Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In conclusion, in the described in vitro mutagenicity tests under the experimental conditions reported, the test item Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts is considered to be non-mutagenic and non-clastogenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name: Phosphoric acid, C14-15 branched and linear alkyl esters, potassium salts
CAS No.: 1893414-79-3
Physical state: white solid at 20 °C
Batch No.: PU61810016
Re-certification date of batch: 09 March 2018
Purity: 100 % (UVCB, lyophilized solid, water content 0.85 % (w/w))
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The S. typhimurium strains are constructed to differentiate between base pair (TA 100, TA 1535, TA 102) and frameshift (TA 98, TA 1537) mutations.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

The toxicity of the test item was determined with tester strains TA 98 and TA 100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test). Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control. The test item was tested in the pre-experiment with the following concentrations:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

2500 µg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed with the following concentrations:

Experiment I:
1.00, 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate
(except TA 98 and TA 100 with and without metabolic activation)

0.316, 1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate
(only TA 98 without metabolic activation)

3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate
(only TA 98 with metabolic activation and TA 100 with and without metabolic activation)

Experiment II:
1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate
(TA 98 and TA 1535 with metabolic activation)

1.00, 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate
(TA 100 with metabolic activation and TA 102 with and without metabolic activation)

0.0316, 0.100, 0.316, 1.00, 3.16, 10.0, 31.6 and 100 µg/plate
(TA 98, TA 100, TA 1535 without metabolic activation and TA 1537 with and without metabolic activation)

Vehicle / solvent:

Aqua destillata

Untreated negative controls:

yes

Remarks:

Aqua destillata

Negative solvent / vehicle controls:

yes

Remarks:

Aqua destillata

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-NOPD (4-nitro-o-phenylene-diamine); 2-AA (2-aminoanthracene)

Details on test system and experimental conditions:

Negative as well as positive controls were included in each experiment. Strain specific positive controls were included in the assay, which demonstrated the effective performance of the test. Negative/solvent controls were treated in the same way as all dose groups.

All Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. They contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain of the lipopolysaccharides of the bacterial cell surface. This increases cell
permeability of larger substances. The other mutation is a deletion of the uvrB gene coding for a protein of the DNA nucleotide excision repair system resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chl) and biotin (bio) genes (bacteria require biotin for growth). The tester strains TA 98, TA 100 and TA 102 contain the R-factor plasmid, pkM101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non R-factor parent strains. pkM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system which is normally present in these organisms. The properties of the S. typhimurium strains with regard to membrane permeability, ampicillin- and tetracycline-resistance as well as normal spontaneous mutation rates are checked regularly according to Ames et al.. In this way it is ensured that the experimental conditions set up by Ames are fulfilled.

Evaluation criteria:

A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Background lawn reduced

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

4-NOPD (-S9) & 2-AA (+S9)

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Background lawn reduced & precipitation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

NaN3 (-S9) & 2-AA (+S9)

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

precipitation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

MMS (-S9) & 2-AA (+S9)

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Background lawn reduced & precipitation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

NaN3 (-S9) & 2-AA (+S9)

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Background lawn reduced & precipitation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

4-NOPD (-S9) & 2-AA (+S9)

The test item Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

Experiment I:

1.00, 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate

(except TA 98 and TA 100 with and without metabolic activation)

0.316, 1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate

(only TA 98 without metabolic activation)

3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate

(only TA 98 with metabolic activation and TA 100 with and without metabolic activation)

Experiment II:

1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate

(TA 98 and TA 1535 with metabolic activation)

1.00, 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate

(TA 100 with metabolic activation and TA 102 with and without metabolic activation)

0.0316, 0.100, 0.316, 1.00, 3.16, 10.0, 31.6 and 100 µg/plate

(TA 98, TA 100, TA 1535 without metabolic activation and TA 1537with and without metabolic activation)

Precipitation of the test item was observed in all tester strains used in experiment I and II at a concentration of 2500 µg/plate and higher (with and without metabolic activation), if tested. Toxic effects of the test item were noted in all tester strains evaluated in experiment I and II. In experiment I toxic effects of the test item were observed in tester strain TA 98 at concentrations of 31.6 µg/plate and higher (without metabolic activation) and at concentrations of 1000 µg/plate (with metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 100 µg/plate and higher (without metabolic activation) and at a concentration of 2500 µg/plate (with metabolic activation). In tester strains TA 1535 and TA 1537 toxic effects of the test item were seen at concentrations of 100 µg/plate and higher (with and without metabolic activation). In tester strain TA 102 toxic effects of the test item were observed at a concentration of 2500 µg/plate (without metabolic activation). In experiment II toxic effects of the test item were noted in tester strain TA 98 at concentrations of 31.6 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation). In tester strain TA 100 toxic effects of the test item were observed at concentrations of 31.6 µg/plate and higher (without metabolic activation) and at a concentration of 2500 µg/plate (with metabolic activation). In tester strain TA 1535 toxic effects of the test item were seen at concentrations of 3.16 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation). In tester strain TA 1537 toxic effects of the test item were observed at concentrations of 3.16 µg/plate and higher (without metabolic activation) and at a concentration of 100 µg/plate (with metabolic activation). In tester strain TA 102 toxic effects of the test item were noted at concentrations of 316 µg/plate and higher (with and without metabolic activation). The reduction in the number of revertants down to a mutation factor of ≤ 0.5 found in experiment II in tester strain TA 1537 at a concentration of 0.316 µg/plate (without metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. All criteria of validity were met.

Conclusions:

Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts is considered to be non-mutagenic in this bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Name: Phosphoric acid, C14-15 branched and linear alkyl esters, potassium salts
CAS No.: 1893414-79-3
Physical state: white solid at 20 °C
Batch No.: PU61810016
Re-certification date of batch: 09 March 2018
Purity: 100 % (UVCB, lyophilized solid, water content 0.85 % (w/w))
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Target gene:

heterozygous TK-locus of mouse lymphoma cell line L5178Y

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

cell bank of Eurofins Munich; each cell batch is routinely checked for mycoplasma infection.
Thawed stock cultures are maintained in plastic culture flasks in RPMI 1640 complete medium and subcultured three times per week.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

The toxicity of the test item was determined in pre-experiment up to a maximum concentration of 5 mg/mL. In the pre-experiment eight concentrations [25, 50, 100, 250, 500, 1000, 2500, 5000 µg/mL] were tested without and with metabolic activation. The experimental conditions in the pre-experiment were the same as described below in the paragraph experimental performance. After a 2-day growth period the relative suspension growth (RSG) of the treated cell cultures was
calculated according to the method of Clive and Spector.

The selection of the concentrations used in the main experiment was based on data from the pre-experiment. In the main experiment 500 µg/mL (without and with metabolic activation) was selected as the highest concentration. The experiment without and with metabolic activation was performed as 4 h short-term exposure assay. The test item was investigated at the following concentrations: without and with metabolic activation: 2.5, 5, 10, 25, 50, 100, 250, and 500 µg/mL
According to OECD Guidelines at least 8 concentrations of the test item were set up in the experiments without and with metabolic activation.

Vehicle / solvent:

Solvent controls: Aqua ad injectabilia 10% v/v

Untreated negative controls:

yes

Remarks:

RPMI 1640 medium supplemented with 5 % horse serum (HS), 100 U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine,25 mM HEPES, 2.5 µg/mL amphotericin B.

Negative solvent / vehicle controls:

yes

Remarks:

Aqua ad injectabilia 10% v/v

Positive controls:

yes

Remarks:

ethylmethanesulfonate; methylmethanesulfonate; benzo[a]pyrene;

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

methylmethanesulfonate

Evaluation criteria:

The test item is considered mutagenic if the following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126
mutants per 10^6 cells and
- a dose-dependent increase in mutant frequency is detected.

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potentialclastogenic effects and/or chromosomal aberrations. According to the OECD guideline, the biological relevance is considered first for the interpretation of result. Statistical methods might be used as an aid in evaluation of the test result. A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Methylmethanesulfonate

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Benzo[a]pyrene

The test item Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts was assessed for a possible potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was conducted with and without metabolic activation. The experiment with metabolic activation was performed by including liver microsomes and NADP for efficient detection of a wide variety of carcinogens requiring metabolic activation. The selection of the concentrations used in the main experiment was based on data from the pre-experiment according to the OECD guideline 490. In the main experiment 500 µg/mL (without and with metabolic activation) was selected as the highest concentration. The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay. The pH-value detected with the test item was within the physiological range. The test item was investigated at the following concentrations:

without and with metabolic activation:

2.5, 5, 10, 25, 50, 100, 250 and 500 µg/mL

Precipitation:

Precipitation of the test item was noted at a concentration of 500 µg/mL (without and with metabolic activation).

Toxicity:

No growth inhibition was observed in the experiment without and with metabolic activation. In the experiment without metabolic activation the relative total growth (RTG) was 83.6% for the highest concentration (500 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 500 µg/mL with a RTG of 94.5%.

Mutagenicity:

The mutant frequencies obtained from all experiments were compared with the Global Evaluation Factor (GEF) and a statistical analysis was performed. The GEF is defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data are gathered from ten laboratories. For the microwell method the GEF was defined to be 126. Criterion for mutagenicity is the extension of the GEF by the induced mutant frequency as well as a dose-dependent increase in mutant frequency. The positive controls EMS (300 µg/mL), MMS (10 µg/mL) and B[a]P (2.5 µg/mL) showed distinct effects in mutation frequency, thus proving the ability of the test system to detect potential mutagenic effects. In the experiment without metabolic activation all validity criteria were met. The negative and solvent controls showed mutant frequencies within the acceptance range of 50-170 mutants/10^6 cells, according to the IWGT criteria. The mutant frequencies of the negative controls were 89.5 and 75.9 mutants/10^6 cells and the mutant frequencies of the solvent controls were 92.6 and 77.6 mutants/10^6 cells, respectively, the positive controls EMS and MMS induced a distinct increase in mutant frequency with 833.5 and 406.5 mutants/10^6 cells. The mutant frequencies induced by the test item did not show a biologically relevant increase. The GEF of 126 was not exceeded in any of the dose groups showing induced mutant frequencies between -25.4 and 2.1 mutants/10^6 cells. None of the observed mutant frequencies was statistically significantly increased over those of the solvent controls. In the experiment with metabolic activation all validity criteria were met. The negative and solvent controls showed mutant frequencies within the acceptance range of 50-170 mutants/10^6 cells, according to the IWGT criteria. The mutant frequencies of the negative controls were 79.9 and 71.0 mutants/10^6 cells and the mutant frequencies of the solvent controls were 98.3 and 62.0 mutants/10^6 cells, respectively, the positive control B[a]P induced a distinct increase in mutant frequency with 627.9 mutants/10^6 cells. The mutant frequencies induced by the test item did not show a biologically relevant increase. The GEF of 126 was not exceeded in any of the dose groups showing induced mutant frequencies between -24.5 and 21.4 mutants/10^6 cells. None of the observed mutant frequencies was statistically significantly increased over those of the solvent controls. All mutant frequencies for negative, solvent and positive controls of the experiment were found within the historical range of the test facility Eurofins Munich.

Clastogenicity:

Colony sizing was performed for the highest concentrations of the test item and for the negative and positive controls.An extension of the GEF by the induced mutant frequency in combination with an increased occurrence of small colonies (defined by slow growth and/or morphological alteration of the cell clone) is an indication for potential clastogenic effects and/or chromosomal aberrations. The positive controls MMS (10 µg/mL) and B[a]P (2.5 µg/mL) induced a significant increase in mutant frequency and a biologically significant increase of small colonies (≥40%), thus proving the ability of the test system to indicate potential clastogenic effects. In the experiment without metabolic activation the percentage of small colonies in the negative controls was found to be 20.3% and 19.6% and in the solvent controls was found to be 21.7% and 11.8%, respectively. The percentage of small colonies of the positive control MMS was found to be 50.5%. In the highest dose groups 22.4% (100 µg/mL), 12.1% (250 µg/mL) and 24.1% (500 µg/mL) of small colonies were found. As none of the values exceeded 40%, all dose groups were considered as not clastogenic. With metabolic activation the percentage of small colonies in the negative controls was found to be 11.3% and 12.8% and in the solvent controls was found to be 22.7% and 13.5%, respectively. The percentage of small colonies of the positive control B[a]P was found to be 53.4%. In the highest dose groups 13.8% (100 µg/mL), 10.4% (250 µg/mL) and 8.2% (500 µg/mL) of small colonies were found. As none of the values exceeded 40%, all dose groups were considered as not clastogenic.

In the experiment with metabolic activation all validity criteria were met. The negative and solvent controls showed mutant frequencies within the acceptance range of 50-170 mutants/10^6 cells, according to the IWGT criteria. The mutant frequencies of the negative controls were 79.9 and 71.0 mutants/10^6 cells and the mutant frequencies of the solvent controls were 98.3 and 62.0 mutants/10^6 cells, respectively, the positive control B[a]P induced a distinct increase in mutant frequency with 627.9 mutants/10^6 cells. The mutant frequencies induced by the test item did not show a biologically relevant increase. The GEF of 126 was not exceeded in any of the dose groups showing induced mutant frequencies between -24.5 and 21.4 mutants/10^6 cells. None of the observed mutant frequencies was statistically significantly increased over those of the solvent controls. All mutant frequencies for negative, solvent and positive controls of the experiment were found within the historical range of the test facility Eurofins Munich.

Clastogenicity

Colony sizing was performed for the highest concentrations of the test item and for the negative and positive controls. An extension of the GEF by the induced mutant frequency in combination with an increased occurrence of small colonies (defined by slow growth and/or morphological alteration of the cell clone) is an indication for potential clastogenic effects and/or chromosomal aberrations. The positive controls MMS (10 µg/mL) and B[a]P (2.5 µg/mL) induced a significant increase in mutant frequency and a biologically significant increase of small colonies (≥40%), thus proving the ability of the test system to indicate potential clastogenic effects. In the experiment without metabolic activation the percentage of small colonies in the negative controls was found to be 20.3% and 19.6% and in the solvent controls was found to be 21.7% and 11.8%, respectively. The percentage of small colonies of the positive control MMS was found to be 50.5%. In the highest dose groups 22.4% (100 µg/mL), 12.1% (250 µg/mL) and 24.1% (500 µg/mL) of small colonies were found (Table 7). As none of the values exceeded 40%, all dose groups were considered as not clastogenic. With metabolic activation the percentage of small colonies in the negative controls was found to be 11.3% and 12.8% and in the solvent controls was found to be 22.7% and 13.5%, respectively. The percentage of small colonies of the positive control B[a]P was found to be 53.4%. In the highest

dose groups 13.8% (100 µg/mL), 10.4% (250 µg/mL) and 8.2% (500 µg/mL) of small colonies were found. As none of the values exceeded 40%, all dose groups were considered as not clastogenic.

Tables

Pre-Experiment for Toxicity, without metabolic activation

Test Group	Concentration [µg/mL]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	Suspension Growth (SG)	Relative Suspension Growth (RSG) [%]
C1	0	330000	871000	1320000	11.5	110.4
C2	0	373000	889000	1290000	11.5	110.2
S1	0	316000	825000	1230000	10.1	100.0
S2	0	349000	821000	1300000	10 .7	100.0
1	25	318000	831000	1480000	12.3	118.1
2	50	342000	853000	1490000	12.7	122.1
3	100	320000	821000	1330000	10.9	104.9
4	250	322000	777000	1410000	11.0	105.2
5 P	500	285000	662000	1460000	9.7	92.8
6 P	1000	154000	180000	755000	2.3	21.8
7 P	2500	1300	2780	3700	0.0	0.1
8 P	5000	1290	1480	2220	0.0	0.1

Pre-Experiment for Toxicity, with metabolic activation

Test Group	Concentration [µg/mL]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	Suspension Growth (SG)	Relative Suspension Growth (RSG) [%]
C1	0	306000	800000	1390000	11.1	108.7
C2	0	319000	789000	1440000	11.4	111.1
S1	0	313000	751000	1310000	9.8	100.0
S2	0	325000	830000	1280000	10.6	100.0
1	25	309000	831000	1260000	10.5	102.3
2	50	329000	888000	1300000	11.5	112.8
3	100	317000	825000	1230000	10.1	99.2
4	250	328000	785000	1300000	10.2	99.7
5 P	500	281000	626000	1410000	8.8	86.3
6 P	1000	253000	451000	1350000	6.1	59.5
7 P	2500	1480	2690	3800	0.0	0.1
8 P	5000	1760	1300	13800	0.0	0.1

Main Experiment - Toxicity Data, without metabolic activation

Test Group	Concentration [µg/mL]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	Suspension Growth (SG)	Relative Suspension Growth (RSG) [%]	Relative Cloning Efficiency (RCE)	Relative Total Growth (RTG)
C1	0	394000	1050000	1410000	14.8	91.0	101.6	92.4
C2	0	387000	1070000	1410000	15.1	92.7	113.6	105.4
S1	0	375000	1060000	1520000	16.1	100.0	100.0	100.0
S2	0	429000	1190000	1380000	16.4	100.0	100.0	100.0
3	2.5	397000	1050000	1480000	15.5	95.5	126.0	120.4
4	5	369000	1080000	1480000	16.0	98.3	123.8	121.6
5	10	362000	1070000	1470000	15.7	96.7	117.5	113.6
6	25	384000	1110000	1470000	16.3	100.3	121.6	122.0
7	50	345000	1050000	1410000	14.8	91.0	111.8	101.7
8	100	411000	1120000	1500000	16.8	103.3	111.8	115.4
9	250	356000	929000	1510000	14.0	86.2	123.8	106.7
10 P	500	361000	828000	1390000	12.2	74.8	111.8	83.6
EMS	300	396000	915000	1390000	12.7	78.2	100.0	78.2
MMS	10	356000	912000	1390000	12.7	77.9	91.3	71.2

Main Experiment - Mutagenicity Data, without metabolic activation

		Cloning Efficiency (CE)			Mutagenicity Data
Test Group	Concentration [µg/mL]	Plate 1	Plate 2	Cloning Efficiency (CE)	Number of cultures / 96 wells					MF      [mutants/ 10^6 cells]	IMF      [mutants/ 10^6 cells]
					Plate 1	Plate 2	Plate 3	Plate 4	Mean
C1	0	71	78	93.5	12	12	18	17	14.8	89.5	/
C2	0	80	76	104.6	18	13	17	8	14.0	75.9	/
S1	0	71	77	92.1	10	16	16	18	15.0	92.6	/
S2	0	75	73	92.1	16	13	10	12	12.8	77.6	/
3	2.5	81	81	116.0	8	9	21	11	12.3	59.7	-25.4
4	5	80	81	114.0	11	14	12	18	13.8	68.1	-17.0
5	10	79	79	108.2	11	12	10	19	13.0	67.7	-17.4
6	25	81	79	112.0	13	15	16	15	14.8	74.5	-10.6
7	50	81	74	102.9	17	13	16	17	15.8	87.2	2.1
8	100	78	77	102.9	14	15	8	21	14.5	80.3	-4.8
9	250	79	82	114.0	20	15	16	15	16.5	82.9	-2.2
10 P	500	79	76	102.9	14	11	11	18	13.5	73.9	-11.2
EMS	300	75	73	92.1	78	79	70	73	75.0	833.5	748.5
MMS	10	76	66	84.1	48	49	49	44	47.5	406.5	321.4

Main Experiment - Colony Sizing, without metabolic activation

Test group	Concentration [µg/mL]	Wells with at least   1 colony	Large colonies	Small colonies	% small colonies
C1	0	59	47	41	20.3
C2	0	56	45	11	19.6
S1	0	60	47	13	21.7
S2	0	51	45	6	11.8
8	100	58	45	13	22.4
9	250	66	58	8	12.1
10 P	500	54	41	13	24.1
MMS	10	190	94	96	50.5

Main Experiment - Toxicity Data, with metabolic activation

Test Group	Concentration [µg/mL]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	Suspension Growth (SG)	Relative Suspension Growth (RSG) [%]	Relative Cloning Efficiency (RCE)	Relative Total Growth (RTG)
C1	0	336000	980000	1390000	13.6	94.2	87.1	82.1
C2	0	358000	993000	1460000	14.5	100.2	85.8	86.0
S1	0	357000	1010000	1450000	14.6	100.0	100.0	100.0
S2	0	343000	972000	1470000	14.3	100.0	100.0	100.0
3	2.5	360000	986000	1470000	14.5	100.2	92.8	93.0
4	5	353000	1000000	1390000	13.9	96.1	88.5	85.0
5	10	353000	1010000	1460000	14.7	101.9	89.9	91.7
6	25	354000	1000000	1420000	14.2	98.2	89.9	88.3
7	50	315000	859000	1420000	12.2	84.3	94.3	79.5
8	100	355000	8810000	1430000	12.6	87.1	99.1	86.3
9	250	356000	850000	1380000	11.7	81.1	108.1	87.7
10 P	500	334000	843000	1420000	12.0	82.7	114.2	94.5
B[a]P	2.5	327000	461000	1100000	5.1	35.1	71.8	25.2

Main Experiment - Mutagenicity Data, with metabolic activation

		Cloning Efficiency (CE)			Mutagenicity Data
Test Group	Concentration [µg/mL]	Plate 1	Plate 2	Cloning Efficiency (CE)	Number of cultures / 96 wells					MF      [mutants/ 10^6 cells]	IMF      [mutants/ 10^6 cells]
					Plate 1	Plate 2	Plate 3	Plate 4	Mean
C1	0	75	74	93.5	15	18	10	10	13.3	79.9	/
C2	0	74	74	92.1	13	11	13	9	11.8	71.0	/
S1	0	71	80	96.5	22	13	13	18	16.5	98.3	/
S2	0	83	80	118.1	13	11	9	19	13.0	62.0	/
3	2.5	75	78	99.6	22	14	20	14	17.5	101.5	21.4
4	5	73	77	95.0	18	14	20	14	16.5	99.6	19.4
5	10	78	73	96.5	13	10	19	12	13.5	79.0	-1.2
6	25	75	76	96.5	12	13	15	13	13.3	77.0	-3.2
7	50	79	75	101.2	14	9	15	16	13.5	75.1	-5.1
8	100	78	79	106.4	13	15	13	17	14.5	77.1	-3.1
9	250	80	82	116.0	25	14	20	18	19.3	97.0	16.9
10 P	500	82	83	122.6	13	13	12	11	12.3	55.7	-24.5
B[a]P	2.5	65	71	77.0	64	61	63	48	59.0	627.9	547.8

Main Experiment - Colony Sizing, with metabolic activation

Test group	Concentration [µg/mL]	Wells with at least   1 colony	Large colonies	Small colonies	% small colonies
C1	0	53	47	6	11.3
C2	0	47	41	6	12.8
S1	0	66	51	15	22.7
S2	0	52	45	7	13.5
8	100	58	50	8	13.8
9	250	77	69	8	10.4
10 P	500	49	45	4	8.2
B[a]P	2.5	236	110	126	53.4

Statistical significance at the 5% level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Biometry - Main Experiment without metabolic activation

Test group	Concentration [µg/mL]	mean mutant frequency	mean induced mutant frequency	p-value	statistical significance
C1	0	89.5	/	/	/
C2	0	75.9	/	/	/
S1	0	92.6	/	/	/
S2	0	77.6	/	/	/
3	2.5	59.7	-25.4	0.135	-
4	5	68.1	-17.0	0.089	-
5	10	67.7	-17.4	0.263	-
6	25	74.5	-10.6	0.521	-
7	50	87.2	2.1	0.766	-
8	100	80.3	-4.8	0.905	-
9	250	82.9	-2.2	0.891	-
10 P	500	73.9	-11.2	0.339	-
EMS	300	833.5	748.4	0.004	+
MMS	10	406.5	321.4	0.003	+

Biometry - Main Experiment with metabolic activation

group	Concentration [µg/mL]	mean mutant frequency	mean induced mutant frequency	p-value	statistical significance
C1	0	79.9	/	/	/
C2	0	71.0	/	/	/
S1	0	98.3	/	/	/
S2	0	62.0	/	/	/
3	2.5	101.5	21.4	0.194	-
4	5	99.6	19.4	0.194	-
5	10	79.0	-1.2	1.000	-
6	25	77.0	-3.2	1.000	-
7	50	75.1	-5.1	1.000	-
8	100	77.1	-3.1	1.000	-
9	250	97.0	16.9	0.438	-
10	500	55.7	-24.5	0.141	-
B[a]P	2.5	627.9	547.8	0.002	+

C: Negative control

S: Solvent control (Aqua ad injectabilia 10% v/v)

P: Precipitation

B[a]P:   Benzo[a]pyrene [µg/mL]

EMS: Ethylmethanesulfonate [µg/mL]

MMS: Methylmethanesulfonate [µg/mL]

Conclusions:

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Executive summary:

The test item Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts was assessed for a possible potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was conducted with and without metabolic activation. No growth inhibition was observed in the experiment without and with metabolic activation.The mutant frequencies obtained from all experiments were compared with the Global Evaluation Factor (GEF) and a statistical analysis was performed. None of the observed mutant frequencies was statistically significantly increased over those of the solvent controls. All mutant frequencies for negative, solvent and positive controls of the experiment were found within the historical range of the test facility Eurofins Munich.

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

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)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: chromosome aberration assay

Specific details on test material used for the study:

Name: Phosphoric acid, C14-15 branched and linear alkyl esters, potassium salts
CAS No.: 1893414-79-3
Physical state: white solid at 20 °C
Batch No.: PU61810016
Re-certification date of batch: 09 March 2018
Purity: 100 % (UVCB, lyophilized solid, water content 0.85 % (w/w))
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Target gene:

Induction of structural chromosome aberrations in Chinese hamster V79 cells.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Cell bank of Eurofins Munich

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

A pre-experiment was conducted under identical conditions as described for the main experiment. The following concentrations were tested without and with S mix: 5, 10, 20, 50, 100, 200, 500, 1000, 2000 and 5000 µg/mL
Cytotoxicity was characterised by the percentages of the relative increase in cell count (RICC) in comparison with the controls.

On the basis of the data and the observations from the pre-experiment and taking into account the recommendations of the guidelines, the following concentrations were selected for the main experiments I and II. The dose group selection for microscopic analyses of chromosomal aberrations was based in accordance with the recommendations of the guidelines.

Concentrations in µg/mL
Experiment I
-S9 Exp. 4h:
2 5 10 25 50 100(P) 200(P)
+S9 Exp. 4h:
2 5 10 25 50 100(P) 200(P)

Experiment II

-S9 Exp. 21h:
2 5 10 25 50 100 200(P)

(P) = Precipitation was observed at the end of treatment

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data

When all of these criteria are met, the test chemical is then considered able to induce chromosomal aberrations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data.

The test chemical is then considered unable to induce chromosomal aberrations in cultured mammalian cells in this test system.

Statistics:

Statistical significance at the 5% level (p < 0.05) was evaluated by the Fischer´s exact test. The p value was used as a limit in judging for significance levels in comparison with the corresponding solvent control. Aberrant cells without gaps were only used for the calculation. Gaps are recorded separately and reported but generally not included in the total aberration frequency calculation according to the guideline.
Statistical significance at the 5% level (p < 0.05) was evaluated by the chi² test for trend. The p value was used as a limit in judging for significance levels.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

4 h treatment time

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation at concentrations of 100 µg/mL and higher

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Ethylmethanesulfonate

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

4 h treatment time

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation at concentrations of 100 µg/mL and higher

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Cyclophosphamide

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

21 h treatment time

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Ethylmethanesulfonate

 Pre-Experiment for Toxicity

According to the guidelines the highest recommended concentration was 5000 µg/mL. The test item was suspended in Aqua ad injectabilia. Precipitation of the test item was noted at concentrations of 100 µg/mL and higher after treatment. The relative increase in cell count (RICC) was used as parameter for toxicity. The concentrations evaluated in the main experiment were based on the results obtained in the pre-experiment.

Precipitation

The test item was suspended in Aqua ad injectabilia and added to cell culture medium. After treatment, precipitate of the test item was noted at 100 µg/mL without and with metabolic activation in experiment I. In experiment II after long-term treatment, precipitation was observed at a concentration of 200 µg/mL.

Toxicity

In experiment I without metabolic activation, a biologically relevant decrease of the relative increase in cell count (decrease below 70% RICC) was noted at 50 µg/mL (66% RICC), 100 µg/mL (26% RICC) and 200 µg/mL (10% RICC). In experiment I with metabolic activation and experiment II without metabolic activation, no biologically relevant decrease of the RICC was detected in all tested concentrations up to 200 µg/mL.

Clastogenicity

There are several criteria for determining a positive result, such as a concentration-related increase or a reproducible increase in the number of cells with chromosome aberrations for at least one of the dose groups, which is higher than the laboratory negative control range. In experiment I without metabolic activation the aberration rate of the negative control (1.7%), solvent control (2.3%) and all evaluated concentrations (10 µg/mL: 2.3%; 25 µg/mL: 1.7% and 50 µg/mL: 2.0%) were within the historical control data of the testing facility (0.0% – 4.0%). With metabolic activation, the aberration rates of the negative control (1.3%), solvent control (2.0%) and all dose groups treated with the test item (25 µg/mL and 50 µg/mL: 2.7%; 100 µg/mL: 2.0%) were within the historical control data of the testing facility (0.0% – 4.3%). In experiment II without metabolic activation the aberration rate of the negative control (1.7%),

solvent control (2.0%) as well as the test item concentrations 50 µg/mL (1.7%), 100 µg/mL (1.0%) and 200 µg/mL (1.3%) were within the historical control data of the testing facility (0.0% – 3.0%). The number of aberrant cells found in the experiments I and II did not show a biologically relevant increase compared to the corresponding negative control. In addition, no dose-response relationship was observed. The Fisher´s exact test was performed to verify the results in the experiment. No statistically significant increase (p < 0.05) of cells with chromosomal aberrations was noted in the dose groups of the test item evaluated in experiment I and II without and with metabolic activation.

The chi² Test for trend was performed to test whether there is a concentration-related increase in chromosomal aberrations. No statistically significant increase was observed in experiment I without and with metabolic activation and in experiment II without metabolic activation. EMS (400 and 600 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induceddistinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the ability of the test system to indicate potential clastogenic effects.

Polyploid Cells

Table below show the number of polyploid metaphases. No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.

Tables:

Experiment I (4 h) - Summary of Cytotoxicity Data without metabolic activation

Dose Group	Concentration [µg/mL]	Polyploid Cells 1	Polyploid Cells 2	Polyploid Cells mean	Cell count C/mL [*10^3] culture 1	Cell count C/mL [*10^3] culture 2	Cell count C/mL [*10^3] culture mean	RICC [%]	Precipitate (+/-)
C	0	0	0	0.0	109.36	108.43	108.9	97	-
S	0	2	0	1.0	117.60	106.58	112.09	100	-
1	2	n.d.	n.d.	-	103.25	102.88	103.07	91	-
2	5	n.d.	n.d.	-	114.08	106.86	110.47	98	-
3	10	0	0	0.0	95.29	105.29	100.29	88	-
4	25	0	0	0.0	101.21	97.79	99.5	88	-
5	50	0	0	0.0	80.93	73.62	77.28	66	-
6	100	n.d.	n.d.	-	45.37	28.43	36.90	26	+
7	200	n.d.	n.d.	-	14.26	25.46	19.86	10	+
EMS	600	0	0	0.0	92.97	88.99	90.98	79	-

300 cells evaluated for each concentration, except for the positive controls EMS (235 cells) and CPA (220 cells).

Experiment I (4h) - Summary of Cytotoxicity Data with metabolic activation

Dose Group	Concentration [µg/mL]	Polyploid Cells 1	Polyploid Cells 2	Polyploid Cells mean	Cell count C/mL [*10^3] culture 1	Cell count C/mL [*10^3] culture 2	Cell count C/mL [*10^3] culture mean	RICC [%]	Precipitate (+/-)
C	0	3	0	1.5	110.26	99.17	104.72	101	-
S	0	1	0	0.5	111.40	95.56	103.48	100	-
1	2	n.d.	n.d.	-	108.14	96.09	102.12	99	-
2	5	n.d.	n.d.	-	101.99	98.29	100.14	99	-
3	10	n.d.	n.d.	-	103.04	98.38	100.71	97	-
4	25	2	0	1.0	103.39	89.49	96.44	92	-
5	50	0	0	0.0	113.16	117.56	115.36	113	-
6	100	0	1	0.5	105.77	109.90	107.84	105	+
7	200	n.d.	n.d.	-	107.88	120.99	114.44	112	+
CPA	0.83	1	0	0.5	92.04	88.26	90.15	86	-

300 cells evaluated for each concentration, except for the positive controls EMS (235 cells) and CPA (220 cells).

Experiment II (21 h) - Summary of Cytotoxicity Data without metabolic activation

Dose Group	Concentration [µg/mL]	Polyploid Cells 1	Polyploid Cells 2	Polyploid Cells mean	Cell count C/mL [*10^3] culture 1	Cell count C/mL [*10^3] culture 2	Cell count C/mL [*10^3] culture mean	RICC [%]	Precipitate (+/-)
C	0	0	2	1.0	226.01	199.90	212.96	100	-
S	0	1	0	0.5	209.52	217.88	213.70	100	-
1	2	0	0	0.0	219.83	235.92	227.88	107	-
2	5	0	0	0.0	269.52	219.71	244.62	115	-
3	10	0	0	0.0	205.62	217.86	211.74	99	-
4	25	0	0	0.0	202.45	196.53	199.49	93	-
5	50	0	0	0.0	215.68	214.66	215.17	101	-
6	100	1	0	0.5	208.35	224.39	216.37	101	-
7	200	0	0	0.0	188.78	236.01	212.40	99	+
EMS	400	1	0	0.0	172.94	164.90	168.92	78	-

The number of polyploid cells was determined in 150 cells per culture of each test group, except for the positive control EMS (175 cells).

RICC: Relative Increase in Cell Count, calculated by the increase in cell number of the test groups compared to the

solvent control groups. The cell count was determined by a cell counter per culture for each test group.

Experiment I (4h) – Summary of Aberration Rates without metabolic activation

Dose Group	Concentration [µg/mL]	Treatment Time	Fixation Interval	mean % aberrant cells - incl.Gaps	mean % aberrant cells - excl. Gaps	Precipitation
C	0	4	21	3.3	1.7	-
S	0	4	21	5.0	2.3	-
3	10	4	21	3.7	2.3	-
4	25	4	21	3.0	1.7	-
5	50	4	21	2.7	2.0	-
EMS	600	4	21	8.5	7.2	-

Experiment I (4h) – Summary of Aberration Rates with metabolic activation

Dose Group	Concentration [µg/mL]	Treatment Time	Fixation Interval	mean % aberrant cells - incl.Gaps	mean % aberrant cells - excl. Gaps	Precipitation
C	0	4	21	3.0	1.3	-
S	0	4	21	5.3	2.0	-
3	25	4	21	6.7	2.7	-
4	50	4	21	5.7	2.7	-
5	100	4	21	2.7	2.0	+
CPA	600	4	21	15.9	12.3	-

Experiment II (21h) – Summary of Aberration Rates without metabolic activation

Dose Group	Concentration [µg/mL]	Treatment Time	Fixation Interval	mean % aberrant cells - incl.Gaps	mean % aberrant cells - excl. Gaps	Precipitation
C	0	21	21	3.0	1.7	-
S	0	21	21	3.0	2.0	-
5	50	21	21	2.7	1.7	-
6	100	21	21	2.0	1.0	-
7	200	21	21	1.7	1.3	+
EMS	400	21	21	17.7	14.3	-

300 cells evaluated for each concentration, except for the positive control EMS (175 cells).

C: Negative Control (Culture Medium)

S: Solvent Control (Aqua ad injectabilia)

EMS: Positive Control (without metabolic activation: Ethylmethanesulfonate)

CPA: Positive Control (with metabolic activation: Cyclophosphamide)

Biometry

Statistical significance at the 5% level (p < 0.05) was evaluated by the Fischer´s exact test. The p value was used as a limit in judging for significance levels in comparison with the corresponding solvent control. Aberrant cells without gaps were only used for the calculation. Gaps are recorded separately and reported but generally not included in the total aberration frequency calculation according to the guideline.

Biometry - Experiment I (4h) without metabolic activation

Solvent Control versus Test Group	Concentration [µg/mL]	Treatment Time [h]	Aberrant Cells (excl. gap)	Significance	p Value
C	0	4	5	-	0.7721
S	0	4	7	/	/
3	10	4	7	-	1.0000
4	25	4	5	-	0 .7721
5	50	4	6	-	1.0000
EMS	600	4	17	+	0.0102

Biometry - Experiment I (4h) with metabolic activation

Solvent Control versus Test Group	Concentration [µg/mL]	Treatment Time [h]	Aberrant Cells (excl. gap)	Significance	p Value
C	0	4	4	-	0.7518
S	0	4	6	/	/
4	25	4	8	-	0.7880
5	50	4	8	-	0 .7880
6	100	4	6	-	1.0000
CPA	0.83	4	27	+	< 0.0001

Biometry - Experiment II (21h) without metabolic activation

Solvent Control versus Test Group	Concentration [µg/mL]	Treatment Time [h]	Aberrant Cells (excl. gap)	Significance	p Value
C	0	21	5	-	1.0000
S	0	21	6	/	/
4	50	21	5	-	1.0000
5	100	21	3	-	0 .5045
6	200	21	4	-	0.7518
CPA	0.83	21	25	+	< 0.0001

Statistical significance at the 5% level (p < 0.05) was evaluated by the chi² test for trend. The p value was used as a limit in judging for significance levels.

Biometry – Trend test

Experiment	Treatment Time [h]	Significance	p Value
Exp. I without metabolic activation	4	-	0.7706
Exp. I with metabolic activation	4	-	0.5970
Exp. II without metabolic activation	21	-	0.7219

+: significant

-: not significant

Conclusions:

In conclusion, it can be stated that during the described in vitro chromosome aberration test and under the experimental conditions reported, the test item Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Therefore, the test item Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts is considered to be non-clastogenic in this chromosome aberration test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available