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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The key studies presented were conducted to internationally recognised testing guidelines with GLP certification.

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Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM medium with glutamine supplemented with 10% (v/v) fetal calf serum (FCS), 1 % (v/v) penicillin/streptomycin (10 000 IU / 10 000 Ng/ml), 1 % (v/v) amphotericine B (250 Ng/ml) - No FCS was used during 4h treatments
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
aroclor induced rat liver S-9 fraction
Test concentrations with justification for top dose:
Mixed Population Method (MP):
24 hours exposure, 24 hours harvest time, without S-9 mix: 0 ; 6.25; 12.5; 25; 50; 75 ug/ml
4 hours exposure, 24 hours harvest time, with S-9 mix: 0 ; 6.25; 12.5; 25; 50; 100 ug/ml

Mitotic Shake Off Method (MSO) (24 hours mitotic shake off):
24 hours exposure, 24 hours mitotic shake off, 27 hours harvest time, without S-9 mix: 0 ; 6 .25; 12.5; 25; 50; 75 ug/ml
4 hours exposure, 24 hours mitotic shake off, 27 hours harvest time, with S-9 mix: 0; 6 .25; 12.5; 25; 50; 100 ug/ml
4 hours exposure, 24 hours mitotic shake off, 27 hours harvest time, with S-9 mix: 0; 80; 100; 120 ; 140 ; 160 ug/ml (not scoreable due to cytotoxicity)
4 hours exposure, 24 hours mitotic shake off, 27 hours harvest time, with S-9 mix: 0; 60; 70; 80; 90 ; 100 ug/ml
Vehicle / solvent:
Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in the V79 in vitro micronucleus assay and for which historical control data is available.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24h (without S9), 4h (with S9)
- Expression time (cells in growth medium): 20h (with S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 24h (mixed population), 27h (mitotic shake off)

STAIN (for cytogenetic assays): Wrights solution (modified May-Gründwald solution)

NUMBER OF REPLICATIONS: 2 per experiment, 4 independent experiments

NUMBER OF CELLS EVALUATED: 1000 per culture, i.e., 2000 per test group

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index, proliferation index
Evaluation criteria:
The in vitro micronucleus assay is considered valid if the following criteria are met:
- The quality of the slides allowed, at least to a large extent, the identification and evaluation of a sufficient number of analyzable cells.
- The proportion of cells with micronuclei in negative control (vehicle control) cultures was within the normal range of the historical control data.
- The positive control chemicals (with and without S-9 mix) induced a significant increase in the number of cells with micronuclei.

The test chemical is considered positive, if:
- A dose-related and reproducible significant increase in the number of cells containing micronuclei is observed
- The proportion of micronucleus-containing cells exceeds both the concurrent negative control range and the negative historical control range.
Species / strain:
Chinese hamster lung fibroblasts (V79)
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not affected
- Effects of osmolality: not affected
- Precipitation: from app. 90-100µg/ml onward

RANGE-FINDING/SCREENING STUDIES:
75µg/ml without S9 and 100µg/ml with S9 were selected as top doses based on cell count, cell attachment, assessment of slides and the proliferation index in a range finding assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A weak and dose dependent suppression of mitotic activity and the proliferation index was reported at and above 25µg/ml without S9. A dose dependent reduction in cell count was observed at and above 50µg/ml withough S9 and at and above 100µg/ml with S9. At the same concentrations, cell attachment was slightly reduced.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Under the experimental conditions of this assay,the substance is considered not to be a chromosome-damaging (clastogenic) agent nor does it to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 1997
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted May 2000
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
his, trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S-9 mix
Test concentrations with justification for top dose:
Experiment 1 (Standard plate test):
20, 100, 500, 2500, 5000µg/plate

Experiment 2 (Preincubation assay):
4, 20, 100, 500, 2500µg/plate

Experiment 3 (Preincubation assay):
0.4, 2, 10, 50, 250µg/plate

Experiment 4 (Preincubation assay, TA 100 only due to technical fault in experiment 2):
4, 20, 100, 500, 2500µg/plate
Vehicle / solvent:
Acetone was selected as the vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: aminoanthracene
Remarks:
with S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylendiamine
Remarks:
without S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period (experiments 2 and 3): 20 min
- Incubation time: 48-72h in the dark at 37°C

SELECTION AGENT (mutation assays):
minimal agar containing 0.5mM histidine and 0.5mM biotine (TA strains)
minimal agar containing 0.5mM tryptophane (e.coli)

NUMBER OF REPLICATIONS: triplicates
Evaluation criteria:
Acceptance criteria:
- The number of revertant colonies in the negative controls is within the range of the historical control data
- The sterility controls revealed no indication of bacterial contamination
- The positive controls induced a significant increase in the number of revertant colonies within the range of the historical control data
- The titer of viable bacteria was > 10^8/ml

The test chemical is considered positive if:
- A dose-related and reproducible increase is observed in the number of revertant colonies, i .e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system

A test substance is generally considered non-mutagenic if:
- The number of revertants for all tester strains are within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from about 500 - 2500µg/plate in the standard plate test, and from 250-500µg/plate onward in the preincubation assay depending on strain and test conditions
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: observed from about 2500µg/plate onward
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
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)
Version / remarks:
adopted July 1997
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted May 2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
adopted August 1998
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium) containing Hank's salt supplemented with 10% FBS, 5µg/mL neomycin, 1% amphotericin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/b-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
The following concentrations were evaluated:
1st experiment:
-S9: 6.3, 12.5, 25, 50µg/mL
+S9: 12.5, 25, 50, 100µg/mL
2nd experiment
-S9: 6.3, 12.5, 25, 37.5
+S9: 12.5, 25, 50, 100
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): about 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): app. 15days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: duplicates

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The gene mutation assay is considered acceptable if it meets the following criteria:
The number of mutant colonies per 10^6 cells found in the solvent controls falls within the laboratory historical control data.
The positive control substances should produce a significant increase in mutant colony frequencies.
The cloning efficiency II (absolute value) of the solvent controls should exceed 50%.

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible positive response for one of the test points.

A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.

A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concentrations a mutation frequency that is three times higher than the spontaneous mutation frequency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study is also taken into consideration.
Statistics:
linear regression (least squares)
Species / strain:
Chinese hamster lung fibroblasts (V79)
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: occured at 412.5µg/mL without, 206.3µg/mL with S9, which is above the maximum concentration that could be tested due to cytotoxicity

RANGE-FINDING/SCREENING STUDIES: concentrations between 25.8 and 3300µg/mL (app. 10mM) were used. Strong cytotoxicity was observed at and above 51.6µg/mL without and 206.3µg/mL with metabolic activation.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
The test material failed to induce statitically significant incidence of gene mutation in chinese hamster lung cells under the conditions of the test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in bacteria

Ethyl phenyl(2,4,6-trimethylbenzoyl)phosphinate was tested in three gene mutation assays in bacteria (Ames) according to or similar to OECD471 (BASF 2002 and 1981, Unilever 2001). No increase in the number of his + or trp+ revertants were observed in both, the standard plate test and in the preincubation assay either without S-9 mix or after the addition of a metabolizing system. Concentrations up to 5000µg/plate were tested in Salmonella strains TA 1535, TA 100, TA 1537, TA 98, TA1538, TA102 and Escherichia coli WP2 uvrA. S9 fraction was prepared from aroclor induced rat liver. Cytotoxicity was observed from 250 - 2500µg/plate onward depending on tester strain and assay conditions. In general, bacteria were more sensitive in the preincubation assay. Precipitations was observed at 2500µg/plate or 5000µg/plate in two of the three studies.

Gene mutation in mammalian cells

The potential of ethyl phenyl(2,4,6 -trimethylbenzoyl)phospinate induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster was investigated in an OECD476 study (BASF 2013). The assay was performed in two independent experiments, using two parallel cultures each with and without liver microsomal activation for a treatment period of 4 hours. The following concentrations were selected based on strong cytotoxicty observed at higher concentrations:

-S9: 6.3, 12.5, 25, 37.5, 50µg/mL

+S9: 12.5, 25, 50, 100µg/mL

The test substance was dissolved in DMSO.

No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Micronucleus test in vitro

Ethyl phenyl(2,4,6-trimethylbenzoyl)phosphinate was assessed in V79 cells in vitro for possible clastogenic or aneugenic activity leading to micronuclei both in the presence and in the absence of a metabolizing system according to the mixed population method and the mitotic shake off method (BASF 2004). According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiment, the following doses were evaluated:

- Mixed Population Method:

24 hours exposure, without S-9 mix: 0 ; 6.25; 12.5; 25; 50; 75 ug/ml

4 hours exposure, with S-9 mix: 0 ; 6.25; 12.5; 25; 50; 100 ug/ml

- Mitotic Shake Off Method (24 hours mitotic shake off):

24 hours exposure, without S-9 mix: 0 ; 6 .25; 12.5; 25; 50; 75 ug/ml

4 hours exposure, with S-9 mix: 0; 6 .25; 12.5; 25; 50; 100 ug/ml

4 hours exposure, with S-9 mix: 0; 80; 100; 120 ; 140 ; 160 ug/ml (not scoreable due to cytotoxicity)

4 hours exposure, with S-9 mix: 0; 60; 70; 80; 90 ; 100 ug/ml

Positive and vehicle controls gave the expected results. Cytotoxicity was observed approximately at and above 50µg/ml without S9 and at and above 100µg/ml with S9. No increase in the number of cells containing micronuclei was observed in cultures treated with the test substance.

In a second MNT in vitro study, human lymphocytes were exposed to the test substance for 3 hours (with and without S9 mix) and for 20h (without S9 mix). Maximum concentrations were selected to yield approximately 50% cytotoxicity. After 20h, cytochalasin was added to inhibit cell division, but not karyokinesis. The frequency of micronuclei is thus independent of the proportion of cells that divide, if only binucleate cells are scored.

Experiment 1:

3h treatment, 17h recovery, without S9: 96.2, 113.1, 133.1µg/ml

3h treatment, 17h recovery, with S9: 81.8, 113.1, 156,6µg/ml

20h treatment, without S9: 26.2, 30.8, 36.3µg/ml

Experiment 2:

3h treatment, 17h recovery, with S9: 113.1, 216.8, 255µg/ml

Experiment 3:

3h treatment, 17h recovery, with S9: 113.1, 156.6, 184.2µg/ml

Treatment of cultures in the absence of S-9 gave no increases above historical control values in the frequency of binucleate cells containing micronuclei at any concentration tested. Treatment of cultures in the presence of S9 resulted in a statistically significant increase at the two highest concentrations of experiment 1 mainly due to low background mutagenicity in the control cells. With the exception of one replicate of the highest dose, all frequency were within the historical control range. The same result, i.e., only one high dose replicate above historical control data, was also obtained in experiments 2 and 3. When analysing the data from these three experiments, the following points should also be noted: Some variation in toxicity was observed between experiments. A small but statistically significant increase in the frequency of binucleate cells containing micronuclei was observed in cultures analysed at 113 .1 µg/mL in Experiment 1, but no such increases were observed at this concentration in Experiments 2 and 3. Furthermore, statistically significant increases in the frequency of binucleate cells containing micronuclei were not observed at 216.8µg/mL in Experiment 2, or at 156.6 µg/mL in Experiment 3. The increases in the frequency of micronuclei were only observed at the maximum dose in each experiment, which was close to 50% CBPI compared to the solvent control values. The results after addition of S9 were thus considered equivocal.

Chromosome aberration in vitro

In an in vitro cytogenetics assay using duplicate human lymphocyte cultures prepared from the pooled blood of three male donors in two independent experiments, cells were incubated with the test substance for 3h (with and without metabolic activation) or 20h (without S9 mix only). The highest dose level used, 600 µg/mL, was in excess of the solubility limit of the test article in culture medium. The highest concentrations chosen for analysis after 3 hours exposure in the first experiment, 196.6 µg/mL and 245.8 µg/mL, induced approximately 56% and 33% mitotic inhibition (reduction in mitotic index) in the absence and presence of S-9 respectively. In the second experiment, 20h incubation without S9 and 3h incubation with S9 cause 64% and 57% mitotic inhibition at 113.1 and 236.2µg/ml, respectively.

Treatment of cultures for 3 hours in the absence and presence of S-9 resulted in frequencies of cells with aberrations (excluding gaps) similar to those in concurrent negative controls and fell within the historical negative control (normal) range. Treatment for 20 hours in the absence of S-9 gave a small, but statistically significant increase in the frequency of cells with aberrations (excluding gaps) at 113.1 gg/mL (the highest concentration analysed). Numbers of aberrant cells in both replicate cultures fell just outside the normal range. It should be noted that the increase in cells with aberrations occurred at a concentration which induced clear cytotoxicity. No increases in aberrant cells were observed at lower concentrations where the range of mitotic inhibition was 0 -55%. Increases in cells with numerical aberrations were seen in most cultures receiving short (3 hour) treatments with Lucirin TPO-L in both the presence and absence of S-9. The effect was attributable to increases in polyploidy. Continuous treatment in the absence of S-9 did not give rise to numerical aberrations. Thus the test was considered equivocal.


Short description of key information:
Ames negative (BASF 2002, OECD 471, GLP)
Ames negative (Unilever 2001, similar to OECD 471, GLP)
Ames negative (BASF 1981, similar to OECD 471 but no cross-linking sensitive strains used)

HPRT negativ (BASF 2013, according to OECD 476, GLP)

MNT in vitro negative (BASF 2004, equivalent to OECD 487, GLP)
MNT in vitro negative w/out S9, equivocal w/ S9 at cytotoxic concentrations (Unilever 2002, equivalent to OECD 487, GLP)
Chromosome aberration equivocal due to polyploidy (Unilever 2001, OECD 473, GLP)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The registered substance failed to induce mutagenic effects in any of the key studies presented. As no positive results were observed in in vitro studies, further in vivo studies are considered to be not scientifically necessary inline with REACH (1907/2006) Annex IX, Column 2, 8.4. The registered substance is therefore considered not to fulfill the criteria for classification for mutagenicity in the EU Classification, Labelling, and Packagin (CLP) regulation (1272/2008).