure in the absence of metabolic activation (S9). 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.

Results

The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus.

The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.

The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

Conclusion

The test item was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of the test.