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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
As per ECHA Decision number: CCH-D-2114449848-30-01/F

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of p-t-butylphenyldiphenyl phosphate and bis(p-t-butylphenyl) phenyl phosphate
EC Number:
939-505-4
Molecular formula:
vary
IUPAC Name:
Reaction mass of p-t-butylphenyldiphenyl phosphate and bis(p-t-butylphenyl) phenyl phosphate
Test material form:
liquid: viscous
Specific details on test material used for the study:
Physical State: liquid
Colour: yellowish
Density: 1.16 - 1.18 g/mL (25 °C)
Active Components: 100%

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:
With metabolic activation:
Tester Strains: S. typhimurium: TA98, TA100, TA1535, TA1537 and TA102
Name: 2-AA; 2-aminoanthracene
CAS No.: 613-13-8
Supplier: Aldrich
Batch No.: STBG0630V
Dissolved in: DMSO
Concentrations: 2.5 µg/plate; 10 µg/plate for TA102

1.1.1. S9 Homogenate
The S9 liver microsomal fraction was prepared at Eurofins Munich. 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.
The following quality control determinations are performed:
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 (Lot: 220219) was 43 mg/mL and was adjusted to 30 mg/mL.

1.1.2. Preparation of S9 Mix
The S9 mix preparation was performed according to Ames et al. [7].
100 mM of sodium-ortho-phosphate-buffer, pH 7.4, was ice-cold added to the following pre-weighed sterilised reagents to give final concentrations in the S9 mix of:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
This solution was mixed with the liver 9000 x g supernatant fluid in the following proportion:
co-factor solution 9.5 parts
liver preparation 0.5 parts
During the experiment the S9 mix was stored on ice.

1.1.3. S9 Mix Substitution Buffer
The S9 mix substitution buffer was used in the study as a replacement for S9 mix, without metabolic activation (-S9).
Phosphate-buffer (0.2 M) contains per litre of purified water:
0.2 M NaH2PO4 x H2O 120 mL
0.2 M Na2HPO4 880 mL
The two solutions were mixed and the pH was adjusted to 7.4. Sterilisation was performed for 20 min at 121 °C in an autoclave.
This 0.2 M phosphate-buffer was mixed with 0.15 M KCl solution (sterile) in the following proportion:
0.2 M phosphate-buffer 9.5 parts
0.15 M KCl solution 0.5 parts
This S9 mix substitution buffer was stored at 4 °C.

Test concentrations with justification for top dose:
Exposure Concentrations
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:
Experiment I (strains TA 98, TA 100):
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate
Experiment I (strains TA 1535, TA 1537, TA 102):
31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Experiment II (all tester strains):
31.6, 100, 316, 1000, 2500 and 5000 µg/plate
As the results of the pre-experiment were in accordance with the criteria described above, these were reported as a part of the main experiment I.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
10.5.3. Experimental Performance
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 µL Overlay agar.
For the pre-incubation method 100 µL of the test item preparation was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
For each strain and dose level, including the controls, three plates (in a few cases only two plates were evaluated, see tables experiment II) were used.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.
Evaluation criteria:
Criteria of Validity
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100, TA102)
- the negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (2016 -2018)):

- S9 + S9
min max min max
TA98 11 58 15 58
TA100 55 155 60 155
TA1535 4 41 3 38
TA1537 3 35 3 34
TA102 142 453 157 547
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.

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 TA98, TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control [11].

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.
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:
no 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:
no 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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no 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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, tBuTPP low TPP did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, tBuTPP low TPP is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

   Summary

In order to investigate the potential of tBuTPP low TPP for its ability to induce gene mutations the plate incorporation test (experiment I) and the pre-incubation test (experiment II) were performed with theSalmonella typhimuriumstrains TA98, TA100, TA1535, TA1537 and TA102.

In two independent experiments several concentrations of the test item were used. Each assay was conductedwithandwithoutmetabolic 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 (strains TA 98, TA 100):

0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate

Experiment I (strains TA 1535, TA 1537, TA 102):

31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Experiment II (all tester strains):

31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Due to a transmission error the experimental design, the pre-experiment was performed with a maximum concentration of 5.0 µL/plate, the remaining experiments were performed with a maximum concentration of 5 mg/plate. Since both options are covered by the OECD 471 guideline, this procedure has no influence on the quality and integrity of the results.

No precipitation of the test item was observed in any tester strain used in experiment I and II (withandwithoutmetabolic activation).

No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (withandwithoutmetabolic activation) in experiment I and II.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with tBuTPP low TPP at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

All criteria of validity were met