Ethyltriphenylphosphonium acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative in all tests conducted:

- Ames test with S. typhimurium TA 98, TA 100, TA 1535, TA 1537, E coli WP2 uvrA (met. act.: with and without) (OECD TG 471, GLP); tested up to cytotoxic concentrations

- In Vitro Mammalian Cell Gene Mutation Assay (CHO/HPRT Assay), (met. act.: with and without) (OECD TG 476, GLP)

- In Vitro Mammalian Micronucleus Assay in Human Peripheral Blood Lymphocytes (HPBL), (met. act.: with and without) (OECD TG 487, GLP)

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:

21 August 2017 to 20 February 2018

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 July 21, 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his

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:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

preliminary toxicity assay: 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 μg per plate. The top dose was the maximum dose per the guidelines.
definitive mutagenicity assay: 66.7, 100, 333, 667, 1000, 3333, and 5000 μg per plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol (for the test substance)
All positive controls were diluted in DMSO, except for sodium azide, which was diluted in sterile water.
- Justification for choice of solvent/vehicle: Ethanol was the vehicle of choice based on the Sponsor’s request and the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in ethanol at a concentration of approximately 100 mg/mL in the solubility test conducted at BioReliance.

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracebe

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: 1 in the preliminary-toxicity assay; 3 in the mutagenicity assay

DETERMINATION OF CYTOTOXICITY
- Method: bacterial background lawn , revertant colony numbers

Evaluation criteria:

For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two consecutive increasing concentrations of test substance as specified below:

Strains TA1535 and TA1537
Data sets were judged positive if the increase in mean revertants at the peak of the dose-response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.

Strains TA98, TA100 and WP2 uvrA
Data sets were judged positive if the increase in mean revertants at the peak of the dose-response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
An equivocal response is an increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited.
A response was evaluated as negative if it was neither positive nor equivocal.

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

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

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

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

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without metabolic activation at 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitate was observed

RANGE-FINDING/SCREENING STUDIES:
a 50.0 μL plating aliquot. No precipitate was observed. Toxicity was observed beginning at 3333 or at 5000 μg per plate with tester strains of Salmonella typhimurium; no toxicity was observed for E. coli WP2.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see attachment
- Negative (solvent/vehicle) historical control data: see attachment

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Toxicity was observed beginning at 3333 or at 5000 μg per plate with tester strains of Salmonella typhimurium. Cytotoxicity was observed for E. coli WP2 only at 5000 µg/plate without metabolic activation.

Conclusions:

The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, ETPPAAc did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9. The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.

Executive summary:

The test substance, ETPPAAc, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system in accordance with OECD guideline 471. Ethanol was used as the vehicle.

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 μg per plate. No precipitate was observed. Toxicity was observed beginning at 3333 or at 5000 μg per plate with tester strains of Salmonella typhimurium. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.

In the mutagenicity assay, the dose levels tested were 66.7, 100, 333, 667, 1000, 3333, and 5000 μg per plate. No precipitate was observed. Toxicity was observed beginning at 3333 or at 5000 μg per plate with most conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

These results indicate that ETPPAAc was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Quality Assurance Audited Draft Report

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 August 2017 - 05 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Reliability 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

updated and adopted 26 September 2014

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes

Remarks:

primary culture

Details on mammalian cell type (if applicable):

Source of cells: Peripheral blood lymphocytes were obtained from a healthy non-smoking individual (no recent history of radiotherapy, viral infection, or the administration of drugs).

Media Used:
Type and identity of media including CO2 concentration if applicable: RPMI 1640 containing 15% fetal bovine serum, 2 mM L glutamine, 100 units penicillin, 100 μg/mL streptomycin, 37 ± 1 degrees C in a humidified atmosphere of 5 ± 1% CO2 in air
- Properly maintained: yes

Cytokinesis block (if used):

cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary test: 0, 0.2, 0.6, 2.0, 6.0, 20, 60, 200, 600, 2000 ug/ml

Concentrations used in the definative tests are stated in the "Any other information on materials and methods" section.

Vehicle / solvent:

Vehicle(s)/solvent(s) used: Ethanol
Justification for choice of vehicle/solvent: Solubility of the test substance, compatibility with the target cells.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Vinblastine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;
- Cell density at seeding (if applicable):

DURATION
- Exposure duration: 4 and 24 hours
- Expression time (cells in growth medium): 24 hr



SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B, 24 hours (± 30 minutes), 1.5 to 2 normal
cell cycle

STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: two

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: cells were collected by
centrifugation, and the suspension of fixed cells was applied to glass microscope slides and air-dried;
stained with acridine orange

NUMBER OF CELLS EVALUATED: A minimum of 2000 binucleated cells from each concentration (if
possible, 1000 binucleated cells from each culture) were examined and scored for the presence of micronuclei.


CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
• the micronucleus should have the same staining characteristics as the main nucleus
• the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic bridges)
• the micronuclei should be of regular shape and approximately 1/3 or less than the diameter of the
main nucleus.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: CBPI relative to vehicle control group

Evaluation criteria:

The test substance was considered to have induced a positive response if all of the following three
criteria are met:
• at least one of the test concentrations exhibited a statistically significant increase when compared
with the concurrent negative control (p ≤ 0.05), and
• the increase was concentration-related (p ≤ 0.05), and
• results were outside the 95% control limit of the historical negative control data.
The test substance was considered to have induced a clear negative response if none of the criteria
for a positive response were met.

Statistics:

Statistical analysis was performed using the Fisher's exact test (p the percentage of micronucleated cells in each treatment group with that of the vehicle control. The
Cochran-Armitage trend test was used to assess dose-responsiveness.

Key result

Species / strain:

lymphocytes: human peripheral blood lymphocytes

Remarks:

primary culture

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the highest dose of test substance in treatment medium was 7.5.
- Effects of osmolality: The osmolality of the test substance doses in treatment medium was considered acceptable
- Precipitation: None
- highest soluble concentration: Soluble at all tested concentrations

RANGE-FINDING/SCREENING STUDIES:
Cytotoxicity [>/= 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was
observed at doses >/= 25 μg/mL in the non-activated and >/= 40 ug/ml S9-activated 4-hour exposure groups, and
at doses >/= 3 μg/mL in the non-activated 24-hour exposure group. Based upon the results of the
preliminary toxicity assay, the doses selected for the micronucleus assay were as follows:

Treatment group Time Recovery Doses (µg/mL)
Non-activated 4 hr 20 hr 5, 20, 25, 30, 35, 40, 45, 50
Non-activated 24 hr 0 hr 0.2, 1, 2, 3, 4, 5, 6
S9-activated 4 hr 20 hr 5, 20, 40, 45, 50, 55, 60, 70

Conclusions:

Under the conditions of the assay described in the study report, ETPPAAc was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes.

Executive summary:

The test substance, ETPPAAc, was tested to evaluate the potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in both the absence and presence of an exogenous metabolic activation system. HPBL cells were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9. Ethanol was used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 µg/mL, which was the limit dose for this assay. Cytotoxicity [greater than of equal to 50% cytokinesis-blocked proliferation index(CBPI) relative to the vehicle control] was observed at doses > / = 60 µg/mL in the non‑activated and S9 -activated 4 -hour exposure groups, and at doses > / = 6 µg/mL in the non‑activated 24‑hour exposure group. Based upon these results, the doses chosen for the micronucleus assay ranged from 5 to 50 µg/mL for the non‑activated 4-hour exposure group; from 5 to 70 µg/mL for the S9-activated 4-hour exposure group; and from 0.2 to 6 µg/mL for the non‑activated 24-hour exposure group.

The initial micronucleus assay was terminated since the dose formulation volume collected for concentration and stability analyses were insufficient, and the micronucleus assay was repeated at the same doses. Since the dose formulation results for this repeat assay did not meet the acceptance criteria, the assay was repeated again at the same dose levels. 

In the second repeat micronucleus assay, cytotoxicity was observed at doses > / = 25 µg/mL in the non‑activated 4 -hour exposure group; at doses > / = 40 µg/mL in the S9 -activated 4 -hour exposure group; and at doses > / =  3 µg/mL in the non‑activated 24‑hour exposure group. The doses selected for evaluation of micronuclei were 5, 20, and 25 µg/mL for the non-activated 4-hour exposure group; 5, 20, and 40 µg/mL for the S9-activated 4-hour exposure group; and 1, 2, and 3 µg/mL for the non-activated 24-hour exposure group.

No significant or dose‑dependent increases in micronuclei induction were observed in any treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

These results indicate ETPPAAc was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system. 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 February 2018 to 31 October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Remarks:

Refer to main study report

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch No.of test material: 70002
- Expiration date of the lot/batch: 27 April 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light and with desiccant
- Stability under test conditions: While the Certificate of Analysis indicates an expiry date (27 April 2019), it does not indicate the acceptable storage parameters for the test article. Thus, the stability of the test substance has not been determined to cover the period of shipment and storage at BioReliance.
- Solubility and stability of the test substance in the solvent/vehicle: Ethanol was determined to be the solvent in study AE97YS.348REACH.BTL. ETPPAAc in ethanol, at nominal concentrations of 25 and 100 mg/mL, was stable at for at least 3.0 hours.

Target gene:

The purpose of this study was to evaluate a test article for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr).

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Properly maintained: yes- Periodically checked for Mycoplasma contamination: yes- Periodically checked for karyotype stability: yes- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

In the preliminary toxicity assay, the concentrations tested were 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL. The maximum concentration evaluated approximated the limit dose for this assay.Based upon these results, the concentrations chosen and the top dose for the definitive mutagenicity assay were 62.5, 125, 250, 500, 750 and 1000 μg/mL with and without S9. In the retest of definitive mutagenicity assay, same concentrations as initial definitive mutagenicity assay were used

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol for test substance. Ethylmethanesulfonate (EMS) was used as the positive control for the non-activated test system. Benzo(a)pyrene [B(a)P] was as the positive control for the S9-activated system- Justification for choice of solvent/vehicle:Ethanol was determined to be the solvent in study AE97YS.348REACH.BTL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 +/- 0.5 hours
- Expression time (cells in growth medium): 7 to 8 days
- Selection time (if incubation with a selection agent): 7 to 10 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: single cultures in the preliminary toxicity assay; duplicate cultures in the repeat of mutagenicity assay

NUMBER OF CELLS EVALUATED: 2.4 x 10e6 cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: adjusted relative survival

Evaluation criteria:

The test substance was considered to have produced a positive response if it induced a statistically significant and dose-dependent statistically significant increase in mutant frequency in comparison to the concurrent vehicle control and an increase exceeding 95% historical vehicle control limits in at least one test dose level(s) as compared with concurrent vehicle control (p<0.01). If only one criterion was met (a statistically significant or dose-dependent increase or an increase exceeding the historical control 95% confidence interval), the results were considered equivocal. If none of these criteria were met, the results were considered to be negative.Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.

Statistics:

Statistical analyses were performed using the method of Snee and Irr (1981), with significance established at the 0.05 level.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The average adjusted relative survival was 43.00 and 18.21% at a concentration of 750 μg/mL with and without S9, respectively.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The test substance did not have an adverse impact on the pH of the cultures (pH 7.0 at the top dose) in each assay.
- Effects of osmolality: The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehicle control by more than 120% in the preliminary toxicity assay.
- Precipitation:Preliminary toxicity assay: Visible precipitate was observed at concentrations ≥1000 μg/mL at the beginning of treatment and at the end of treatment without S9
Retest of definitive mutagenicity assay:Visible precipitate was observed at concentrations ≥750 μg/mL by the end of treatment without S9.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Under the conditions of the assay described in this report, ETPPAAc was concluded to be negative for the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro mammalian cell forward gene mutation (CHO/HPRT) assay.

Executive summary:

The test substance, ETPPAAc, was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr). Ethanol was used as the vehicle.

In the preliminary toxicity assay, the concentrations tested were 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL. The maximum concentration evaluated approximated the limit dose for this assay. Visible precipitate was observed at concentrations ≥1000 μg/mL at the beginning of treatment and at the end of treatment without S9. Adjusted relative survival was 68.18 and 10.01% at a concentration of 500 μg/mL with S9 and 1000 μg/mL without S9, respectively. Adjusted relative survival was or approximated 0% at all higher concentrations with and without S9. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 62.5, 125, 250, 500, 750 and 1000 μg/mL with and without S9.

In the initial definitive mutagenicity assay appropriate tissue culture flasks were not available to continue with the assay, therefore, the assay was aborted and repeated in the retest of definitive mutagenicity assay. In the retest of definitive mutagenicity assay, same concentrations as initial definitive mutagenicity assay were used. No visible precipitate was observed at the beginning of treatment. Visible precipitate was observed at concentrations ≥750 μg/mL by the end of treatment without S9. The average adjusted relative survival was 43.00 and 18.21% at a concentration of 750 μg/mL with and without S9, respectively. Cultures treated at concentrations of 62.5, 125, 250, 500 and 750 μg/mL with and without S9 were chosen for mutant selection. In the treatment condition with S9 activation, mutant frequency at the dose 500 μg/mL (13.52) was observed to be outside the historical control limit. However the response was within the 99% historical range and the highest dose level (750 μg/mL) was also within the historical control ranges. Therefore, this response was not considered to be biologically significant. No other significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p > 0.01). The positive controls induced significant increases in mutant frequency (p<0.01).

These results indicate ETPPAAc was negative for the ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial reverse mutation assay

The test substance, ETPPAAc, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system in accordance with OECD guideline 471. Ethanol was used as the vehicle.

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 μg per plate. No precipitate was observed. Toxicity was observed beginning at 3333 or at 5000 μg per plate with tester strains of Salmonella typhimurium. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.

In the mutagenicity assay, the dose levels tested were 66.7, 100, 333, 667, 1000, 3333, and 5000 μg per plate. No precipitate was observed. Toxicity was observed beginning at 3333 or at 5000 μg per plate with most conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

These results indicate that ETPPAAc was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

 

Mammalian cell gene mutation assay

The test substance, ETPPAAc, was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr). Ethanol was used as the vehicle.

In the preliminary toxicity assay, the concentrations tested were 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL. The maximum concentration evaluated approximated the limit dose for this assay. Visible precipitate was observed at concentrations ≥1000 μg/mL at the beginning of treatment and at the end of treatment without S9. Adjusted relative survival was 68.18 and 10.01% at a concentration of 500 μg/mL with S9 and 1000 μg/mL without S9, respectively. Adjusted relative survival was or approximated 0% at all higher concentrations with and without S9. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 62.5, 125, 250, 500, 750 and 1000 μg/mL with and without S9.

In the initial definitive mutagenicity assay appropriate tissue culture flasks were not available to continue with the assay, therefore, the assay was aborted and repeated in the retest of definitive mutagenicity assay. In the retest of definitive mutagenicity assay, same concentrations as initial definitive mutagenicity assay were used. No visible precipitate was observed at the beginning of treatment. Visible precipitate was observed at concentrations ≥750 μg/mL by the end of treatment without S9. The average adjusted relative survival was 43.00 and 18.21% at a concentration of 750 μg/mL with and without S9, respectively. Cultures treated at concentrations of 62.5, 125, 250, 500 and 750 μg/mL with and without S9 were chosen for mutant selection. In the treatment condition with S9 activation, mutant frequency at the dose 500 μg/mL (13.52) was observed to be outside the historical control limit. However the response was within the 99% historical range and the highest dose level (750 μg/mL) was also within the historical control ranges. Therefore, this response was not considered to be biologically significant. No other significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p > 0.01). The positive controls induced significant increases in mutant frequency (p<0.01).

These results indicate ETPPAAc was negative for the ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system.

 

in vitro mammalian cell micronucleus test

The test substance, ETPPAAc, was tested to evaluate the potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in both the absence and presence of an exogenous metabolic activation system. HPBL cells were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9. Ethanol was used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 µg/mL, which was the limit dose for this assay. Cytotoxicity [greater than of equal to 50% cytokinesis-blocked proliferation index(CBPI) relative to the vehicle control] was observed at doses > / = 60 µg/mL in the non‑activated and S9 -activated 4 -hour exposure groups, and at doses > / = 6 µg/mL in the non‑activated 24‑hour exposure group. Based upon these results, the doses chosen for the micronucleus assay ranged from 5 to 50 µg/mL for the non‑activated 4-hour exposure group; from 5 to 70 µg/mL for the S9-activated 4-hour exposure group; and from 0.2 to 6 µg/mL for the non‑activated 24-hour exposure group.

 

The initial micronucleus assay was terminated since the dose formulation volume collected for concentration and stability analyses were insufficient, and the micronucleus assay was repeated at the same doses. Since the dose formulation results for this repeat assay did not meet the acceptance criteria, the assay was repeated again at the same dose levels. 

In the second repeat micronucleus assay, cytotoxicity was observed at doses > / = 25 µg/mL in the non‑activated 4 -hour exposure group; at doses > / = 40 µg/mL in the S9 -activated 4 -hour exposure group; and at doses > / =  3 µg/mL in the non‑activated 24‑hour exposure group. The doses selected for evaluation of micronuclei were 5, 20, and 25 µg/mL for the non-activated 4-hour exposure group; 5, 20, and 40 µg/mL for the S9-activated 4-hour exposure group; and 1, 2, and 3 µg/mL for the non-activated 24-hour exposure group.

No significant or dose‑dependent increases in micronuclei induction were observed in any treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

These results indicate ETPPAAc was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system. 

Justification for classification or non-classification

Based on reliable, relevant and adequate data the substance is considered to be not mutagenic and not clastogenic. According to Regulation EC No 1272/2008 no classification and labelling for mutagenicity is required.