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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an in vitro bacterial reverse mutation assay the test material was negative for mutagenicity in S. typhimurium TA98, TA100, TA 1535, TA1537 and E. Coli WP2 uvrA.

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.

The test material contains between 20 - 40% of the parent alcohol Alcohols, C11-14-iso-, C13-rich (EC 271-235-6). In consideration of this, in the REACH registration for that substance, it was shown not to be genotoxic in a battery of in vitro assays. These data support the findings, i.e. no genotoxicity, in this dossier for Phosphoric Acid, Esters with Alcohols, C11-14 iso, C13-rich.

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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:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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.

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:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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.

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, Esters with Alcohols, C11-14 iso, C13-rich [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:
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
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, Esters with Alcohols, C11-14 iso, C13-rich [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 Prime Source substance was also not mutagenic. Likewise, the Prime 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:
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:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24th May - 21 October, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 7703200
- Expiration date of the lot/batch: 2019-02-28

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, below 70 RH%)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Based on the results of a Preliminary Concentration Range Finding Test, the test item concentrations in the Initial Mutation Test (5 strains) were, 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate, the test item concentrations in the Confirmatory Mutation Test (5 strains) in all Salmonella typhimurium strains were 1581, 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate and in Escherichia coli WP2 uvrA strain were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate, the test item concentrations in the Complementary Initial Mutation Test in Escherichia coli WP2 strain were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg test item/plate. Examined concentrations in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA100 strain without metabolic activation were 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test item/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of the test item was examined using distilled water, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF) and acetone. At 100 mg/mL concentration opalescence, dense emulsion was observed using distilled water as vehicle and clear solution was observed using DMSO, DMF and acetone as vehicles. Due to the better biocompatibility, DMSO was selected as vehicle (solvent) for the study.
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylenediamine; TA 98; 4 µg/plate
Remarks:
-S9
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene; all Salmonella strains; 2 µg/plate
Remarks:
+S9
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-amino anthracene; E. coli WP2 uvrA; 50 µg/plate
Remarks:
+S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: Initial Mutation Test - Plate Incorporation Method; Mutation Test - Pre-Incubation Method; Confirmatory Mutation Test - Pre-Incubation Method; Complementary Initial Mutation Test - Plate Incorporation Method; Complementary Confirmatory Mutation Test - Pre-Incubation Method.

Procedure for Exposure in the Initial Mutation Test and in the Complementary Initial Mutation Test:
A standard plate incorporation procedure was performed as an Initial Mutation Test. The same method was used in the Complementary Initial Mutation Test. Bacteria (cultured in Nutrient Broth No.2 as described in Section 5.3.6.) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45°C. The equivalent number of minimal glucose agar plates (three plates per concentration and for each control) was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).

The content of the tubes:
top agar: 2000 μL
vehicle (solvent) or test item solution (or reference controls): 50 μL
overnight culture of test strain: 100 μL
phosphate buffer (pH 7.4) or S9 mix: 500 μL
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48±1 hours.

Procedure for Exposure in the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test:
A pre-incubation procedure was performed as a Confirmatory Mutation Test since in the Initial Mutation Test no positive effect was observed. The same method was used in the Complementary Confirmatory Mutation Test.
For the pre-incubation method, bacteria (cultured in Nutrient Broth No.2. as described in 5.3.6.) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.
Before the overlaying, 50 μL of test item formulations or its vehicle (or positive reference controls or their solvent), 100 μL of the overnight culture of bacterial cells and the 0.5 mL of S9 mix (activated test conditions) or phosphate buffer pH 7.4 (nonactivated test conditions) were added into the appropriate tubes to provide direct contact between bacteria and the test item. These tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37ºC in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48±1 hour.
Evaluation criteria:
The colony numbers on the untreated / negative (vehicle/solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.
Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.
Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions was more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions was more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
Criteria for a Negative Response:
The test item was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproduciblevbiologically relevant positive response at any of the dose groups, with or without metabolic activation.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Initial mutation test: 1581 µg/plate (+/- S9) Confirmatory mutation test: 1581 µg/plate (+S9); 1581 and 500 µg/plate (-S9)
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:
cytotoxicity
Remarks:
Initial mutation test: 1581 and 500 µg/plate (-S9); 1581 µg/plate (+S9) Confirmatory mutation test: 1581, 500, 158.1 and 50 μg/plate (-S9); 1581 and 500 μg/plate (+S9)
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:
cytotoxicity
Remarks:
Initial mutation test: 1581 and 500 μg/plate (-S9); 1581 μg/plate (+S9) Confirmatory mutation test: 1581, 500 and 158.1 μg/plate (-S9); 1581 μg/plate (+S9)
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:
cytotoxicity
Remarks:
Initial mutation test: 1581, 500 and 158.1 μg/plate (-S9); 1581 μg/plate (+S9) Confirmatory mutation test: 1581, 500, 158.1 and 50 μg/plate (-S9); 1581 and 500 μg/plate (+S9) Complementary confirmatory mutation test: 158.1 and 50 μg/plate (-S9)
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No precipitate was detected on the plates in the main tests in all examined bacterial strains with and without metabolic activation.
lower numbers of revertant colonies may indicate inhibitory, cytotoxic effect of the test item in Escherichia coli WP2 uvrA without metabolic activation at 5000 μg test item/plate.

Tables are attached.

In the Initial Mutation Test and in the Complementary Initial Mutation Test (using the plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 50 μg/plate and 5 μg/plate concentrations without metabolic activation (the observed mutation factor value was 1.64). However, there was no clear dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.

In the Confirmatory Mutation Test and in the Complementary Confirmatory Mutation Test (using the pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 at 0.5 μg/plate concentration without metabolic activation (the observed mutation factor value was 2.14). However, there was no clear dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.

Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.

Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

Conclusions:
The test item had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.
Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavoneinduced rats.

The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), a Confirmatory Mutation Test (Pre-Incubation Method), a Complementary Initial Mutation Test (Plate Incorporation Method, during the Experimental Period II) and a Complementary Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of a solubility test, the test item was formulated in Dimethyl sulfoxide (DMSO). Concentrations of 5000; 2500; 1000; 316; 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test (5 strains) were, 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate, the test item concentrations in the Confirmatory Mutation Test (5 strains) in all Salmonella typhimurium strains were 1581, 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate and in Escherichia coli WP2 uvrA strain were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate, the test item concentrations in the Complementary Initial Mutation Test in Escherichia coli WP2 strain were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg test item/plate. Examined concentrations in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA100 strain without metabolic activation were 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test item/plate.

In the Preliminary Concentration Range Finding Test and in the main tests, none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies did not exceed the biological relevance when compared to the vehicle control and were within the normal biological variability of the test system.

No precipitate was detected on the plates in the Preliminary Concentration Range Finding Test and in the main tests in all examined bacterial strains with and without metabolic activation.

Inhibitory, cytotoxic effect of the test item was observed in the Preliminary Concentration Range Finding Test and in the main tests in all Salmonella typhimurium strains with and without metabolic activation at several concentrations. Furthermore in the Confirmatory Mutation Test the lower numbers of revertant colonies may indicate inhibitory, cytotoxic effect of the test item in Escherichia coli WP2 uvrA without metabolic activation at 5000 μg test item/plate. The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.

Endpoint:
in vitro gene mutation study in bacteria
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(Prime): Phosphoric acid, 2-ethylhexyl ester [EC 235-741-0; CAS 12645-31-7]
Target: Phosphoric Acid, Esters with Alcohols, C11-14 iso, C13-rich [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 Prime Source substance was also not mutagenic. Likewise, the Prime 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:
read-across source
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Initial mutation test: 1581 µg/plate (+/- S9) Confirmatory mutation test: 1581 µg/plate (+S9); 1581 and 500 µg/plate (-S9)
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:
cytotoxicity
Remarks:
Initial mutation test: 1581 and 500 µg/plate (-S9); 1581 µg/plate (+S9) Confirmatory mutation test: 1581, 500, 158.1 and 50 μg/plate (-S9); 1581 and 500 μg/plate (+S9)
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:
cytotoxicity
Remarks:
Initial mutation test: 1581 and 500 μg/plate (-S9); 1581 μg/plate (+S9) Confirmatory mutation test: 1581, 500 and 158.1 μg/plate (-S9); 1581 μg/plate (+S9)
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:
cytotoxicity
Remarks:
Initial mutation test: 1581, 500 and 158.1 μg/plate (-S9); 1581 μg/plate (+S9) Confirmatory mutation test: 1581, 500, 158.1 and 50 μg/plate (-S9); 1581 and 500 μg/plate (+S9) Complementary confirmatory mutation test: 158.1 and 50 μg/plate (-S9)
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro bacterial reverse mutation assay:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), a Confirmatory Mutation Test (Pre-Incubation Method), a Complementary Initial Mutation Test (Plate Incorporation Method, during the Experimental Period II) and a Complementary Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of a solubility test, the test item was formulated in Dimethyl sulfoxide (DMSO). Concentrations of 5000; 2500; 1000; 316; 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test (5 strains) were, 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate, the test item concentrations in the Confirmatory Mutation Test (5 strains) in all Salmonella typhimurium strains were 1581, 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate and in Escherichia coli WP2 uvrA strain were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate, the test item concentrations in the Complementary Initial Mutation Test in Escherichia coli WP2 strain were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg test item/plate. Examined concentrations in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA100 strain without metabolic activation were 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test item/plate.

In the Preliminary Concentration Range Finding Test and in the main tests, none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies did not exceed the biological relevance when compared to the vehicle control and were within the normal biological variability of the test system.

No precipitate was detected on the plates in the Preliminary Concentration Range Finding Test and in the main tests in all examined bacterial strains with and without metabolic activation.

Inhibitory, cytotoxic effect of the test item was observed in the Preliminary Concentration Range Finding Test and in the main tests in all Salmonella typhimurium strains with and without metabolic activation at several concentrations. Furthermore in the Confirmatory Mutation Test the lower numbers of revertant colonies may indicate inhibitory, cytotoxic effect of the test item in Escherichia coli WP2 uvrA without metabolic activation at 5000 μg test item/plate. The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.

Justification for classification or non-classification

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

The test material contains between 20 - 40% of the parent alcohol Alcohols, C11-14-iso-, C13-rich (EC 271-235-6). In consideration of this, in the REACH registration for that substance, it was shown not to be genotoxic in a battery of in vitro assays. These data support the findings, i.e. no genotoxicity, in this dossier for Phosphoric Acid, Esters with Alcohols, C11-14 iso, C13-rich.