Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

3 In vitro Genetic Toxicity studies were performed on the registered substance.

1. Reverse Mutation Assay (AMES)-

In order to investigate the potential of tBuTPP low TPP to induce gene mutations in bacteria cells, with and without metabolic activation, using Salmonella typhimuriumstrains TA98, TA100, TA1535, TA1537 and TA102. The study was conducted according to OECD testing guideline 471 and following GLP.

Results:

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic 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 (with and without metabolic 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.

In conclusion, 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.

2.      In vitroMammalian Micronucleus Assay in Human Lymphocytes –

 In order to investigate the potential of tBuTPP low TPP to induce micronuclei in human lymphocytes with and without metabolic activation. The study was conducted according to OECD testing guideline 487 and following GLP.

Results:

 tBuTPP low TPP did not induce structural and/or numerical chromosomal damage in human lymphocytes in both short term exposure (4 hours) and long term exposure (44 hours). Therefore, tBuTPP low TPP is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the in vitro Mammalian Cell Micronucleus Test.

3.      In vitroMammalian Cell Gene Mutation (HPRT locus) in Chineese Hamster V79 cells-

The test item tBuTPP low TPP was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster with and without metabolic activation. The study was conducted according to OECD testing guideline 476 and following GLP.

Results:

In the experiments no biologically relevant increase of mutants was found after treatment with the test item (withoutandwithmetabolic activation). Most of the mutant values are within the historical data base of the test facility.

 

 

Link to relevant study records

Referenceopen allclose all

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
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
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%
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.
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.
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
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

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EU Method B.49 "In vitro Mammalian cell Micronucleus assay", 2017
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: tBuTPP low TPP
Chemical Name: Reaction mass of p-t-butylphenyldiphenyl phosphate and bis(p-t-butylphenyl) phenyl phosphate
Batch No.: 18 235 H 0299
Molecular Weight:approx. 382 g/mol
Physical State:liquid
Colour:yellowish
Density:1.16 - 1.18 g/mL (25 °C)
Active Components:100%
Expiry Date: 28 August 2020
Storage Conditions: room temperature, protected from light
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Human peripheral blood lymphocytes from healthy and non-smoking donors
Cytokinesis block (if used):
Cytokinesis Block Proliferation Index
As an assessment of the cytotoxicity, a cytokinesis block proliferation index (CBPI) was determined from 500 cells according to the following formula:
CBPI= (c1 x 1) + (c2 x 2) + (cx x 3)
n
c1: mononucleate cells
c2: binucleate cells
cx: multinucleate cells
n: total number of cells

The CBPI was used to calculate the % cytostasis, which indicates the inhibition of cell growth of treated cultures in comparison to control cultures:
% Cytostasis= 100 – 100 x ((CBPIT – 1) / (CBPIC – 1))

CBPIT: Cytokinesis Block proliferation index of treated cultures
CBPIC: Cytokinesis Block proliferation index of control cultures
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Homogenate
Metabolic activation of substances can be achieved by supplementing the cell cultures with liver microsome preparations (S9 mix).
The S9 liver microsomal fraction was prepared at Eurofins Munich GmbH. 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 preparation was performed according to Ames et al. 1997.
The following quality control determinations were 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.

S9 Mix
An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the concentrations below:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-phosphate-buffer pH 7.4. During the experiment the S9 mix was stored on ice.
The final concentration of S9 mix in the cultures was 5%.
Test concentrations with justification for top dose:
Exposure Concentrations
Duplicate cultures were treated at each concentration. The selection of the concentrations used in experiment I and II based on data from the pre-experiment (below). The following concentrations were used in the main experiments:
Experiment I:
without metabolic activation: 10, 25, 50, 60, 80, 100, 125, 150 and 200 µg/mL
with metabolic activation: 50, 100, 200, 250, 300, 350, 400, 450 and 500 µg/mL
Experiment II:
without metabolic activation: 5, 10, 25, 30, 35, 40, 45, 50, 75 and 100 µg/mL
The following concentrations were selected in the main experiment for the microscopic analyses. The selection was based on cytotoxicity for all experimental conditions.
Experiment I with short-term exposure (4 h):
without metabolic activation: 25, 50 and 60 µg/mL
with metabolic activation: 50, 100 and 200 µg/mL
Experiment II with long-term exposure (44 h):
without metabolic activation: 30, 40, 45 and 50 µg/mL

Pre-Experiment for Toxicity
A pre-experiment was conducted under identical conditions as described for the main experiment I (4 h incubation) in order to determine the toxicity of the test item. The CBPI was used for the quantification of cytotoxicity. The following concentrations were tested without and with S9 mix:
7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2500 and 5000 µg/mL
The concentration of 5000 µg/mL was considered to be the highest test concentration used in this test system following the recommendation of the corresponding OECD testing guideline 487


Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
colchicine
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
Preparation of the Test Item
A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 5000 µg/mL.
The test item was suspended in cell culture medium (RPMI) within 1 hour prior to treatment.
After an ultrasonication treatment for 10 min at 37°C a stable suspension was obtained. The solvent was compatible with the survival of the cells and the S9 activity.
For the maximum concentration without metabolic activation the osmolality (in comparison to negative (solvent) control) and pH value were determined:

Concentration (µg/mL) Osmolality (mOsm/kg) pH value
Exp. I Negative control - 287 -
Test item 200 286 7.4
Exp. II Negative control - 291 -
Test item 100 291 7.4


Experimental Design
Pre-Experiment for Toxicity
A pre-experiment was conducted under identical conditions as described for the main experiment I (4 h incubation) in order to determine the toxicity of the test item. The CBPI was used for the quantification of cytotoxicity. The following concentrations were tested without and with S9 mix: 7.8, 15.6, 31.3, 62.5,
125, 250, 500, 1000, 2500 and 5000 µg/mL The concentration of 5000 µg/mL was considered to be the highest test concentration used in this test system following the recommendation of the corresponding OECD testing guideline 487.

Experimental Performance
Experiment I
Whole blood samples were treated with anti-coagulant (Heparin) and were pre-cultured (44 to 48 h) in presence of PHA prior to exposure to the test item. It is recommended to test human lymphocytes 44 to 48 h after PHA stimulation, when the cell cycle synchronisation is disappeared. The lymphocytes were incubated with the test item for 4 h in presence or absence of metabolic activation. At the end of the incubation, the treatment medium was removed and the cells were washed twice with PBS + 10% FBS. Subsequently the cells were incubated in complete culture medium + 6 µg/mL cytochalasin B for 40 h to 42 h at 37°C and 5% CO2 [9].

Experiment II
If negative or equivocal results are obtained, they should be confirmed using continuous treatment (long-term treatment) without metabolic activation. The whole blood cultures were pre-cultured in the presence of PHA for 44 to 48 h prior to exposure to the test item. Then the test item was added in complete culture medium. 1 h later 6 µg/mL cytochalasin B were added and the cells were incubated for further 43 h at 37 °C. At the end of the treatment the cell culture medium was removed and the cells were prepared for microscopic analysis.

Number of Cultures
Duplicate cultures were performed at each concentration level except for the pre-experiment.
Preparation of the Cultures
At the end of the cultivation, the complete culture medium was removed. Subsequently, the cells were treated with cold hypotonic solution (0.075 M KCl) for some minutes at room temperature and immediately centrifuged. The pellet was resuspended with a solution consisted of fixation solution + NaCl 0.9% (1+1) and centrifuged. After that the cells were fixed with methanol + glacial acetic acid (3+1). The cells were resuspended gently and the suspension was dropped onto clean glass slides. Consecutively, the cells were dried on a heating plate. The cells were stained with acridine orange solution.

Analysis of Micronuclei
All slides, including those of positive and negative controls were independently coded before microscopic analysis. For each dose group at least 2000 binucleated cells (if possible) per concentration (1000 binucleated cells per slide) were analysed for micronuclei according to the criteria of Fenech [7], i.e. clearly surrounded by a nuclear membrane, having an area of less than one-third of that of the main nucleus, being located within the cytoplasm of the cell and not linked to the main nucleus via nucleoplasmic bridges. Mononucleated and multinucleated cells and cells with more than six micronuclei were not considered [8].
Rationale for test conditions:
As per relevant testing guidleines
Evaluation criteria:
Acceptability of the Assay
A mutation assay is considered acceptable if it meets the following criteria:
- The concurrent negative/solvent control is considered acceptable for addition to the laboratory historical negative/solvent control database.
- Concurrent positive controls should induce responses that are compatible with those generated in the laboratory’s historical positive control data base and produce a statistically significant increase compared with the concurrent negative/solvent control.
- Cell proliferation criteria in the negative/solvent control should be fulfilled.
- All experimental conditions are tested unless one resulted in positive results.
- Adequate number of cells and concentrations are analysable.
- Criteria for the selection of top concentration are fulfilled.

Evaluation of Results
A test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative/solvent control
- the increase is concentration-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical negative/solvent control data (e.g. Poisson-based 95% control limits).
When all of these criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
A test item is considered to be clearly negative if in all experimental conditions examined none of the criteria mentioned above are met.



Key result
Species / strain:
lymphocytes: Human peripheral blood lymphocytes from healthy and non-smoking donors
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
experiment I (short exposure 4 hours)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
59% only at top concentration 60ug/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: ocytes from healthy and non-smoking donors
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
experiment I (short exposure 4 hours
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: ocytes from healthy and non-smoking donors
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
experiment II (long trerm exposure 44 hours)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
56% only at top concentration 50ug/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: ocytes from healthy and non-smoking donors
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
experiment II (long trerm exposure 44 hours)
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item tBuTPP low TPP did not induce structural and/or numerical chromosomal damage in human lymphocytes.
Therefore, tBuTPP low TPP is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the in vitro Mammalian Cell Micronucleus Test.
Executive summary:

Summary

In order to investigate a possible potential of tBuTPP low TPP to inducemicronuclei in human lymphocytesanin vitromicronucleus assay was carried out. The following study design was performed:

 

Without S9

With S9

Exp. I

Exp. II

Exp. I

Exposure period

4 h

44 h

4 h

Cytochalasin B exposure

40 h

43 h

40 h

Preparation interval

44 h

44 h

44 h

Total culture period*

92 h

92 h

92 h

*Exposure started 48 h after culture initiation

The selection of the concentrations was based on data from the pre-experiment. In the first main experimentwithoutandwithmetabolic activation 60 µg/mL and 200 µg/mL test item, respectively, and in experiment II 50 µg/mL test item was selected as the highest concentration for microscopic evaluation.

The following concentrations were evaluated for micronuclei frequencies:

Experiment Iwith short-term exposure (4 h):

withoutmetabolic activation: 25, 50 and 60 µg/mL

withmetabolic activation: 50, 100 and 200 µg/mL

Experiment IIwith long-term exposure (44 h):

withoutmetabolic activation: 30, 40, 45 and 50 µg/mL

 

No precipitate of the test item was noted in the cultures at the end of treatment in any concentration group evaluated in experiment I and II.

 

 


 

Table1:  Summary: Experiment I and II, without metabolic activation

 

Dose Group

Concentration [µg/mL]

Number of cells evaluated

Cytostasis

[%]

Relative Cell Growth
[%]

Micro-nucleated
Cells Frequency
[%]

Historical Control Limits Negative Control

P

Statistical Significant Increasea

Exp. I

4 h treatment, 44 h fixation interval

C

0

2000

0

100

0.65

0.29% - 1.16%

/

/

2

25

2000

14

86

0.55

-

-

3

50

2000

27

73

0.35

-

-

4

60

2000

59

41

0.60

-

-

MMS

50

2000

 0*

122

2.85

-

+

Colc

0.4

1366

79

21

8,63

-

+

 

 

 

 

Exp. II

44 h treatment, 44 h fixation interval

C

0

2000

0

100

0.80

0.29% - 1.16%

/

/

4

30

2000

20

80

0.40

-

-

6

40

2000

28

72

0.60

-

-

7

45

2000

50

50

0.35

-

-

8

50

2000

56

44

0.40

-

-

MMS

50

2000

8

92

2.30

-

+

Colc

0.02

1705

81

19

1.69

-

+

 

 

Table2:  Summary: Experiment I, with metabolic activation

 

Dose Group

Concentration [µg/mL]

Number
 of cells evaluated

Cytostasis

[%]

Relative Cell Growth
[%]

Micro-

Nucleated
Cells
Frequency
[%]

Historical Control Limits Negative Control

P

Statistical Significant Increasea

Exp. I

4 h treatment,

44 h fixation interval

C

0

2000

0

100

0.95

0.28% - 1.26%

/

/

1

50

2000

 0*

120

0.75

-

-

2

100

2000

6

94

0.50

-

-

3

200

2000

53

47

0.45

-

-

CPA

12.5

2000

39

61

5.35

-

+

 

 

C:                                         Negative Control (Culture medium)

P:                                           Precipitation (+: precipitation, -: no precipitation)

a:                                          statistically significant increase compared to negative control (c² test , p<0.05).

               +: significant; -: not significant

MMS:                                    Methylmethanesulfonate, Positive Control (without metabolic activation)

Colc.:                                    Colchicine, Positive Control (without metabolic activation)

CPA:                                     Cyclophosphamide, Positive Control (with metabolic activation)

 

Relative Cell Growth:         100 x ((CBPITest conc– 1) / (CBPIcontrol -1))

 

Cytostasis [%] = 100- Relative Cell Growth [%]

*: the cytostasis is defined 0, when the relative cell growth exceeds 100%

 

 In experimentIwithoutmetabolic activationno increase of the cytostasis above 30% was noted up to 50µg/mL. At 60µg/mLa cytostasis of 59% was noted. In experimentIwithmetabolic activationno increase of the cytostasis above 30% was noted up to 100µg/mL. At 200µg/mLa cytostasis of 53% was observed.

In experimentIIwithoutmetabolic activationno increase of the cytostasis above 30% was noted up to 40µg/mL. At 45µg/mLa cytostasis of 50% and at 50 µg/mL a cytostasis of 56% was observed.

In experiment I and IIwithoutandwithmetabolic activation no biologically relevant increase of the micronucleus frequency was noted after treatment with the test item.

The nonparametricc² Test was performed to verify the results in both experiments. No statistically significant enhancement (p<0.05) of cells with micronuclei was noted in the concentration groups of the test item evaluated in experiment I and IIwithandwithoutmetabolic activation.

Thec² Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions. No statistically significant increase in the frequency of micronucleated cells under the experimental conditions of the study was observed in experiment I and II .

Methylmethanesulfonate (MMS, 50 µg/mL) and cyclophosphamide (CPA, 12.5  µg/mL) were used as clastogenic controls. Colchicine (Colc, 0.02 and 0.4 µg/mL) was used as aneugenic control. All induced distinct and statistically significant increases of the micronucleus frequency. This demonstrates the validity of the assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
Name: tBuTPP low TPP
Chemical Name: Reaction mass of p-t-butylphenyldiphenyl phosphate and bis(p-t-butylphenyl)
phenyl phosphate
EC No.: 939-505-4
Batch No.: 9395054-1-2019 (COA attached)
Molecular Weight: approx. 382 g/mol
Physical State: liquid
Colour: yellowish
Density: 1.16 - 1.18 g/mL (25 °C)
Active Components: 100%
Expiry Date: not provided by the sponsor
Storage Conditions: room temperature, protected from light
Safety Precautions: The routine hygienic procedures will be sufficient to assure personnel health and safety.
Target gene:
HPRT Locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Mix
An advantage of using in vitro cell cultures is the accurate control of the concentration and exposure time of cells to the test item under study. However, due to the limited capacity of cells growing in vitro for metabolic activation of potential mutagens, an exogenous metabolic activation system is necessary [16]. Many substances only develop mutagenic potential when they are metabolized by the mammalian organism. Metabolic activation of substances can be achieved by supplementing the cell cultures with liver microsome preparations (S9 mix).
The S9 liver microsomal fraction was obtained from Trinova Biochem GmbH, Giessen, Germany. Male Sprague Dawley rats were induced with phenobarbital / β naphthoflavone.
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 (Lot: 4180) was 39.2 mg/mL.

S9 Mix
An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the concentrations below:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-phosphate-buffer pH 7.4. During the experiment the S9 mix was stored on ice.
The percentage of S9 mix in the final treatment medium was 5% (v/v).
Test concentrations with justification for top dose:
Exposure Concentrations
The test item was tested at the following concentrations:
without metabolic activation:
50, 75, 100, 125, 150, 175, 200, 300, 350 and 400 µg/mL
and with metabolic activation:
10, 25, 50, 100, 200, 250, 300, 400, 500 and 1000 µg/mL
According to OECD Guidelines more than 4 concentrations may be particularly important when using single cultures. Therefore, 5 - 6 concentrations (as indicated above in bold) were selected on the basis of cytotoxicity, for evaluating mutagenicity in the main experiment.

The experiment with and without metabolic activation was performed as 4 h short-term exposure assay.
Vehicle / solvent:
yes
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
Test System
Cells
V79 cells in vitro have been widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells are characterized by their high proliferation rate (12 - 14 h doubling time of the Eurofins BioPharma Product Testing Munich GmbH stock cultures) and their high cloning efficiency of untreated cells (usually more than 50%). These features of the cells are necessary for the appropriate performance of the study.
The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins BioPharma Product Testing Munich GmbH. This allows the repeated use of the same cell culture batch in experiments. Each cell batch was routinely checked for mycoplasma infections (via PCR), stable spontaneous mutant frequency as well as stability of the modal chromosome number. Freshly thawed cells from stock cultures were maintained in plastic culture flasks in minimal essential medium (MEM) and cultured at a humidified atmosphere of 5% CO2 and at 37 °C incubation temperature.
For purifying the cell population of pre-existing HPRT- mutants cells were exposed to HAT medium containing 10 µM hypoxanthine, 3.2 µM aminopterin, 5 µM thymidine and 10 µM glycine for several cell doublings (2 - 3 days) with a subsequent recovery period in medium supplemented with 10 µM hypoxanthine and 5 µM thymidine [14], [15].

Medium
Complete Culture Medium
MEM medium supplemented with
10 % fetal bovine serum (FBS)
100 U/100 µg/mL penicillin/streptomycin
2 mM L-glutamine
25 mM HEPES
2.5 µg/mL amphotericin B
Treatment Medium
MEM medium supplemented with
0 % fetal bovine serum (FBS): short-term exposure
100 U/100 µg/mL penicillin/streptomycin
2 mM L-glutamine
25 mM HEPES
2.5 µg/mL amphotericin B
Selective Medium
MEM medium supplemented with
10 % fetal bovine serum (FBS)
100 U/100 µg/mL penicillin/streptomycin
2 mM L-glutamine
25 mM HEPES
2.5 µg/mL amphotericin B
11 µg/mL 6-thioguanine (TG)
The selective agent (TG) was obtained from Sigma-Aldrich (Lot No.: SLBZ6113).

Pre-Test for Toxicity
The toxicity of the test item was determined in a pre-experiment. Eight concentrations [25, 50, 100, 250, 500, 1000, 1500 and 2500 µg/mL] were tested with and without metabolic activation for the 4 h short-term exposure assay.
Toxicity of the test item was evaluated using the relative survival (RS). A cytotoxic effect is considered when the relative survival decreases below 70% (formula for calculation see Section 10.5.4).
Approximately 10 x 106 cells were exposed to designated concentrations of the test item either in the presence or absence of metabolic activation in the mutation experiment. After 4 h exposure the treatment medium (MEM without serum) containing the test item was removed and the cells were washed twice with PBS, trypsinised and counted with a cell counter. For each treatment group two 25 cm2 flasks have been seeded with approximately 200 cells to determine cloning efficiencies (viability). After incubation for an appropriate time (7 - 9 days) colonies were fixed with methanol, stained with Giemsa and counted [17].
The relative survival was calculated based on the cloning efficiency of the cells plated immediately after treatment adjusted by any loss of cells during treatment (formula for calculation see Section 10.5.4).


A detailed table of the Experimental Design is attached below.
Evaluation criteria:
Acceptability of the Assay
A mutation assay is considered acceptable if it meets the following criteria:
- Negative and/or solvent controls fall within the performing laboratories 95th control limits of the historical control data range: 8.3 - 43.9 mutants/106 cells (without metabolic activation) and
8.6 - 46.4 mutants/106 cells (with metabolic activation) (January 2015 - December 2019).
-S9 +S9
Mean 26 28
Min 5 7
Max 49 54
SD 8.88 9.44
RSD [%] 34.04 34.30
n = 95 104
LCL 8.3 8.6
UCL 43.9 46.4
S9: metabolic activation
Mean: mean of mutants/106 cells
Min.: minimum of mutants/106 cells
Max.: maximum of mutants/106 cells
SD: standard deviation
RSD: relative standard deviation
n: number of control values
LCL: lower control limit
UCL: upper control limit

- The absolute cloning efficiency ([number of positive cultures x 100] / total number of seeded cultures) of the negative and /or solvent controls is > 50%.
- The positive controls (EMS and DMBA) induce a statistically significant increase compared with the concurrent negative control and are compatible with the laboratory historical data base.
- Two experimental conditions (e.g. with and without metabolic activation) are tested unless one results in a positive response.

Evaluation of Results
A test chemical is considered to be clearly negative if, in all experimental conditions examined,
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend-test, and
- all results are inside the distribution of the historical negative control data.

Statistics:
Statistical Analysis
The non-parametric Mann-Whitney test was applied to the mutation data to prove the concentration groups for any significant difference in mutant frequency compared to the solvent controls. Mutant frequencies of the solvent controls were used as reference.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
the relative survival was 15.5, 13.5 and 17.5% for the highest concentrations (150, 200 and 300 µg/mL) evaluated (Table 4-attached).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
The highest biologically relevant concentration evaluated with metabolic activation was 500 µg/mL with a relative survival of 58.4% (Table 6).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: the test item tBuTPP low TPP is considered to benon-mutagenicat the HPRT locus using V79 cells of the Chinese Hamster.
Conclusions:
In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item tBuTPP low TPP is considered to benon-mutagenicat the HPRT locus using V79 cells of the Chinese Hamster.
Executive summary:

Summary

The test item tBuTPP low TPP was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster.The selection of the concentrations was based on data from the pre-experiments. The experiment with and without metabolic activation was performed as a 4 h short-term exposure assay.

The test item was investigated at the following concentrations:

without metabolic activation:

50, 75, 125, 150, 200 and 300 µg/mL

and with metabolic activation:

50, 100, 200, 300 and 500 µg/mL

Precipitation of the test item was noted in the pre experiments at concentrations of 500 µg/mL and higher (without and with metabolic activation). In the main experiment precipitation was observed at concentrations of 500 µg/mL and higher (with metabolic activation) and at concentrations of 350 µg/mL and higher (without metabolic activation).

Biologically relevant growth inhibition (relative survival RS < 70%) was observed in the experiment with and without metabolic activation. In the experiment without metabolic activation the relative survival was 15.5, 13.5 and 17.5% for the highest concentrations evaluated (150, 200 and 300 µg/mL). The highest biologically relevant concentration evaluated with metabolic activation was 500 µg/mL with a relative survival of 58.4%.

In the experiments no biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation). Most of the mutant values are within the historical data base of the test facility.

In the experiment with metabolic activation at concentration 200 µg/mL the mutant frequency induced by the test item did show a statistical significantly increase above the historic control range of 8.6 - 46.4 mutants per 106 cells. In addition, at concentration 300 µg/mL the mutant frequency was also above the historical control data, but was not statistical significantly increased. However, for the adjoining concentrations no significantly increase was observed and since no statistical significantly concentration-related increase was determined in the ² test for trend, these effects were considered as not biologically relevant.

In the experiment without metabolic activation at the lowest tested concentration (50 µg/mL) the mutant frequency induced by the test item did show a statistical significantly increase above the historic control range of 8.3 - 43.9 mutants per 106 cells. However, this effect was only observed at the lowest concentration evaluated and since no statistical significantly concentration-related increase was determined in the ² test for trend, this effect was considered as not biologically relevant.DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

For this endpoint 3 in vitro tests, performed similar to OECD test guidelines, were selected as key studies:

- One Bacterial Reverse Mutation Assays (Ames test). The results of the tests conducted on the compound in the absence and in the presence of a metabolic activation system were all negative. The test compound, S-154 Lot QH-30701 B0-78-82, did not demonstrate genetic activity in any of the assays conducted in this evaluation and was considered not mutagenic under these test conditions.

- A Sister Chromatid Exchange (SCE) assay in mammalian cells and In vitro mammalian Chromosome Aberration test. Chromosome aberration results were negative at all dose levels both with and without the enzyme activation system. No significant increases were observed in the frequency of cells with aberrations, and so the compound is concluded to be non-clastogenic. SCE frequencies also failed to increase with dose, both with and without the activation system, and so the test compound was clearly negative in this assay. In conclusion, Phosflex 51B failed to induce visible chromosome damage under the conditions of these assays.

- An vitro Mammalian Cell Gene Mutation Test. The test compound, Phosflex 51B, did not induce an increase in mutations at the TK locus in L5178Y mouse lymphoma cells at concentrations of 0.975 to 125.0 nl/ml with and without activation. The concentration of 125 nl/ml was highly toxic and insufficient survivors were obtained at 250 nl/ml to perform the assay. Therefore the test compound is considered to be inactive in this Mouse Lymphoma Forward Mutation Assay.

The following 2 in vitro tests are considered supporting studies:

- In vitro Mammalian Cell Gene Mutation Test (similar to OECD 476): S-154 BO-78-86 was not mutagenic in the mouse lymphoma L5178Y cells when tested under the conditions outlined in this report.

- In vitro Mammalian Cell Transformation assay: The test material, Phosflex 51B, did not induce a significant increase in transformed foci over the applied concentration range of 0.00125 ul/ml to 0.02 ul/ml. This concentration range corresponded to approximately 90% to 52% survival in the cytotoxicity test. Therefore, the test material is considered to be inactive in this Balb/3T3 In Vitro Transformation Assay.


Short description of key information:
- Gene mutation in bacteria (Bacterial Reverse Mutation Assay/Ames) (similar to OECD 471): not mutagenic.
- In vitro cytogenicity test (Sister Chromatid Exchange (SCE) assay in mammalian cells and In vitro Mammalian Chromosome Aberration test) (similar to OECD 479): negative.
- In vitro Mammalian Cell Gene Mutation Test (Mouse lymphoma assay) (similar to OECD 476): not mutagenic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In all available key and supporting studies t-BuTTP Low TPP did not show any genotoxic potential. Therefore, it can be concluded that the substance is not mutagenic and therefore does not need to be classified for mutagenicity according to the criteria outlined in 1272/2008/EC (CLP/EU-GHS).