Phosphoric acid, C12-18-alkyl esters, potassium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of the full set of in-vitro genotoxicity tests required by REACH regulation including read-across information form structural related source substances, Phosphoric acid, C12-18-alkyl esters, potassium salts does not need to be classified for germ cell mutagenicity according to CLP, EU GHS (Regulation (EC) No 1272/2008).

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:

2017-07-07 to 2017-07-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(adopted 1997)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus

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:

rat liver S9-mix

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II:
TA 98: 3; 10; 33; 100; 333; 1000; and 2500 µg/plate
TA 100: 0.3; 1; 3; 10; 33; 100; 333; and 1000 µg/plate
all remaining strains: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Precipitation was observed from 333 to 5000 µg/plate in Experiment I and from 1000 to 5000 µg/plate in Experiment II.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD -TA 1537 and TA 98; 2-aminoanthracene, 2-AA With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) experiment I and the pre-incubation test (experiment II)
- Cell density at seeding (if applicable): (10E8-10E9 cells/mL).

DURATION
- Preincubation period: 60 min at 37 °C
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: three plates/strain/concentration

Evaluation criteria:

The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce an increase above the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle 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

Vehicle 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

Vehicle 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

Vehicle 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:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Precipitation: yes
The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate in Experiment I and from 1000 to 5000 µg/plate in Experiment II.

HISTORICAL CONTROL DATA
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:

Strain without S9 mix with S9 mix
Mean SD Min Max Mean SD Min Max
Solvent control 12 2.5 6 25 12 2.5 7 26
TA 1535 Untreated control 12 3.1 6 28 12 2.9 7 26
Positive control 1130 143.1 334 1816 388 58.2 176 668

Solvent control 10 2.2 6 19 13 3.5 7 30
TA1537 Untreated control 11 2.7 5 21 14 4.0 7 31
Positive control 82 12.7 43 157 191 60.8 83 434

Solvent control 25 4.4 13 43 34 6.2 15 58
TA 98 Untreated control 27 4.9 12 43 37 6.5 11 57
Positive control 378 73.7 211 627 3949 771.8 360 6586

Solvent control 156 26.0 78 209 148 32.3 73 208
TA 100 Untreated control 176 23.6 79 217 172 25.4 85 218
Positive control 1966 293.2 498 2767 3798 830.4 536 6076

Solvent control 41 5.6 27 63 50 6.8 28 72
WP2 uvrA Untreated control 42 5.8 30 63 52 6.8 36 88
Positive control 798 362.7 319 4732 378 112.6 167 1265

Mean = mean value of revertants/plate
SD = standard deviation
Min = minimal value/Max = maximal value

In Experiment II in strain TA 1537 with S9 mix the positive control did not reach the lower limit of the historical control data. Since the deviation is rather small (82±8 colonies vs. 83) and the threshold of thrice the number the corresponding solvent control was exceeded by far (induction factor of 8.2) the test was considered to be valid.

Conclusions:

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Phosphoric acid, C12-18 alkyl esters, potassium salts at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix).
Phosphoric acid, C12-18 alkyl esters, potassium salts is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997), Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA were exposed to Phosphoric acid, C12-18 alkyl esters, potassium salts in water at the following concentrations in the absence and presence of mammalian metabolic activation (rat liver S9 mix):

Experiment I             3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:      

TA 98:                     3; 10; 33; 100; 333; 1000; and 2500 µg/plate
TA 100:                   0.3; 1; 3; 10; 33; 100; 333; and 1000 µg/plate
all remaining strains:  10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment I was conducted using the plate incorporation method, experiment II using the pre-incubation method.

The test substance was tested up to cytotoxic concentrations. Toxic effects were observed in all strains with and without metabolic activation, based on reduced number of spontaneous revertants.

The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate in Experiment I and from 1000 to 5000 µg/plate in Experiment II.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Phosphoric acid, C12-18 alkyl esters, potassium salts at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, Phosphoric acid, C12-18 alkyl esters, potassium salts is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 2

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:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to attached document

Reason / purpose for cross-reference:

read-across source

Key result

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 examined

Positive controls validity:

valid

Conclusions:

Available data from Phosphoric acid, dodecyl ester, potassium salt were used to evaluate the clastogenic potential of Phosphoric acid, C12-18-alkyl esters, potassium salts. Phosphoric acid, dodecyl ester, potassium salt is considered to be non-clastogenic in a chromosome aberration test according to OECD guideline 473.
Based on a read-across approach there is no evidence of a clastogenic potential for Phosphoric acid, C12-18-alkyl esters, potassium salts based on information from Phosphoric acid, dodecyl ester, potassium salt.

Reference 3

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:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to attached document

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Available data from an OECD 476 guideline study with Phosphoric acid, dodecyl ester, potassium salt were used to evaluate the mutagenicity in mammalian cells of Phosphoric acid, C12-18-alkyl esters, potassium salts. Phosphoric acid, dodecyl ester, potassium salt is considered to be non-mutagenic in HPRT locus using V79 cells of the Chinese Hamster.
Based on a read-across data there is no evidence of a mutagenic potential in mammalian cells for Phosphoric acid, C12-18-alkyl esters, potassium salts based on information from Phosphoric acid, dodecyl ester, potassium salt.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In-vitro studies

Gene mutation in bacteria

Phosphoric acid, C12-18 alkyl esters, potassium salts

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997), Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA were exposed to Phosphoric acid, C12-18 alkyl esters, potassium salts in water at the following concentrations in the absence and presence of mammalian metabolic activation (rat liver S9 mix):

 

Experiment I             3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:      

TA 98:                       3; 10; 33; 100; 333; 1000; and 2500 µg/plate

TA 100:                     0.3; 1; 3; 10; 33; 100; 333; and 1000 µg/plate

all remaining strains:  10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

 

Experiment I was conducted using the plate incorporation method, experiment II using the pre-incubation method. The test substance was tested up to cytotoxic concentrations. Toxic effects were observed in all strains with and without metabolic activation, based on reduced number of spontaneous revertants. The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate in Experiment I and from 1000 to 5000 µg/plate in Experiment II. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Phosphoric acid, C12-18 alkyl esters, potassium salts at any dose level, neither in the presence nor absence of metabolic activation (S9 mix).

 

Phosphoric acid, C12-18 alkyl esters, potassium salts is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

 

Phosphoric acid, dodecyl ester, potassium salt

In a reverse gene mutation assay in bacteria according to OECD guideline 471 the potential of Phosphoric acid, dodecyl ester, potassium salt to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA was evaluated.

 

The assay was performed in two independent experiments both with and without liver microsomal activation at concentrations up to 5000 µg/plate. Toxic effects occurred in all strains at higher concentrations.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with at any dose level, neither in the presence nor absence of metabolic activation. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Mammalian cell gene mutation assay

Phosphoric acid, dodecyl ester, potassium salt

In an in vitro gene mutation study in mammalian cells according to OECD guideline 476 Phosphoric acid, dodecyl ester, potassium salt was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster. Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as 20 h long time exposure assay.

 

The test item was investigated at the following concentrations:

Experiment I

without metabolic activation: 50, 100,250,500,750, 1000, 1250, 1500 and 2000 µg/ml,

with metabolic activation: 50, 100,250,500, 750, 1000, 1250 and 1500 µg/ml.

Experiment II

without metabolic activation: 7.5,10,25,50,75,100,125,150 and 175 µg/ml,

with metabolic activation: 150,200,300,600,800, 1000, 1200 and 1400 μg/mL

 

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed.

DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

 

In conclusion, under the experimental conditions reported, Phosphoric acid, dodecyl ester, potassium salt is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Chromosome Aberration:

Phosphoric acid, dodecyl ester, potassium salt

In an in vitro chromosome aberration assay, Phosphoric acid, dodecyl ester, potassium salt was investigated for the potential to induce structural chromosomal aberrations in Chinese hamster V79 cells in the absence and presence of metabolic activation with S9 homogenate.

The chromosomes were prepared 20 h after start of treatment with the test item. The treatment intervals were 4 h with and without metabolic activation (experiment I) and 4 h with and 20 h without metabolic activation (experiment II). Two parallel cultures were set up. At least 200 metaphases per concentration were scored for structural chromosomal aberrations. 

 

The following concentrations were selected for microscopic analysis:

Experiment I:

without metabolic activation: 15 .6, 250, 500 and 1000 µg/mL

with metabolic activation: 15.6, 500, 1000 and 1500 µg/mL

Experiment II:

without metabolic activation: 15.6, 31.3, 62.5 and 125.0 µg/mL

with metabolic activation: 900, 1600 and 1800 µg/mL

 

Precipitation of the test item was observed with and without metabolic activation in both experiments.

Toxic effects of the test item were observed in experiment I without metabolic activation at concentrations of 500 µg/mL and higher, with metabolic activation at concentrations of 1000 µg/mL and higher. In experiment II (long time exposure) without metabolic activation toxic effects of the test item were observed at concentrations of 62.5 µg/mL and higher, with metabolic activation at concentrations of 1600 µg/mL and higher.

 

In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test item with and without metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.

No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item in neither of the experiments.

Positive controls induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.

 

Phosphoric acid, dodecyl ester, potassium salt is considered to be non-clastogenic in this chromosome aberration test.

 

Justification for read-across

Evaluation of structure-activity relationship is based on data from structural similar substances with the same basic structure, salts of phosphoric acid alkyl ester, varying mainly in the chain length of fatty acid moiety. A detailed justification document for the read-across is attached in the respective target records of IUCID.

Conclusion

Negative data from gene mutation studies in bacteria were available for the target substance Phosphoric acid, C12-18 alkyl esters, potassium salts and the source substancePhosphoric acid, dodecyl ester, potassium salt.

Information from in-vitro mammalian cell gene mutation assays fromPhosphoric acid, dodecyl ester, potassium salt, indicates no mutagenic potential. 

Further data from in-vitro chromosome aberration assay are available for Phosphoric acid, dodecyl ester, potassium salt, no statistically significant increase in aberration frequency was noted.

 

Based on the results of the full set of in-vitro genotoxicity tests required by REACH regulation, including those form the structurally closely related read-across substances Phosphoric acid, dodecyl ester, potassium salt,Phosphoric acid, C12-18 alkyl esters, potassium saltsis considered to be non-mutagenic in mammalian cells.

Justification for classification or non-classification

Based on the results of the full set of in-vitro genotoxicity tests required by REACH regulation including read-across information form a structural related source substance, Phosphoric acid, C12-18-alkyl esters, potassium salts does not need to be classified for germ cell mutagenicity according to CLP, EU GHS (Regulation (EC) No 1272/2008).