Phosphoric acid, mixed esters with biphenyl-4,4'-diol and phenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization. Study is adequate for assessment with acceptable restrictions. Stability of the test material was the responsibility of the sponsor and therefore an expiry date was not reported for the batch of test material used.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: essential amino acid requiring strains

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male Sprague Dawley rats treated by oral gavage on 3 consecutive days with phenobarbitone and β-naphthoflavone (80 and 100 mg/kg/day, respectively) for enzyme induction. (S9 mix = 10% liver S9 in standard cofactors)

Test concentrations with justification for top dose:

Preliminary Toxicity Study (TA100, WP2 uvrA without or with metabolic activation (S9))
Doses: 0 (vehicle control); 0.15; 0.5; 1.5; 5; 15; 50; 150; 500; 1500 and 5000 μg/plate

Experiments 1 and 2,
(direct plate incorporation method, TA 98, TA100, TA1535, TA1537, WP2 uvrA, each without and with metabolic activation (S9) tested in triplicate)
Doses: 0 (vehicle control); 50; 150; 500; 1500 and 5000 μg/plate.

Vehicle / solvent:

Dimethyl sulphoxide (DMSO) (solvent was dried using molecular sieves, sodium alumino-silicate i.e. 2 mm pellets, nominal pore diameter 4 A)

Justification for choice of solvent/vehicle:
Test material was insoluble in water according to information of the sponsor, but it was soluble in dimethyl sulphoxide (DMSO) at the required concentration of 50 mg/mI in solubility checks performed in this laboratory. Vehicle (DMSO) control plates produced counts of revertant colonies within the normal range.

Untreated negative controls:

yes

Remarks:

In addition, sterility controls were included to assess sterility of the test material

Negative solvent / vehicle controls:

yes

Remarks:

without and with S9 mix

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Positive control substances for tests without metabolic activation (S9 mix). All of them are well established reference mutagens.

Untreated negative controls:

yes

Remarks:

In addition, sterility controls were included to assess sterility of the test material and S9 mix

Negative solvent / vehicle controls:

yes

Remarks:

without and with S9 mix

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene for TA 100, TA 1535, TA 1537 & WP2 uvrA

Remarks:

Positive control substance for tests with metabolic activation (S9 mix). Both are well established reference mutagen.

Details on test system and experimental conditions:

Direct Plate Incorporation Tests, both without and with metabolic activation, were performed in both experiments (Experiments 1 and 2).

Prior to use the master strains were checked for characteristics, viability and spontaneous reversion rate and were all found to be satisfactory.

The following positive controls were used to check mutability of the bacteria and activity of the S9 mix:

Without metabolic activation (S9 mix):

N-ethyl-N'-nitro-N-nitrosoguanidine:
- 2 μg/plate: - strain WP2 uvrA,
- 3 μg/plate: - strain TA 100,
- 5 μg/plate: - strain TA 1535

9-Aminoacridine:
- 80 μg/plate: - strain TA 1537

4-Nitroquinoline-1-oxide:
- 0.2 μg/plate: - strain TA 98

With metabolic activation (S9 mix):

2-Aminoanthracene:
- 1 μg/plate: - strain TA 100,
- 2 μg/plate: - strains TA 1535 and TA 1537,
- 10 μg/plate: - strain WP2 uvrA,

Benzo(a)pyrene:
- 5 μg/plate: - strain TA 98

Evaluation criteria:

The test chemical was considered to exhibit mutagenic activity in this assay if the following criteria were met:
A dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain in the presence and/or absence of the S9 microsomal enzymes.

The test chemical was considered to be non-mutagenic (negative) if the above criteria were not met.

Positive controls were considered to be valid if their mean revertant count (per strain without or with metabolic acitvation) was at least two times the respective vehicle control value.

Statistics:

The study report does not clearly indicate whether or not the data were analysed for statistically significant differences of revertant counts although reference is given to:
Kirkland DJ (Ed) 1989: Statistical Evaluation of Mutagenicity Test Data.
UKEMS Sub-committee on Guidelines for Mutagenicity Testing, Report - Part Ill, Cambridge University Press.

Comment:
However, the study report states that biological relevance of the results would be considered first and the study result was unequivocal. Statistical analysis of the data was not required to determine the result of the test.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Preliminary Toxicity study

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Preliminary Toxicity study

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Preliminary Toxicity study

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Preliminary Toxicity study

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

S9 mix used in each experiment and the test material formulations tested for sterility in the preliminary toxicity test showed no evidence of contamination.
An opaque film was noted from 1500 µg/plate and an associated oily precipitate observed at 5000 µg/plate. Neither of these observations interfered with the scoring of revertant colonies.

Conclusions:

negative without and with metabolic activation (S9 mix)

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

of 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Reference detailed in Robust study summary section "Any other information on materials and methods incl. tables"

Deviations:

not specified

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: Chinese hamster lung fibroblasts (CHL/IU cells)

Details on mammalian cell type (if applicable):

- Source of CHL/IU cells: Health Science Research Resources Bank, Japan Health Sciences Foundation on April 17, 2002
- Number of chromosomes: 25 per cell.
- Doubling time: Ca. 15 hours.
- Mycoplasma check: Cells were mycoplasma free in the testing facility.
- Spontaneous frequency of cells with structural
aberrations and numerical aberation cells: < 5%
- Storage: Cells suspended in Eagle's minimum essential medium (from Nissui Pharmaceutical Co., Ltd.)
supplemented with 10 vol% heatinactivated newborn calf serum (NBCS, fromHyClone) including
10% (v/v) DMSO frozen in liquid nitrogen.
- Culture condition: CO2 incubator set at 37°C and 5% CO2 under humid condition.
- Subculture: 90 mm diameter Petri dishes, twice a week.
Passage number of cells: 8 for cell growth inhibition test, 12 for chromosomal aberration test.
- Type and identity of media:
Basal medium (MEM) = L-Glutamine (final concentration: 0.292 g/L) and sodium hydrogen carbonate (final concentration: ca. 1.85 g/L) added to
Eagle's minimum essential medium (from Nissui Pharmaceutical Co., Ltd.). MEM was then supplemented with 10 vol% heatinactivated newborn calf
serum (NBCS, fromHyClone).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male Sprague Dawley rats treated with phenobarbital and 5,6-benzoflavone for enzyme induction.

Test concentrations with justification for top dose:

CELL GROWTH INHIBITION TEST, harvest at 24 h: ---> 6 h treatment/18 h recovery (-/+S9) and 24 h continuous treatment (-S9):
Concentrations prepared: 0*, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 µg/mL
Microscopically examined (metaphase analysis):
- 6 h treatment/18 h recovery (-/+S9): 0*, 1250, 2500 and 5000 µg/mL
- 24 h continuous treatment (-S9): 0*, 313, 625 and 1250 µg/mL


MAIN TEST 1, harvest at 24 h: ---> 6 h treatment/18 h recovery (-/+S9):
Concentrations prepared (-/+S9): 0*, 1250, 2500 and 5000 µg/mL
Microscopically examined (metaphase analysis) (-/+S9): 0*, 1250, 2500 and 5000 µg/mL

MAIN TEST 2, harvest at 24 h: ---> 24 h continuous treatment (-S9):
Concentrations prepared (-S9): 0*, 78.1, 156, 313, 625, 1250, 2500 and 5000 µg/mL
Microscopically examined (metaphase analysis) -S9: 0*, 625, 1250 and 2500 µg/mL

(-/+S9) = without and with metabolic activation
(- S9) = without metabolic activation
* 0 μg/mL = vehicle control (DMSO)

CRITERIA FOR SELECTING APPROPRIATE TEST CONCENTRATIONS FOR METAPHASE ANALYSIS:
see field "Any other information on materials and methods incl. tables"

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Justification for choice of solvent/vehicle:
The test material was insoluble in water but was dissolved in DMSO at 500 mg/mL without indication of an exothermic reaction or change in colour within 2 hours after preparation. This concentration produced a test substance concentration in culture medium of 5000 µg/mL when administering this DMSO test substance solution to the culture medium at 1% v/v.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Dissolved in the vehicle dosed into cell culture medium;

DURATION:
Treatment durations, durations of subsequent recovery in fresh culture medium (free from test material) and the harvest time point are specified in the field "Test concentrations"

- Final concentration of S9 in culture medium: 5% v/v.
- Fixation time (treatment start up to fixation or harvest of cells): 24 hours

SPINDLE INHIBITOR (cytogenetic assays):
Demecolcine was added to the cultures (0.1 µg/mL culture medium) 2 hours before the end of the culture.

FURTHER PROCESSING OF THE CELLS:
The cells were trypsinised to detach them from the tissue culture flask.

For determination of cell growth rate and 50% cell growth inhibition concentration (IC50), 200 µL cell suspension were diluted at 2% v/v in Cell Pack (Sysmex Corporation).

For cytogenetic testing, the remaining cells were collected by centrifugation and treated with hypotonic solution (0.075 M KCl) for 15 min at 37 °C. After incubation in the hypotonic solution, the cells were fixed with 3 + 1 methanol + glacial acetic acid.

STAIN (for cytogenetic assays):
After fixation the cells were stained with 2% (v/v) Giemsa in 1/15 M phosphate buffer (pH 6.8).

NUMBER OF REPLICATIONS:
Duplicate dishes were prepared per dose and condition.

NUMBER OF CELLS EVALUATED:
In the growth inhibition test 50 metaphases per dose and condition, in the main tests, a total of 200 metaphases per selected dose concentration and condition (50 cells per specimen), were analysed for chromosomal aberrations.

DETERMINATION OF CYTOTOXICITY:
Cell count with Microcell counter (CDA 500, Sysmex Corporation) for determination of relative cell growth rate (relative to that of the vehicle control group) and IC50.

Microscopic examination of the metaphases included the recording of the following parameters:
- Aberrant cells (structural aberration, excluding gaps)
- Number of gaps
- Types of aberrations:
Chromatid break, Chromosome break, Chromatid exchange, Chromosome exchange, Others (e.g. multiples with number of aberrations of the same
category > 10)

Determination of polyploidy (numerical aberration):
- Cells with greater than 38 chromosomes were classified as polyploids

Evaluation criteria:

An assay was considered to be acceptable because:
- The frequencies of cells with chromosomal aberrations did not fluctuate markedly between two culture dishes.
- The frequencies of aberrant cells in the vehicle control group were < 5%.
- The frequencies of cells with structural aberrations in the positive control groups were ≥ 20%.

The findings were judged to be positive if the frequencies of cells with structural aberrations or numerical aberrations were 10% or more with a dose-related increase, or the frequencies of aberrant cells were 5% or more in both main tests.

A negative response was claimed if the criteria for a positive response were not met.

Statistics:

Statistical analysis of the data was not performed. The study results were unambiguously negative.

Key result

Species / strain:

mammalian cell line, other: Chinese hamster lung fibroblasts (CHL/IU cells)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

In all tests, following 6 h or 24 h continuous treatment

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Only at 5000 µg/mL, in both cell growth inhibition tests and following 24 h continuous treatment in the main test.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mammalian cell line, other: Chinese hamster lung fibroblasts (CHL/IU cells)

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

In all tests, following 6 h or 24 h continuous treatment

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Only in the cell growth inhibition test following 6 h treatment at 5000 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Precipitate was macroscopically seen in the growth inhibition test at 156 µg/mL and above at all doses and conditions at the start and end of treatment. By culture end, precipitate was seen at 313 µg/mL and above in the 6 h treatment / 18 h recovery tests. In addition, precipitate was macroscopically seen in the main tests at all conditions and test concentrations prepared for the 6 h treatment / 18 h recovery tests at treatment start and end and at culture end, and at 156 µg/mL and above in the 24 h treatment test at treatment start and end.

Conclusions:

negative Without and with metabolic activation (-/+S9)

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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)

Version / remarks:

of 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

of 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (1996) Guideline S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media, in general used for cell culture:
R10p, i.e. medium R0 supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v,
whereby medium R0 is RPMI 1640 buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 μg/mL gentamicin.

- Type and identity of media, used for cloning efficiency plating:
R20p prepared by mixing equal volumes of R10p and R30p,
whereby R30p is medium R0 supplemented with 0.02% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 30% v/v.

HiDHS = heat-inactivated donor horse serum

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male Sprague Dawley derived rats treated with phenobarbital and 5,6-benzoflavone for enzyme induction.

Test concentrations with justification for top dose:

PRELIMINARY TOXICITY TESTING (suspension growth relative to that of vehicle controls)
Test concentrations at 3 h exposure with (+S9) and without (–S9) metabolic activation and at 24 h exposure without metabolic activation (–S9):
9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg/mL

MUTATION TESTS
Experiment 1, 3 h exposure (–S9):
Exposure concentrations: 156.25, 312.5, 625, 1250 and 2500 μg/mL
Mutant phenotype determination at: 312.5, 625, 1250 and 2500 μg/mL

Experiment 1, 3 h exposure (+S9):
Exposure concentrations: 156.25, 312.5, 625, 1250 and 2500 μg/mL
Mutant phenotype determination at: 312.5, 625, 1250 and 2500 μg/mL

Experiment 2, 24 h exposure (–S9):
Exposure concentrations: 156.25, 312.5, 625, 1250 and 2500 μg/mL
Mutant phenotype determination at: 312.5, 625, 1250 and 2500 μg/mL

CRITERIA FOR SELECTING APPROPRIATE TEST CONCENTRATIONS FOR MUTANT PHENOTYPE DETERMINATION:
The highest concentration tested was one that allowed the maximum exposure up to 5000 µg/mL or 10 mM for freely soluble compounds, or the limit of toxicity (i.e. relative total growth reduced to approximately 10 to 20% of the concurrent vehicle control) or the limit of solubility. For a toxic substance, at least 4 analysable concentrations should have been achieved which ideally spanned the toxicity range of 100 to 10% relative total growth (RTG).

Vehicle / solvent:

Dimethyl sulphoxide (DMSO, 1% v/v final concentration in the medium)

Justification for choice of solvent/vehicle:
DMSO was chosen as a vehicle to maximise exposure of cultures in the test system to ADK STAB FP-800. ADK STAB FP-800 was shown to be soluble in DMSO at 500 mg/mL. This concentration produced a test substance concentration in culture medium of 5000 µg/mL when administering this DMSO test substance solution to the culture medium at 1% v/v. At 5000 µg/mL fluctuation in osmolality was > 50 mOsm/kg compared with the vehicle control. At 2500 µg/mL fluctuation in osmolality and in pH were within accepptable limits leading to the choice of 2500 µg/mL as the maximum concentration tested in the preliminary toxicity test.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (1% v/v final concentration in the medium)

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (1% v/v final concentration in the medium)

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment 1: 3 h exposure with (+S9) and without (–S9) metabolic activation
Experiment 2: 24 h exposure without metabolic activation (–S9)

- Selection time: At 48 h after the end of exposure addition of the selection agent trifluorothymidine (TFT)
then allowing 10-14 days for cells to grow with TFT.

SELECTION AGENT: Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2 cultures at each concentration,
[from each culture two vials for assessment of growth in suspension, two 96-well plates for assessment of cloning efficiency
and two 96-well plates for assessment of mutant potential; vehicle controls in quadruplicate].

NUMBER OF CELLS EVALUATED: 2000 cells/well x 192 wells = 384000 cells per culture

DETERMINATION OF CYTOTOXICITY: Relative total growth; (in preliminary toxicity test Relative suspension growth)

Evaluation criteria:

The mutation test result was regarded as negative if:
The mean mutant frequency of all test concentrations was less than the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF = 126 x 10^–6, Moore et al. 2006, detailed reference see below).

If the mutant frequency of any test concentrations exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, a linear trend test was applied: If the linear trend test was negative, the result was regarded as negative. If the linear trend test was positive, this indicated a positive, biologically relevant response.

Reference for GEF:
Moore, M.M., Honma, M., Clements, J., Bolcsfoldi, G., Burlinson, B. Cifone, M., Clarke, J., Delongchamp, R., Durward, R., Fellows, M., Gollapudi, B., Hou, S., Jenkinson, P., Lloyd, M., Majeska, J., Myhr, B., O’Donovan, M, Omori, T, Riach, C., San, R., Stankowski. JR. L.F., Thakur, A.K., Van Goethem, F., Wakuri, S. and Yoshimura, I. (2006). Mouse lymphoma thymidine kinase gene mutation assay: Follow-up meeting of the international workshop on Genotoxicity testing – Aberdeen, Scotland, 2003 – Assay acceptance criteria, positive controls, and data evaluation. Environmental and Molecular Mutagenesis. 47, 1-5.

Statistics:

The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al. 1989 using a one-sided F-test, where p<0.001.
Robinson, W.D., Green, M.H.L., Cole, J., Healy, M.J.R., Garner, R.C., and Gatehouse, D. (1989). Statistical evaluation of bacterial/mammalian fluctuation tests. In: Kirkland, D. J. (Ed). UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report. Part 111. Statistical Evaluation of Mutagenicity Test Data, p.102-140. Cambridge University Press, Cambridge.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

not determined

Remarks:

Preliminary Toxicity Testing: 3 h exposure (–/+S9) and 24 h exposure (–S9)

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Experiment 1, 3 h exposure (–/+S9)

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

Experiment 2, 24 h exposure (–S9)

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
At 5000 µg/mL fluctuation in osmolality was unacceptably high (i.e. > 50 mOsm/kg compared with the vehicle control) leading to the choice of 2500 µg/mL as the maximum concentration tested in the preliminary toxicity test and in both mutation main tests.

JUSTIFICATION FOR NOT CONFIRMING THE NEGATIVE RESULT ATTAINED WITH METABOLIC ACTIVATION (+S9)
In the presence of S9, there were no increases in mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. All mean mutant frequencies of the test concentrations were within the historical vehicle control values and there were no increases in mean mutant frequencies of any test concentration assessed that were associated with a linear trend (P>0.05). Therefore, it was considered unnecessary to perform a direct repeat of the assay in the presence of S9. In the absence of S9, the negative mutagenicity results attained after 24 h of exposure (Experiment 2) confirmed those attained after 3 hours of exposure (Experiment 1).

In the Preliminary Toxicity Test, precipitate (observed by eye) was seen at ADK STAB FP-800 concentrations in final medium of 312.5 µg/mL and greater in the absence and presence of S9 mix, following a 3 hour exposure, and at concentrations of 156.25 µg/mL and greater in the absence of S9 mix following a 24 hour exposure.

In Main Experiments 1 and 2, precipitate was seen at the end of treatment at any exposure concentration selected for metaphase analysis, i.e. at 312.5 µg/mL and greater.

Conclusions:

negative without and with metabolic activation (-/+S9)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the negative results attained in all in vitro genotoxicity studies with the read-across source substance ADK STAB FP-800 the read-across target substance ADK STAB FP-900L is considered not to be genotoxic and does not warrant any classification regarding mutagenicity according to European classification rules [REGULATION (EC) 1272/2008].