Phosphorous acid, mixed 2,4-bis(1,1-dimethylpropyl)phenyl and 4-(1,1-dimethylpropyl)phenyl triesters

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3rd July 2009 to 9th November 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 19/08/2008 Date of signature: 04/03/2009

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of bis[2,4-bis(2-methylbutan-2-yl)phenyl] 4-(2-methylbutan-2-yl)phenyl phosphite and 2,4-bis(2-methylbutan-2-yl)phenyl bis[4-(2-methylbutan-2-yl)phenyl] phosphite and tris[4-(2-methylbutan-2-yl)phenyl] phosphite.

EC Number:

700-485-5

Cas Number:

939402-02-5

Molecular formula:

Mixture of 4 components, the molecular formulae of which are: C33 H45 O3 P, C38 H55 O3 P, C43 H65 O3 P and C48 H75 O3 P

IUPAC Name:

Reaction mass of bis[2,4-bis(2-methylbutan-2-yl)phenyl] 4-(2-methylbutan-2-yl)phenyl phosphite and 2,4-bis(2-methylbutan-2-yl)phenyl bis[4-(2-methylbutan-2-yl)phenyl] phosphite and tris[4-(2-methylbutan-2-yl)phenyl] phosphite.

Test material form:

liquid: viscous

Details on test material:

Description: Clear colourless viscous liquid
Date received: 25 June 2009
Storage conditions: Approximately 40°C, in the dark, under nitrogen

Method

Target gene:

thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Type and identity of media: RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 µg/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/ml) and 10% donor horse serum (giving R10 media) at 37°C with 5% CO2 in air.

- Properly maintained: yes

- Periodically checked for Mycoplasma contamination: yes

- Periodically checked for karyotype stability: no

- Periodically "cleansed" against high spontaneous background: yes
Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/ml), Hypoxanthine (15 µg/ml), Methotrexate (0.3 µg/ml) and Glycine (22.5 µg/ml). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/beta-naphthoflavone induced rat liver, S9 mix

Test concentrations with justification for top dose:

Preliminary toxicity test:
0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/ml

Mutagenicity test
Experiment 1
4-hours without S9: 0, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/ml
4-hours with S9: 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 937.5 and 1250 µg/ml

Experiment 2
24-hours without S9: 0, 39.06, 78.13, 156.25, 312.5, 625, 1250, 1875 and 2500 µg/ml
24-hours with S9: 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 937.5 and 1250 µg/ml

Vehicle and positive controls were used in parallel with the test material. Solvent (acetone) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) at 400 µg/ml and 150 µg/ml for Experiment 1 and Experiment 2 respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) 2 µg/ml was used as the positive control in the presence of metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

- Justification for choice of solvent/vehicle: not stated in report

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (acetone) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Used at 400 and 150 µg/ml for Exp. 1 and Exp. 2, respectively, in absence of S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (acetone) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Used at 2 µg/ml in presence of S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

Preparation of Test and Control Materials
The test material was accurately weighed and dissolved in acetone before the appropriate dilutions were made. The test material was considered to be a mixture; therefore the maximum dose level investigated in the preliminary toxicity test was 5000 µg/ml, the maximum recommended dose level. The purity of the test material was 97% and, therefore, a correction factor was applied when formulating the dosing solutions. There was no marked change in pH when the test material was dosed into media and the osmolality did not increase by more than 50 mOsm. Analysis for concentration, homogeneity and stability of the test material preparations were not a requirement of the test method and were therefore not performed.

Microsomal Enzyme Fractions
PB/Beta-NF S9 was prepared in-house on 07 June 2009 and 13 September 2009 from the livers of male Sprague Dawley rats weighing ~250g. These had each received, orally, three consecutive daily doses of phenobarbital/Beta-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on the fourth day. The S9 was stored at -196°C in a liquid nitrogen freezer.

S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0.

20% S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1. In Experiment 2, 10% S9-mix (i.e. 1% final concentration of S9), was added.

Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/ml, using a 4 hour exposure time both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/ml using a 24 hour exposure without S9. The dose range used in the preliminary toxicity test was 19.53 to 5000 µg/ml for all three of the exposure groups. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted using a coulter counter and then serially diluted to 2 x 10^5 cells/ml.

The cultures were incubated and sub-cultured after 24 hours by counting and diluting to 2 x 10^5 cells/ml. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth values (SG). The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a Percentage Relative Suspension Growth Value (%RSG).

Results from the preliminary toxicity test were used to set the test material dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) Maximum recommended dose level, 5000 µg/ml or 10 mM.
ii) The presence of excessive precipitate where no test material-induced toxicity was observed.
iii) Test material-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al 2002).

Mutagenicity Test
Experiment 1
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10^6 cells/ml in 10 ml aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate (A + B), both with and without metabolic activation (S9-mix) at eight dose levels of the test material (39.06 to 5000 µg/ml in the absence of metabolic activation, and 19.53 to 1250 µg/ml in the presence of metabolic activation), vehicle and positive controls. To each universal was added 2 ml of S9-mix if required, 0.2 ml of the treatment dilutions, (0.2 ml for the positive control) and sufficient R0 medium to bring the total volume to 20 ml. The treatment vessels were incubated at 37°C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.

Experiment 2
As in Experiment 1, an exponentially growing stock culture of cells was established. The cells were counted and processed to give 1 x 10^6 cells/ml in 10 ml duplicate cultures in R10 medium for the 4-hour treatment with metabolic activation cultures. In the absence of metabolic activation the exposure period was extended to 24 hours therefore 0.3 x 10^6 cells/ml in 10 ml duplicate cultures were established in 25 cm2 tissue culture flasks. To each culture 2 ml of S9 mix was added if required, 0.2 ml of the treatment dilutions (0.2 ml for the positive control) and sufficient R0 medium to give a final volume of 20 ml (R10 is used for the 24-hour exposure group). The dose range of the test material was 39.06 to 2500 µg/ml in the absence of metabolic activation, and 19.53 to 1250 µg/ml in the presence of metabolic activation. The treatment vessels were incubated at 37°C with continuous shaking using an orbital shaker for 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.
At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/ml. The cultures were incubated and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 10^5 cells/ml. On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/ml and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/ml 5 trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/ml and plated (2 cells/well) for viability (%V) in non-selective medium. The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Evaluation criteria:

Please see "Any other information on materials and methods"

Statistics:

Please see "Any other information on materials and methods"

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no marked changes in pH
- Effects of osmolality: Osmality did not increase by more than 50 mOsm
- Evaporation from medium: none stated in report
- Water solubility: not water soluble
- Precipitation: none described in report

RANGE-FINDING/SCREENING STUDIES: In all three of the exposure groups there were marked dose related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. A precipitate of the test material was observed at and above 78.13 µg/ml in all three of the exposure groups. In the subsequent first experiment the maximum dose level in the absence of metabolic activation was the maximum recommended dose level (5000 µg/ml), and the maximum dose level in the presence of metabolic activation was limited by toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA: not stated in report

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment 1

There was evidence of toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was also evidence of a marked decrease in viability (%V) at 5000 µg/ml in the absence of metabolic activation, therefore indicating that residual toxicity had occurred. However, it should be noted that the significant decrease was only observed at a dose level that exceeded the usual acceptable upper limit of toxicity. Optimum levels of toxicity were achieved in the presence of metabolic activation. The RTG value observed at 5000 µg/ml in the absence of metabolic activation exceeded the usual upper limit of acceptable toxicity. However, the %RSG value indicated that the optimum level of toxicity had been achieved at this dose level. Therefore, it was considered that with no evidence of any statistically significant increases in mutant frequency at this dose level or any of the dose levels in this exposure group, or the 4 hour exposure group in the presence of metabolic activation where optimum levels of toxicity were achieved, the test material had been adequately tested. Acceptable levels of toxicity were seen with both positive control substances.

Neither of the vehicle control mutant frequency values were markedly outside the acceptable range of 50 to 200 x 10^-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell at any of the dose levels (including a dose level that exceeded the usual acceptable upper limit of toxicity) in either the absence or presence of metabolic activation. Precipitate of test material was observed at and above 78.13 µg/ml in both of the exposure groups.

Experiment 2

As was seen previously, there was evidence of a marked dose-related reduction in %RSG and RTG values in cultures dosed with the test material in the both the absence and presence of metabolic activation. There was no evidence of any significant reductions in viability (%V), therefore indicating that no residual toxicity had occurred in either the absence or presence of metabolic activation on this occasion. Optimum levels of toxicity were achieved in the in the presence of metabolic activation. The RTG value observed at 2500 µg/ml in the absence of metabolic activation marginally exceeded the usual upper limit of acceptable toxicity. However, the %RSG value indicated that the optimum level of toxicity had been achieved at this dose level. It was therefore considered that with no evidence of any toxicologically significant responses, in either the first or second experiment, using a dose range where optimum levels of toxicity were achieved or exceeded, the test material had been adequately tested. It was also considered that the heterogeneity (poor correlation between A and B plates) observed at 2500 µg/ml in the absence of metabolic activation was due to toxicity. Both positive controls induced acceptable levels of toxicity.

The 24 hour exposure without metabolic activation demonstrated that the extended time point had a marked effect on the toxicity of the test material.

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10^-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell in the presence of metabolic activation. In the absence of metabolic activation, a small dose related (linear trend) statistically significant response was observed. However, none of the individual dose levels were of statistical significance, the GEF was not exceeded at any of the individual dose levels (including a dose level that marginally exceeded the usual upper limit of acceptable toxicity), and all of the mutant frequency values observed were within the acceptable range for vehicle controls. Therefore, the response was considered to be spurious and of no toxicological significance. A precipitate of test material was observed at and above 78.13 µg/ml in the absence of metabolic activation, and at and above 39.06 µg/ml in the presence of metabolic activation.

Key for Tables 1 -4

%RSG = Relative Suspension Growth

RTG = Relative Total Growth

%V = Viability Day 2

CP = Cyclophosphamide

EMS = Ethylmethanesulphonate

MF§ = 5-TFT resistant mutants/10⁶viable cells 2 days after treatment

Ø = Not plated for viability or 5-TFT resistance

$$ = Evidence of heterogeneity

* = p<0.05

X = Treatment excluded from test statistics due to toxicity

Table 1: Statistical analysis: Experiment 1 (-S9) 4 -hour exposure

Treatment (µg/ml)	% RSG	% V	RTG	MF§
0	100	65.55	1.00	128.41
39.06 Ø	101
78.13 Ø	97
156.25	100	85.83	1.33	106.21
312.5	106	64.83	1.06	105.28
625	95	66.78	0.97	174.92
1250	65	83.01	0.82	134.80
2500	48	79.06	0.58	143.60
5000 X	12	10.70	0.02	85.94
Positive Control EMS
400	77	30.90	0.36	1553.93

Test for linear trend

Slope	1.133E-008
Variance	1.056E-016
b²/Sb	1.214

Table 2: Statistical analysis: Experiment 1 (+S9) 4-hHour exposure

Treatment (µg/ml)	% RSG	% V	RTG	MF§
0	100	79.18	1.00	126.08
19.53 Ø	108
39.06 Ø	102
78.13	102	65.80	0.85	124.43
156.25	89	75.40	0.84	118.83
312.5	60	80.34	0.61	94.32
625	18	75.99	0.19	140.85
937.5	13	77.81	0.12	125.24
1250	26	98.08	0.26	109.13
Positive Control CP
2	57	33.63	0.25	933.24

Test for linear trend

Slope	-4.368E-009
Variance	2.569E-016
b²/Sb	0.074

Table 3: Statistical analysis: Experiment 2 (-S9) 24 -hour exposure

Treatment (µg/ml)	% RSG	% V	RTG	MF§
0	100	66.04	1.00	136.86
39.06  Ø	83
78.13   Ø	81
156.25	85	73.67	0.95	106.99
312.5	80	63.43	0.78	121.86
625	78	62.06	0.73	136.89
1250	50	66.78	0.42	167.57
1875	65	62.06	0.45	172.48
2500   $ $, X	10	50.09	0.08	163.44
Positive control EMS
150	81	48.70	0.59	1128.04

Test for linear trend

Slope	2.831E-008
Variance	2.369E-016
b²/Sb	3.383*

Table 4: Statistical analysis: Experiment 2 (+S9) 4 -hour exposure

Treatment (µg/ml)	% RSG	% V	RTG	MF§
0	100	73.67	1.00	82.60
19.53  Ø	106
39.06  Ø	103
78.13	95	75.40	0.98	80.70
156.25	86	76.59	0.89	85.23
312.5	67	66.78	0.62	129.53
625	29	77.20	0.25	100.12
937.5	17	110.65	0.28	102.60
1250	22	81.00	0.26	91.69
Positive Control CP
2	77	59.42	0.62	461.07

Test for linear trend

Slope	1.209E-008
Variance	1.931E-016
b²/Sb	0.756

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476), Method B17 of Commission Regulation (EC) No. 440/2008 of and is acceptable to the Japanese New Chemical Substance Law (METI).

Methods

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at eight dose levels using a 4 hour exposure group in the presence of metabolic activation (1% S9) and a 24 hour exposure group in the absence of metabolic activation. The dose range of test material was selected following the results of a preliminary toxicity test. The dose range for Experiment 1 was 39.06 to 5000 µg/ml in the absence of metabolic activation and 19.53 to 1250 µg/ml in the presence of metabolic activation. The dose range for Experiment 2 was 39.06 to 2500 µg/ml in the absence of metabolic activation and 19.53 to 1250 µg/ml in the presence of metabolic activation.

Results

The maximum dose level used was limited by test material induced toxicity in all but the 4 hour exposure group in the absence of metabolic activation of Experiment 1, where the maximum recommended dose level (5000 µg/ml) was used. Precipitate of test material was observed at and above 78.13 µg/ml in Experiment 1. In Experiment 2, a precipitate of test material was observed at and above 78.13 µg/ml in the absence of metabolic activation, and at and above 39.06 µg/ml in the presence of metabolic activation. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Conclusion

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.