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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
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

Test material

Constituent 1
Reference substance name:
Phosphoric acid, C12-18-alkyl esters, potassium salts
EC Number:
291-905-1
EC Name:
Phosphoric acid, C12-18-alkyl esters, potassium salts
Cas Number:
90506-43-7
Molecular formula:
Not applicable for UVCB substance
IUPAC Name:
Phosphoric acid, C12-18-alkyl esters, potassium salts
Test material form:
solid - liquid: suspension

Method

Target gene:
Histidine locus
Species / strain
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.
Controls
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.

Results and discussion

Test resultsopen allclose all
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


Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.

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