Phosphoric acid, mono and bis-linear butyl esters, potassium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No evidence of mutagenic activity was detected during a bacterial reverse mutation assay (OECD 471/472 and EU Method B.13/14).

The test item did not induce a statistically significant increase 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.

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.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 May 1997 to 22 May 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: US EPA 40 CFR 798.5265 and 798.5100

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MAFF Japan (59 NohSan No 4200)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Physical characteristics: Water white liquid
- Purity: 50% active ingredient in water
- Haskell number: 22586
- Stability: Test substance appeared to be stable under the conditions of the study; no evidence of instability was observed.

Target gene:

Histidine and tryptophan

Species / strain / cell type:

S. typhimurium, other: TA100, TA1535, TA97a and TA98

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced S9

Test concentrations with justification for top dose:

RANGE-FINDING STUDY
- Nominal test item concentrations of 10, 25, 50, 100, 250, 500, 1000, 2500 and 5000 μg/plate

DEFINITIVE STUDY
- Nominal test item concentrations of 25, 100, 500, 1000, 2500 and 5000 μg/plate

Vehicle / solvent:

Sterile water

Negative solvent / vehicle controls:

yes

Remarks:

sterile water

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

NAAZ (2 μg/plate for TA100 without metabolic activation

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2AA (1 μg/plate for TA100 with metabolic activation)

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

NAAZ (2 μg/plate for TA1535 without metabolic activation)

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2AA (2 μg/plate for TA1535 with metabolic activation)

Positive controls:

yes

Positive control substance:

other: ICR 191 Acridine

Remarks:

ICR 191 (2 μg/plate for TA97a without metabolic activation)

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2AA (1 μg/plate for TA97a with metabolic activation)

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

2NF (25 μg/plate for TA98 without metabolic activation)

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2AA (2 μg/plate for TA98 with metabolic activation)

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

MMS (1000 μg/plate for WP2 uvrA (pKM101) without metabolic activation)

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2AA (25 μg/plate for WP2 uvrA (pKM101) with methabolic activation)

Details on test system and experimental conditions:

TEST SUBSTANCE AND NEGATIVE CONTROL
- Preparation of the stock solution of the test substance at 50 mg/mL resulted in a homogeneous, colourless, solution.
- Additional dilutions of the test substance were performed at room temperature in sterile water.
- The negative control was assumed to be stable during the study and no evidence of instability was observed.
- Any impurities were not expected to have interfered with the study.

POSITIVE INDICATORS
- Deionised water was used as the solvent for NAAZ, ICR 191 and MMS.
- The solvent for other positive indicators was DMSO.
- Positive indicators were assumed to be stable in this study and no evidence of instability was observed.
- Any impurities were not expected to have interfered with the study.

TESTER STRAIN CHARACTERISATION, STORAGE AND CULTURE
- S typhimurium tester strains were obtained from Dr Bruce Ames, Berkeley, CA.
- The phenotypic characteristics and mutational sensitivites of the S typhimurium strains are described in the attached document.
- E coli WP2 uvrA (pKM101) was obtained from the National Collection of Industrial Bacteria, Torrey Research Station, Scotland. Because tryptophan biosynthesis is blocked by an ochre nonsense mutation, revertants arise as a result of base pair substitution. A second class of mutants may arise as a result of nonsense suppressor mutations in genes coding for tRNAs. Frameshift mutagens are not generally expected to be detected by this strain.

TESTER STRAIN STORAGE AND CULTURE
- All bacterial strains were stored frozen in 8 % DMSO in Oxoid nutrient broth at approximately -70 °C.
- Overnight cultures were prepared by inoculating 20 mL of Oxoid nutrient broth with 0.1 mL of thawed bacterial suspension and incubating at 37 °C with shaking.
- Overnight cultures were then stored on ice until used for mutagenesis assays.
- Bacterial strain phenotypes were confirmed concurrently with each trial. Results demonstrated the appropriate responses (Table 1).

METABOLIC ACTIVATION SYSTEM
- Because the tester strains lack many of the enzymes required to convert various promutagens to a reactive state, the assay was performed with and without a rat liver homogenate activation system (S9 mix) similar to the method of Maron and Ames (1983).
- The S9 mix consisted of MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM), NADP (4 mM), sodium phosphate pH 7.4 (100 mM) and S9 protein/1.0 mL S9 mix.
- The S9 purcahsed from MOLTOX was the 9000 x g supernatant of liver homogenate (1 g wet liver; 3.0 mL KCl).
- Livers were from young male Sprague Dawley rats injected i.p. with Aroclor 1254 (500 mg/kg) five days before sacrifice.

DOSE SELECTION
- In accordance with EPA, OECD and MAFF Japan test guidelines, the highest concentration evaluated in the study was a suspension of the test substance at a nominal concentration of 5000 μg/plate.
- Solubility information was confirmed empirically as needed during the study.
- Concentrations were calculated with the assumption that addition of the test substance to the solvent did not change the volume of the resulting solution.

STABILITY AND CONCENTRATION VERIFICATION
- Solutions of the test substance were prepared immediately prior to treatment and were presumed to be stable under the conditions of the study.
- Treatment and control dosing solutions were not analysed for concentration, uniformity or stability.
- Top agar was not assayed for stability or concentration of the test substance or control articles since this assessment was not considered necessary to achieve the objectives of the study.

BACTERIAL MUTAGENICITY ASSAYS
- The study consisted of one trial with and without activation.
- Three replicates were plated for each tester strain, test concentration and condition.
- Positive indicators and negative controls were included in all assays.
- Treatments with activation were conducted by adding 0.1 mL of an overnight culture containing at least 1 x 10E08 bacteria to 2 mL of top agar (0.6 % agar w/v and 0.6 % NaCl w/v) supplemented with 0.05 mM L-histidine and 0.05 mmM D-biotin for S typhimurium strains or 0.05 mM L-tryptophan for the E coli strain.
- The components were mixed and poured onto a plate containing 25 mL of Davis minimal agar with dextrose (minimal agar plates purchased from MOLTOX).
- Treatments without activation were identical to those with activation with the exception that the S9 mix was replaced with 0.5 mL of sterile water.
- Revertant colonies were counted after the individually labelled plates were incubated at approximately 37 °C for about 48 hours.
- When necessary, plates were refrigerated prior to counting.

Evaluation criteria:

- Acceptability criteria for the study are attached.
- Classification guidelines relating to the study are attached.

Statistics:

STATISTICAL ANALYSIS
- For each tester strain, the average number of revertants and the standard deviation at each concentration (with and without S9 activation) were calculated.

Key result

Species / strain:

other: Salmonella typhimurium TA100, TA1535, TA97a and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- Mutagenic activity of the tester strains is shown in Tables 2 to 6 (attached) together with a list of abbreviations used in the tables.

Conclusions:

No evidence of mutagenic activity was detected using Salmonella typhimurium strains TA100, TA1535, TA97a and TA98 plus Escherichia coli strain WP2 uvrA (pKM101).

Executive summary:

The test substance was evaluated for mutagenicity in accordance with OECD 471 and other appropriate test guidelines using Salmonella typhimurium strains TA100, TA1535, TA97a and TA98 and in Escherichia coli strain WP2 uvrA (pKM101) with and without an exogenous metabolic activation system (S9). Concentrations of 10, 25, 50, 100, 250. 500, 1000 and 5000 μg/plate were evaluated in comparison to negative (solvent) controls in a range-finding study using Salmonella typhimurium strains TA97a and TA100 plus Escherichia coli strain WP2 uvrA (pKM101). Concentrations of 25, 100, 500, 1000, 2500 and 5000 μg/plate were subsequently used in Salmonella typhimurium strains TA100, TA1535, TA97a and TA98 plus Escherichia coli strain WP2 uvrA (pKM101). No evidence of mutagenic activity was detected under the conditions of the study. The substance was considered negative.

Reference 2

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

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

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 / 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.

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%.

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.

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.

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.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 April 2011 and 18 August 2011

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate

Target gene:

Not applicable.

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 determin
ed 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, at 37°C with 50/0 C02 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 mix

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 (μg/ml)0*, 9.77, 19.53, 39.06*, 78.13*, 156.25*, 312.5, MMC 0.4*
4(20)-hour with S9 (μg/ml) 0*, 9.77, 19.53, 39.06*, 78.13*, 156.25*, 312.5, CP 5*
Experiment 2 24-hour without S9 (μg/ml) 0*, 9.77, 19.53*, 39.06*, 78.13*, 156.25*, 312.5, MMC 0.2*
4(20)-hour with S9 (μg/ml) 0*, 9.77, 19.53*, 39.06*, 78.13*, 156.25*, 312.5, CP 5*
*Dose levels selected for metaphase analysis
MMC: Mitomycin
CCP: Cyclophosphamide

Vehicle / solvent:

Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was selected as the solvent because the test item was readily miscible in it.
Preparation of Test Item and Control Items:The test item was accurately weighed, dissolved in dimethyl sulphoxide (DMSO) and serial dilutions prepared. The test item was considered to be a mixture, therefore the maximum dose level was 5000 μg/ml, the maximum recommended dose level. There was a modest decrease in pH of less than 1 pH unit when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm at the dose levels investigated. The decrease in pH was within acceptable limits.

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.Migrated to IUCLID6: (CP)

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 for 4(20)-hour and 24-hour culture respectiv ely.Migrated to IUCLID6: (MMC)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 48 hrs
- Exposure duration: Experiment 1 - 4 hrs with and without S9. Experiment 2 - 24 hrs without S9, 4 hrs with S9.
- Expression time (cells in growth medium): 20 hrs for 4 hrs exposure.
- Selection time (if incubation with a selection agent): Not applicable.
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs.
SPINDLE INHIBITOR (cytogenetic assays): Demecolcine (Colcemid 0.1 μg/ml)
STAIN (for cytogenetic assays): When the 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 and 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 was approximately 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. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.

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 doserelationship. 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.

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle 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). It can be seen that the test item showed clear evidence of dose-related toxicity in all three exposure groups. A greasy oily precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure period, at and above 2500 μg/ml, in all three of the exposure groups.Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to 156.25 μg/ml, in all three of the exposure groups. Dose selection for Experiments 1 and 2 was based on test item induced toxicity.
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 Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate (μg/ml)
4(20)-hour without S90*, 9.77, 19.53, 39.06*, 78.13*, 156.25*, 312.5, MMC 0.4*
4(20)-hour with S90*, 9.77, 19.53, 39.06*, 78.13*, 156.25*, 312.5, CP 5*
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 156.25 μg/ml in both the absence and presence of metabolic activation (S9). No metaphases suitable for scoring were observed at 312.5 μg/ml in either exposure group.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 that 59% growth inhibition was achieved at 156.25 μg/ml in the absence of S9 and 40% growth inhibition was achieved at 156.25 μg/ml in the presence of S9.No precipitate of the test item was observed at the end of the treatment period in either exposure group.The maximum dose level selected for metaphase analysis was base d on the maximum surviving dose level and toxicity, and was 156.25 μg/ml in both of the exposure groups.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. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.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 (see attached background material - 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:GroupFinal concentration of Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate (μg/ml)
24-hour without S90*, 9.77, 19.53*, 39.06*, 78.13*, 156.25*, 312.5, MMC 0.2*
4(20)-hour with S90*, 9.77, 19.53*, 39.06*,78.13*, 156.25*, 312.5, CP 5*
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present up to the test item dose level of 156.25 μg/ml in both the absence and presence of metabolic activation (S9). No metaphases suitable for scoring were observed at 312.5 μg/ml in either of the exposure groups.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 that 75% growth inhibition was achieved at 156.25 μg/ml in the absence of S9 and 50% growth inhibition was achieved at 156.25 μg/ml in the presence of S9.No precipitate of the test item was observed at the end of the treatment period in either of the exposure groups.The maximum dose level selected for metaphase analysis was the same as Experiment 1, and was based on toxicity at 156.25 μg/ml, for both exposure groups. In the absence
of S9 the toxicity observed at 156.25 μg/ml was high at 75%, however the dose level was selected for metaphase analysis because the next dose level down demonstrated no 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. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.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.

Conclusions:

Interpretation of results (migrated information):negativeThe test item did not induce a statistically significant increase 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 al, 1990). The method used was designed to be compatible with 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, UKDoH Guidelines for the Testing of Chemicals for Mutagenicity as detailed in the UKEMS Recommended Procedures for Basic Mutagenicity Test (1990), US EPA OPPTS 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.

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 the satisfactory performance of the test and of the activity of the metabolising system.

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 the optimum 50% mitotic inhibition.

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

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across hypothesis proposed is that the organism is not exposed to common compounds but rather, because of structural similarity, that different compounds have similar toxicological and fate properties. In this case the ECHA Read-Across Assessment Framework (RAAF) Scenario 2 is used.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Phosphoric acid, 2-ethylhexyl ester [EC 235-741-0; CAS 12645-31-7]
Target: Phosphoric acid, mono and bis-linear butyl esters, potassium salts [EC not yet assigned; CAS not assigned]
3. ANALOGUE APPROACH JUSTIFICATION
Ames tests conducted on the Source substance and the Target substances showed they were not mutagenic. For the in vitro cytogenicity study in mammalian cells endpoint the Source substance was also not mutagenic. Likewise, the Source substance was not mutagenic in an in vitro gene mutation study in mammalian cells. The weight of evidence from both Source and Target substance strongly suggests that these molecules would not be considered as mutagens. Thus, endpoint data gaps for the Target substance could be adequately predicted from the test studies previously conducted.
4. DATA MATRIX
Please refer to attached justification.

Reason / purpose for cross-reference:

read-across source

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

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across hypothesis proposed is that the organism is not exposed to common compounds but rather, because of structural similarity, that different compounds have similar toxicological and fate properties. In this case the ECHA Read-Across Assessment Framework (RAAF) Scenario 2 is used.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Phosphoric acid, 2-ethylhexyl ester [EC 235-741-0; CAS 12645-31-7]
Target: Phosphoric acid, mono and bis-linear butyl esters, potassium salts [EC not yet assigned; CAS not assigned]
3. ANALOGUE APPROACH JUSTIFICATION
Ames tests conducted on the Source substance and the Target substances showed they were not mutagenic. For the in vitro cytogenicity study in mammalian cells endpoint the Source substance was also not mutagenic. Likewise, the Source substance was not mutagenic in an in vitro gene mutation study in mammalian cells. The weight of evidence from both Source and Target substance strongly suggests that these molecules would not be considered as mutagens. Thus, endpoint data gaps for the Target substance could be adequately predicted from the test studies previously conducted.
4. DATA MATRIX
Please refer to attached justification.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: Human

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial reverse mutation assay

The test substance was evaluated for mutagenicity in accordance with OECD 471 and other appropriate test guidelines using Salmonella typhimurium strains TA100, TA1535, TA97a and TA98 and in Escherichia coli strain WP2 uvrA (pKM101) with and without an exogenous metabolic activation system (S9). Concentrations of 10, 25, 50, 100, 250. 500, 1000 and 5000 μg/plate were evaluated in comparison to negative (solvent) controls in a range-finding study using Salmonella typhimurium strains TA97a and TA100 plus Escherichia coli strain WP2 uvrA (pKM101). Concentrations of 25, 100, 500, 1000, 2500 and 5000 μg/plate were subsequently used in Salmonella typhimurium strains TA100, TA1535, TA97a and TA98 plus Escherichia coli strain WP2 uvrA (pKM101). No evidence of mutagenic activity was detected under the conditions of the study. The substance was considered negative.

Justification for classification or non-classification

All in vitro studies were negative, therefore no classification is necessary.