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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:
June - August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Phosphoric acid, mixed esters with alcohols, C7-9-iso-, C8-rich and alcohols, C11-14-iso-, C13-rich, ethoxylated, potassium salt
EC Number:
947-738-8
Molecular formula:
Molecular formulas of exemplary components: C33H68KO10P C49H100KO16P C24H49K2O10P C24H50KO10P C16H34KO4P C8H17K2O4P C8H18KO4P
IUPAC Name:
Phosphoric acid, mixed esters with alcohols, C7-9-iso-, C8-rich and alcohols, C11-14-iso-, C13-rich, ethoxylated, potassium salt
Test material form:
liquid

Method

Species / strain
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:
S9 liver microsomal fraction
Test concentrations with justification for top dose:
Pre-experiment
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

Main experiment
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5000 µg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed with the following concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

All concentrations used - per active ingredient (90%, conversion factor 1.1)
Vehicle / solvent:
Aqua destillata
Controls
Untreated negative controls:
yes
Remarks:
A. dest., Eurofins Munich, Lot No. 170619, 170627,170710
Negative solvent / vehicle controls:
yes
Remarks:
A. dest., Eurofins Munich, Lot No. 170619, 170627,170710
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 1. 4-nitro-o-phenylene-diamine; 2. 2-aminoanthracene
Details on test system and experimental conditions:
Bacteria
Five strains of S. typhimurium with the following characteristics were used:
TA 98: his D 3052; rfa-; uvrB-; R-factor: frame shift mutations
TA 100: his G 46; rfa-; uvrB-; R-factor: base-pair substitutions
TA 1535: his G 46; rfa-; uvrB-: base-pair substitutions
TA 1537: his C 3076; rfa-; uvrB-: frame shift mutations
TA 102: his G 428 (pAQ1); rfa-; R-factor: base-pair substitutions

Tester strains TA 98, TA 1535 and TA 102 were obtained from MOLTOX, INC., NC 28607, USA. Tester strains TA 100 and TA 1537 were obtained from Xenometrix AG, Switzerland. They were stored as stock cultures in ampoules with nutrient broth (OXOID) supplemented with DMSO (approx. 8% v/v) over liquid nitrogen.

Samples of each tester strain were grown by culturing for 12 h at 37 °C in Nutrient Broth to the late exponential or early stationary phase of growth (approx. 109 cells/mL). A solution of 125 µL ampicillin (10 mg/mL) (TA 98, TA 100, TA 102) was added in order to retain the phenotypic characteristics of the strain.

S9 Homogenate
The S9 liver microsomal fraction was prepared at Eurofins Munich and obtained from Trinova Biochem GmbH, Giessen, Germany. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route (Eurofins Munich) and male Sprague Dawley rats were induced with phenobarbital / β-naphthoflavone (Trinova).

The following quality control determinations were performed by Eurofins Munich:
a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene
b) Sterility Test
A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4 mL and stored at <= -75 °C.
The protein concentration in the S9 preparation was 34.2 mg/mL.

The following quality control determinations were performed by Trinova Biochem GmbH:
a) Alkoxyresorufin-0-dealkylase activities
b) Test for the presence of adventitious agents
c) Promutagen activation (including biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene)
A stock of the supernatant containing the microsomes is frozen in aliquots of 5 mL and stored at <= -75 °C.
The protein concentration in the S9 preparation was 33.7 mg/mL.
Evaluation criteria:
Evaluation of cytotoxicity
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "N" or "B", respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

Evaluation of mutagenicity
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
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.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
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.

Results and discussion

Test resultsopen allclose all
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
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
During the described mutagenicity test and under the experimental conditions reported, the substance did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, it is considered to be non-mutagenic in this bacterial reverse mutation assay.

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