Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro tests are available: An Ames test performed according to OECD guideline and GLP principles with Klimisch score 2, a chromosome aberration study performed according to OECD 473 guideline and GLP principles and an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells performed according to OECD 476 guideline and GLP principles, all three with Klimisch score 1.
The test substance was found to be mutagenic in the MLA test, but negative in the AMES and in the in vitro chromosome aberration test.

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
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:
S9-mix prepared from male Wistar rats livers
Test concentrations with justification for top dose:
0; 33; 100; 333; 1000; 2500 and 5000 µg/plate (Standard plate test and preincubation test)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: 2-aminoanthracene; N-methyl-N`-nitro-N-nitrosoguanidine; 4-nitro-o-phenylenediamine
Details on test system and experimental conditions:
Standard plate test
Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)

After mixing, the samples were be poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were be poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

Preincubation Test

0.1mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The
colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perseptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Positive controls:
With S9 mix
2-aminoanthracene (2-AA); 2.5 μg/plate, dissolved in DMSO; strains: TA 1535, TA 100, TA 1537, TA 98; 60 μg/plate, dissolved in DMSO; strain: Escherichia coli WP2 uvrA
Without S9 mix
N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 5 μg/plate, dissolved in DMSO; strains: TA 1535, TA 100
4-nitro-o-phenylenediamine (NOPD); 10 μg/plate, dissolved in DMSO; strain: TA 98
9-aminoacridine (AAC); 100 μg/plate, dissolved in DMSO; strain: TA 1537
4-nitroquinoline-N-oxide (4-NQO); 5 μg/plate, dissolved in DMSO; strain: E. coli WP2 uvrA
Evaluation criteria:
The test substance was considered positive in this assay if the following criteria were met:
A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

TOXICITY

A bacteriotoxic effect (decrease in the number of trp+ revertants) was observed in the standard plate test only in the tester strain E. coli WP2 uvrA with and without S9 mix from about 2500 μg/plate onward. In the preincubation assay bacteriotoxicity (slight decrease in the number of his+ revertants) was occasionally observed depending on the strain only without S9 mix from about 2500 μg/plate onward.

SOLUBILITY

No test substance precipitation was found with and without S9 mix.

According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within or nearby the range of the historical negative control data for each tester strain (see Appendix 5). In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within, slightly below or above the range of the historical positive control data (see Appendix 5).

Thus, under the experimental conditions chosen here, it is concluded that Reaction mass of 2,2’-[(4-methylphenyl)imino]bisethanol and Ethanol 2-[[2-(2-hydroxyethoxy)ethyl](4- methylphenyl)amino]- is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 28th, 2012 - March 14th, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: Cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.
- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable, immediately after blood collection lymphocyte cultures were started.
- Periodically "cleansed" against high spontaneous background: not applicable, immediately after blood collection lymphocyte cultures were started.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.
Test concentrations with justification for top dose:
Dose range finding test:
Without S9-mix, 3 hr exposure; 24 hr fixation: 33, 100, 333, 1000, 1956 µg/mL
Without S9-mix, 24 hr exposure; 24 hr fixation: 33, 100, 333, 1000, 1956 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 33, 100, 333, 1000, 1956 µg/mL
With S9-mix, 3hr exposure; 24 hr fixation: 33, 100, 333, 1000, 1956 µg/mL

First cytogenetic test:
Without S9-mix, 3 h exposure time, 24 h fixation time: 33, 333, 1000, 1500, 1956 µg/mL
With S9-mix, 3 h exposure, 24 h fixation time: 33, 333, 1000, 1500, 1956 µg/ mL

Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 30, 100, 250, 500, 750, 1000, 1250 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 30, 100, 250, 500, 750, 1000, 1250 µg mL
With S9-mix, 3 hr exposure; 48 hr fixation: 30, 300, 1000, 1500, 1956 µg/mL
Vehicle / solvent:
- Solvent used: DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
At 0.5 and 0.75 µg/ml for a 3 h exposure period, 0.2 and 0.3 µg/ml for a 24 h exposure period and 0.1 and 0.15 µg/ml for a 48 h exposure period.
Positive control substance:
mitomycin C
Remarks:
Without metabolic activation
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
At 10 µg/ml.
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- The highest concentration analysed was selected based on toxicity, inhibition of the mitotic index of about 50% or greater (24 h and 48 h continuous exposure time) or the recommended 0.01 M (3 h exposure time).
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.
Key result
Species / strain:
lymphocytes: Cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- No effects of pH or osmolarity were reported: The pH and osmolarity of a concentration of 1956 µg/ml were 7.46 and 415 mOsm/kg respectively (compared to 7.42 and 433 mOsm/kg in the solvent control).

- Precipitation: No precipitation was observed up to and including the top dose of 1956 µg/mL (=0.01 M).

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the highest tested dose without metabolic activation with 3 h exposure time/ 24 h fixation time.
- Cytotoxicity was observed without metabolic activation at 1000 µg/mL and above with 24 h exposure time/ 24 h fixation time.
- Cytotoxicity was observed without metabolic activation at 333 µg/mL and above with 48 h exposure time/ 48 h fixation time.
- Cytotoxicity was observed with metabolic activation at 1956 µg/mL awith 3 h exposure time/ 24 h fixation time.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses.
Conclusions:
A chromosome aberration study with Accelerator (PT 25E or PT 25E/2) was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that Accelerator (PT 25E or PT 25E/2) is not clastogenic in human lymphocytes.
Executive summary:

A chromosome aberration study with Accelerator (PT 25E or PT 25E/2) was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. No precipitation of the substance was observed, but the test substance was tested up to and beyond cytotoxic concentrations. Both in the absence and presence of S9-mix

Accelerator (PT 25E or PT 25E/2) did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. It can be concluded that Accelerator (PT 25E or PT 25E/2) does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May - August 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
- RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3 hours treatment: 100, 333, 1000, 3330, 5000 µg/mL
Without S9-mix, 24 hours treatment: 100, 333, 1000, 3330, 5000 µg/ml
Experiment 1:
Without S9-mix, 3 hours treatment: 10, 33, 100, 333, 1000, 1250, 1500 µg/mL
With S9-mix, 3 hours treatment: 10, 33, 100, 333, 1000, 1250, 1500, 1675 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 1, 3, 10, 30, 100, 200, 300 µg/mL
With S9-mix, 3 hours treatment: 30, 100, 300, 1000, 1250, 1500, 1600, 1650 μg/mL
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent/vehicle:
Test compound was stable and soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines.
Positive control substance:
methylmethanesulfonate
Remarks:
15 and 5 μg/ml for a 3 and 24 hours treatment period, resp.
Positive control substance:
cyclophosphamide
Remarks:
7.5 and 10 μg/ml with 4 and 8% (v/v) S9 fraction resp.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (10^6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 per 10^6 survivors, and for CP not below 700 per 10^6 survivors.

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.
Statistics:
The global evaluation factor (GEF) has been defined by the IWTGP as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 3330 µg/mL (3 hours treatment); at and above 250 µg/mL (24 hours treatment)
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 1650 µg/mL (3 hours treatment)
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- The pH and osmolarity of a concentration of 5000 μg/ml were 7.4 and 0.382 Osm/kg respectively (compared to 7.4 and 0.289 Osm/kg in the solvent control).
- Precipitation: No precipitation was observed up to the highest dose level of 5000 µg/mL.

RANGE-FINDING/SCREENING STUDIES:
- Cytotoxicity was observed at and above dose levels of 3330 µg/mL in the absence and presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

In the absence of S9-mix, Accelerator (PT 25E or PT 25E/2) did not induce a significant increase in the mutation frequency in the first experiment. However the test substance induced a 5.3-fold increase in the mutation frequency at the prolonged treatment period of 24 hours. Verification of the results in a third experiment, showed that the test substanceinduced an up to 3.3-fold increase in the mutation frequency. The increases observed in the second and third experiment were above the laboratory’s historical solvent control range in both experiments as well as abovethe GEF + MF(controls)(187 and 271 per 106survivorsin the second and third experiment, respectively). The responses observed in the absence S9-mix fulfilled the criteria for a positive response.

Conclusions:
The mutagenic activity of Accelerator (PT 25E or PT 25E/2) was evaluated in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells, performed according to OECD 476 and following GLP principles.The test item induced mutations in the absence of metabolic activation, but not with metabolic activation. This was confirmed in an independent repeat experiment.
It is concluded that the test substance is positive in the in vitro mammalian cell gene mutation test.
Executive summary:

An in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells, performed according to OECD 476 and following GLP principles was performed. The test substance was tested up to cytotoxic concentrations. The positive control data and the solvent control data validated the study. In the presence of S9-mix, Accelerator (PT 25E or PT 25E/2) did not induce a significant increase in the mutation frequency in two experiments. In the absence of S9-mix, the test substance induced a 5.3-fold increase in the mutation frequency at the prolonged treatment period of 24 hours. This was verified in a further experiment, in which the substance induced an up to 3.3-fold increase in the mutation frequency. The mutation frequency of both the small and large colonies was significantly increased.

Based on these data, it is concluded that the test substance is positive in the in vitro mammalian cell gene mutation test.

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Two alkaline in vivo Comet assays have been performed: the first assay in liver, stomach and duodenum of rats and the second assay in stomach and duodenum of rats (according to OECD 489 guideline and GLP principles)

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Endpoint:
genetic toxicity in vivo, other
Remarks:
DNA strand breaks in liver, stomach and duodenum
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 2016 - February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
adopted September 26, 2014 (A new version of the guideline has been adopted by OECD. The study procedures described in this report are also in compliance with this new guideline:
Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for the Testing of Chemicals, Guideline No. 489: In Vivo Mammalian Alkaline Comet Assay (adopted July 29, 2016).
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic items. Moreover, historical control background data has been generated with this strain.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6-8 weeks old
- Weight at study initiation: 149 ± 7.9 g (range 131 – 162 g) in experiment 1 and 149 ± 7.8 g (range 137 – 168 g) in the repeat experiment.
- Assigned to test groups randomly: yes
- Fasting period before study: no (A limited quantity of food was supplied during the night before dosing (approximately 7 g/rat)

- Housing: Group housing of maximum 5 animals per sex in labeled Macrolon cages (type MIV) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Diet (e.g. ad libitum): free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): free access to tap-water
- Acclimation period: at least 6 days before the start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2– 21.4
- Humidity (%): 30 - 73
- Air changes (per hr): approximately 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17 May 2016 To: 16 February 2017
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Stable for 6 hours in the range of 20-200 mg/mL in propylene glycol.
No correction was made for the purity/composition of the test compound.
Accelerator (PT 25E or PT 25E/2) was dissolved in propylene glycol. The specific gravity of propylene glycol is 1.036 g/ml. Accelerator (PT 25E or PT 25E/2) concentrations were treated with ultra-sonic waves to obtain a homogeneous solution (sonication: max. time
12 min, max. temp 28°C in experiment 1 and max. time 14 min, max. temp 32°C in the repeat experiment). Accelerator (PT 25E or PT 25E/2) concentrations were dosed within 3 hours after preparation in experiment 1 and within 2 hours in the repeat experiment.
Duration of treatment / exposure:
three consecutive days
Post exposure period:
once daily
Dose / conc.:
187.5 mg/kg bw/day (actual dose received)
Dose / conc.:
375 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
at least 5 males per treatment group
Control animals:
yes, concurrent vehicle
Positive control(s):
ethylmethanesulphonate
- Route of administration: oral
- Doses / concentrations: 10 mL/kg bw; 200 mg/kg bw
Tissues and cell types examined:
liver, stomach and duodenum
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Selection of an adequate dose range for the in vivo Comet main test was based on a dose range finding study. The test procedure and conditions were similar to those applied in the main test. In a sub-acute toxicity study with the test substance no gender difference was observed. Based on these data the dose range finding study and the main studies were conducted in males only.
Two dose groups, one comprising of 1 male (600 mg/kg bw) and the other comprising of 3 males (750 mg/kg bw) were dosed for three consecutive days (once daily) with Accelerator (PT 25E or PT 25E/2). The group comprised of 3 males were dosed with the highest concentration that was used for the main study. The observation period after dosing was one to 3 days. During this period mortality and physical condition were recorded at least once a day.
The dose range study was started with a dose of 600 mg/kg bw. This dose was selected for ethical reasons and based on the results of the 28-day repeated dose toxicity study in rat.



TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Approximately 3-4 hours after the third treatment with the test compound or vehicle and second treatment with EMS liver (first experiment only), stomach and duodenum tissue was collected/isolated and examined for DNA damage with the alkaline Comet assay.

DETAILS OF SLIDE PREPARATION:To 20 µL of the cell suspension, 280 µL melted low melting point agarose (LMAgarose; Trevigen, Gaithersburg, USA) was added. The cells were mixed with the LMAgarose and 50 µL was layered on a precoated Comet slide (Trevigen) in duplicate. Three slides per tissue were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for at least 10 minutes (actual time 15-24 minutes in experiment 1 and 16-23 minutes in the repeat experiment) in the refrigerator in the dark until a clear ring appeared at the edge of the Comet slide area.
The cells on the slides were overnight (approximately 16-18 h in both experiments) immersed in prechilled lysis solution (Trevigen) in the refrigerator. After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4) for approximately 5 minutes. The slides were then placed in freshly prepared alkaline solution for 25-56 minutes in experiment 1 and for 20-32 minutes at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 1 Volt/cm. The electrophoresis was performed for 30 minutes under constant cooling (actual temperature 5.0 – 7.5°C in experiment 1 and 4.5 – 6.0 °C). After completion of electrophoresis, the slides were immersed/rinsed in the neutralization buffer. The slides were subsequently immersed for approximately 5 minutes in absolut ethanol (≥99.6%) and allowed to dry at room temperature. The slides were stained for approximately 5 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Bleiswijk, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark.

METHOD OF ANALYSIS:
To prevent bias, slides were randomly coded (per tissue) before examination of the Comets. An adhesive label with study identification number and code were placed over the marked slide. The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system (Perceptive instruments Ltd, Suffolk, United Kingdom). One hundred fifty Comets (50 comets of each replicate LMAgarose circle) were examined per sample. On a few slides, one of the agorose circles was damaged, therefore an agarose circle from the second backup slide was used for scoring.
The following criteria for scoring of Comets were used:
• Only horizontal orientated Comets were scored, with the head on the left and the tail on the right.
• Cells that showed overlap or were not sharp were not scored.

OTHER:One experiment was perfomed for liver and two experiments were performed for glandular stomach and duodenum since the first experiment did not pass the acceptance criteria for the control group.
Evaluation criteria:
A test compound is considered positive in the Comet assay (in a tissue) if the following criteria are met:
It induces a biologically as well as a statistically significant (Dunnett’s test, one-sided,
p < 0.05) dose-dependent increase in percentage Tail Intensity. In case of other non-dose-dependent significant increases the data interpretation will be on a case by case base.

A test compound is considered as negative in the Comet assay (in a tissue) if the following criteria are met:
None of the tested concentrations show a statistically significant (Dunnett’s test, one-
sided, p < 0.05) dose-dependent increase in percentage Tail Intensity.

Data was normally distributed thus no transformation (y = 1/y) of the data was necessary. In addition no Cochran Armitage trend test (p < 0.05) was performed.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis.
Statistics:
Dunnett’s test, one-sided; ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
high dose animals: lethargic and tremors first hour after dosing; one animal died after the third dose
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 600 (1 male) and 750 (3 males) mg/kg bw/day
- Clinical signs of toxicity in test animals: The dose range finding study was started with a dose of 600 mg/kg. At 600 mg/kg bw/day the effects were minor: the male animal was only lethargic after the first dose and recovered thereafter and no treatment related clinical signs were observed after the second and third dose. It was therefore concluded that a dose of 600 mg/kg bw/day was too low as maximum concentration in the main assay.
Subsequently three male animals were dosed with 750 mg/kg bw/day. The following clinical signs were observed during the duration of the study (days 1-3): lethargy, hunched posture, rough coat and tremors. One animal died after the first dosing with 750 mg/kg bw/day.


RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: Based on the results of the dose range finding study dose levels of 187.5, 375 and 750 mg/kg body weight were selected as appropriate doses for the Comet main test.
Five male animals were used in each treatment group. Three additional male animals, treated with 750 mg Accelerator (PT 25E or PT 25E/2)/kg body weight, were used to correct for possible deaths.
After treatment, single cell suspensions were prepared from the liver, stomach and duodenum tissue. The viability of one single cell suspension per tissue per group was assessed by using trypan blue. The viability of the single suspension was 90-100% in experiment 1 and 97 – 100% in the repeat experiment. The viability was assessed for one animal per group.
- Statistical evaluation:
No statistically significant increase in the mean Tail Intensity (%) was observed in liver cells, stomach cells and duodenum cells of Accelerator (PT 25E or PT 25E/2)-treated male animals at any of the dose levels tested compared to the vehicle treated animals.


The concentrations analysed in the formulations were considered in agreement with target concentrations (i.e. mean accuracies between 102% and 106%). No test item was detected in the vehicle control samples.

Liver

The mean Tail Intensity (%) in liver cells of vehicle treated male rats was 4.66 ± 1.60% (Mean± SD). The mean vehicle control Tail Intensity was within the historical control data range. The positive control EMS showed a mean Tail Intensity of 98.05 ± 0.48% (Mean± SD,21-fold induction; p<0.001). Overall it was concluded that the comet assay in liver was valid.

Stomach (Experiment 1)

No statistically significant increase in the mean Tail Intensity (%) was observed in stomach cells of Accelerator (PT 25E or PT 25E/2)-treated male animals at any of the dose levels tested compared to the vehicle treated animals.

The mean Tail Intensity (%) in stomach cells of vehicle treated male rats was 78.50 ± 7.92% (Mean± SD). The mean vehicle control Tail intensity was above the historical control data range. The positive control EMS showed a mean Tail Intensity of 99.01 ± 0.83% (Mean± SD,1.3-fold induction; p<0.001).

Overall the acceptability criteria of the test were not met; the percentage tail intensity of the solvent control outside the laboratory historical control data range. Therefore although no increase in Tail intensity (%) is observed no conclusion can be drawn about the potential DNA damaging properties of Accelerator (PT 25E or PT 25E/2) in glandular stomach. A repeat experiment was performed.

Stomach (Repeat)

No statistically significant increase in the mean Tail Intensity (%) was observed in stomach cells of Accelerator (PT 25E or PT 25E/2)-treated male animals at any of the dose levels tested compared to the vehicle treated animals. The mean Tail Intensity (%) in stomach cells of vehicle treated male rats was 70.33 ± 3.42% (Mean± SD). The mean vehicle control Tail intensity was within the historical control data range. The positive control EMS showed a mean Tail Intensity of 95.47 ± 0.62% (Mean± SD,1.4-fold statistically significant induction; Studentsttest p<0.001). Overall it was concluded that the comet assay (repeat) in stomach was valid.

Duodenum (Experiment 1)

No statistically significant increase in the mean Tail Intensity (%) was observed in duodenum cells of Accelerator (PT 25E or PT 25E/2)-treated male animals at any of the dose levels tested compared to the vehicle treated animals.

The mean Tail Intensity (%) in duodenum cells of vehicle treated male rats was 74.13 ± 11.25% (Mean± SD). The mean vehicle control Tail intensity was above the historical control data range.The positive control EMS showed a mean Tail Intensity of 99.58 ± 0.25% (Mean± SD,1.3-fold induction; p<0.001).

Overall the acceptability criteria of the test were not met; the percentage tail intensity of the solvent control is outside the laboratory historical control data range. Therefore although no increase in Tail intensity (%) is observed no conclusion can be drawn about the potential DNA damaging properties of Accelerator (PT 25E or PT 25E/2) in duodenum. A repeat experiment was performed.

Duodenum (Repeat)

No statistically significant increase in the mean Tail Intensity (%) was observed in duodenum cells of Accelerator (PT 25E or PT 25E/2)-treated male animals at any of the dose levels tested compared to the vehicle treated animals.

The mean Tail Intensity (%) in duodenum cells of vehicle treated male rats was 55.34 ± 15.40% (Mean± SD). The mean vehicle control Tail intensity was within the historical control data range.The positive control EMS showed a mean Tail Intensity of 97.03 ± 0.51% (Mean± SD,1.8-fold induction; p<0.001). Overall it was concluded that the comet assay (repeat) in duodenum was valid.

Overall the results in liver, stomach and duodenum show that there is no statistically significant induction and thus also no trend of induction in the Tail Intensity (%) in Accelerator (PT 25E or PT 25E/2)-treated male animals.

Conclusions:
It is concluded that the comet assay in liver, duodenum and glandular stomach were valid and Accelerator (PT 25E or PT 25E/2) does not provoke DNA damage in the Comet assay up to and including a dose of 750 mg/kg bw/day (the maximum tolerated dose in accordance with current regulatory guidelines).
Executive summary:

Accelerator (PT 25E or PT 25E/2) was tested in the alkaline in vivo Comet assay in male rats, to evaluate its potential genotoxic effect in liver, glandular stomach and duodenum cells. The study procedures described in this report were based on the most recent OECD guidelines. The test item was dissolved in propylene glycol. Formulation analysis was performed to determine the accuracy of preparation of the test substance in formulations. The concentrations analysed in the formulations were in agreement with target concentrations (i.e. mean accuracies between 102% and 106%) . No test item was detected in the vehicle control samples. Based on the dose range finding study a dose of 750 mg/kg bw/day was selected as highest dose for the main study (maximum tolerated dose).

One experiment was perfomed for liver and two experiments were performed for glandular stomach and duodenum since the first experiment did not pass the acceptance criteria for the control group.

In the main studies, groups of 5 male animals were dosed once daily via oral gavage with vehicle or with 187.5, 375 and 750 mg Accelerator (PT 25E or PT 25E/2) per kg body weight for three consecutive days. Three additional satellite animals were treated with the highest test concentration to replace possible deads. A positive control group (5 male rats) for the comet assays was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) per kg body weight. The following treatment related clinical signs were observed the groups treated with 750 mg Accelerator (PT 25E or PT 25E/2)/kg body weight in experiment 1: lethargy, tremors, hunched posture, quick breathing, convulsions and ventral recumbency. One animal died within 15 minutes after the third dose. This animal was replaced by a satellite animal. The treatment related clinical signs in the repeat assay were similar. The following treatment related clinical signs were observed in the groups treated with 750 mg Accelerator (PT 25E or PT 25E/2)/kg body weight in the repat assay: lethargy, tremors, ataxia and ventral recumbency. Approximately 3-4 hours after the second dose of EMS and third dose of the vehicle or Accelerator (PT 25E or PT 25E/2), liver, glandular stomach, and duodenum tissue were collected. Single cell suspensions were made followed by Comet slide preparation and assessment of Tail Intensity (%). No biologically relevant increase in the mean Tail Intensity (%) was observed in liver cells of Accelerator (PT 25E or PT 25E/2) treated male animals compared to the vehicle treated animals (3.23%, 3.55% and 2.62% at 187.5 mg/kg bw, 375 mg/kg bw and 750 mg/kg bw, respectively) . The mean Tail Intensity (%) in liver cells of vehicle treated male rats was 4.66 ± 1.60% (Mean± SD). The mean Tail Intensity was within the historical control data range for the vehicle control group. The positive control EMS showed a mean Tail Intensity of 98.05 ± 0.48% (Mean± SD,21-fold induction; statistically significant according to Studentsttest p<0.001). Overall it was concluded that the comet assay in liver was valid.

No statistically significant increase in the mean Tail Intensity (%) was observed in glandular stomach and duodenum cells of Accelerator (PT 25E or PT 25E/2)-treated male animals at any of the dose levels tested compared to the vehicle treated animals in experiment 1.

The vehicle control Tail intensities in glandular stomach and duodenum, were above the historical control data range in experiment 1. Therefore the acceptability criteria of these assays were not met; the percentage tail intensity of the solvent control is outside the laboratory historical control data range.

The mean Tail Intensity (%) in stomach cells of vehicle treated male rats was 78.50 ± 7.92% (Mean± SD) in experiment 1. The positive control EMS showed a mean Tail Intensity of 99.01 ± 0.83% (Mean± SD,1.3-fold induction; statistically significant according to Studentsttest p<0.001).

The mean Tail Intensity (%) in duodenum cells of vehicle treated male rats was 74.13 ± 11.25% (Mean± SD). The positive control EMS showed a mean Tail Intensity of 99.58 ± 0.25% (Mean± SD,1.3-fold induction; statistically significant according to Studentsttest p<0.001). Although no increase in Tail intensity (%) in glandular stomach and duodenum was observed for any treatment group in experiment 1 (82.53%, 73.25% and 65.93% for stomach and 80.78%, 55.08% and 41.77% for duodenum at 187.5 mg/kg, 375 mg/kg and 750 mg/kg, respectively), no conclusion could be drawn about the potential DNA damaging properties of Accelerator (PT 25E or PT 25E/2) in duodenum and glandular stomach since the acceptance criteria for the control group were not passed. The mean vehicle control Tail intensity was above the historical control data range in stomach and duodenum.

Therefore a repeat experiment was performed for glandular stomach and duodenum. No statistically significant increase in the mean Tail Intensity (%) was observed in glandular stomach (70.33%) and duodenum cells (55.34%) of Accelerator (PT 25E or PT 25E/2)-treated male animals at any of the dose levels tested compared to the vehicle treated animals in the repeat experiment.

The mean Tail Intensity (%) in stomach cells of vehicle treated male rats was 70.33 ± 3.42% (Mean± SD). The mean vehicle control Tail intensity was within the historical control data range. The positive control EMS showed a mean Tail Intensity of 95.47 ± 0.62% (Mean± SD,1.4-fold induction; p<0.001). Overall it was concluded that the comet assay (repeat) in glandular stomach was valid.

The mean Tail Intensity (%) in duodenum cells of vehicle treated male rats was 55.34 ± 15.40% (Mean± SD). The mean vehicle control Tail intensity was within the historical control data range. The positive control EMS showed a mean Tail Intensity of 97.03 ± 0.51% (Mean± SD,1.8-fold induction). Overall it was concluded that the comet assay (repeat) in duodenum was valid.

Overall the results in liver, stomach and duodenum show that there is no statistically significant induction and thus also no trend of induction in the Tail Intensity (%) in Accelerator (PT 25E or PT 25E/2)-treated male animals.

It is concluded that the comet assay in liver, duodenum and glandular stomach is valid and Accelerator (PT 25E or PT 25E/2) does not provoke DNA damage in the Comet assay up to and including a dose of 750 mg/kg bw (the maximum tolerated dose in accordance with current regulatory guidelines).

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 July 2018 - 16 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This study was specifically requested by ECHA (Decision Number TPE-D-2114310297-55-01-/F).
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Wistar
Remarks:
WI (Han)
Details on species / strain selection:
These rats are recommended by international guidelines (OECD, EC).
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: mean body weights 146 ± 11 g - 165 ± 1 g
- Assigned to test groups randomly: yes
- Fasting period before study: A limited quantity of food was supplied during the night before dosing
- Housing: Group housing of maximum 5 animals per sex in labeled Macrolon cages
- Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: free access to tap-water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 22
- Humidity (%): 50 - 64
- Air changes (per hr): 10 or greater
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03 July 2018 To: 05 July 2018
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: propylene glycol
- Justification for choice of vehicle: The substance dissolved in propylene glycol and the test substance is reported to be stable in the vehicle in the range of 2 - 200 mg/mL for 6 hours (as determined during WIL Research project 501370, summarized under 7.5.1). Furthermore, the results of this repeated dose study indicate that propylene glycol is an adequate solvent for this test item in an animal test with oral exposure.
- Concentration of test material in vehicle: 37.5, 75 and 150 mg/kg body weight
- Amount of vehicle: 5 mL/kg bw
- The specific gravity of propylene glycol is 1.036 g/mL.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The substance was dissolved in propylene glycol and the concentrations were treated with ultra-sonic waves to obtain a homogeneous solution, concentrations were dosed within 2.5 hours after preparation.
The individual dosing volume (mL) were calculated on the basis of the body weight measured at the time of assignment to dose groups.

Duration of treatment / exposure:
Three consecutive days.
Frequency of treatment:
Once daily.
Post exposure period:
3 - 4 hours
Dose / conc.:
187.5 mg/kg bw/day (actual dose received)
Dose / conc.:
375 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Five
Control animals:
yes, concurrent vehicle
Positive control(s):
EMS (ethylmethanesulphonate)
- Route of administration: oral
- Doses / concentrations: 200 mg/kg body weight dissolved in physiological saline, the dosing volume was 10 mL/kg body weight.
Tissues and cell types examined:
Stomach and duodenum tissue.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on the previous Comet assay (CRL project 511920), three doses were selected for the main study.

TREATMENT AND SAMPLING TIMES:
The animals were dosed with vehicle or test substance for three consecutive days and twice with EMS. Approximately 3-4 hours after the third treatment with the test item or vehicle and second treatment with EMS the stomach and duodenum were collected/isolated and examined for DNA damage with the alkaline Comet assay.

DETAILS OF SLIDE PREPARATION (In order to comply with the information requested by ECHA for glandular stomach and duodenum this new study was initiated with a modified method):
Isolation of glandular stomach cells:
The stomach was cut open and washed free from food using cold Hank’s Balanced Salt Solution (HBSS; Ca++, Mg++ free, Life Technologies, Breda, the Netherlands). The fore-stomach was removed and discarded. The glandular stomach was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA (Merck, Darmstadt, Germany)).
The glandular stomach was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The surface epithelia of the glandular epithelia was gently scraped 3-4 times with a cell scraper, as requested by ECHA. This layer was discarded since the lifetime of these cells is very short in the body with a maximum of 3 days. Therefore this layer contains a high amount of apoptotic cells which disturb the interpretation in the Comet assay. Moreover, since the lifetime of these cells is very short it is unlikely that these cells play a role in carcinogenesis.
The glandular stomach was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. The stomach was then scraped multiple times with a cell scraper and the cells were collected in the mincing buffer present in the petri-dish. The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution, pH 7.5 (DMSO (Merck) was added immediately before use).
The cell suspension was filtered through a 100 µm Cell Strainer (Falcon, Corning life Sciences, Tewksbury, United States) to purify the cell suspension and collected in a tube and stored on ice.

Isolation of duodenum cells:
The duodenum was cut open and washed free from food using Hank’s Balanced Salt Solution (HBSS; Ca++, Mg++ free). The duodenum was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA).
The duodenum was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The surface epithelia of the glandular epithelia was gently scraped 3-4 times with a cell scraper, as requested by ECHA, to remove apoptotic cells in the upper cell layer. This layer was discarded.
The duodenum was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. The duodenum was then scraped multiple times with a cell scraper and the cells are collected in the mincing buffer present in the petri-dish.
The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution (HBSS) (Ca++, Mg++ free, and phenol red free if available), pH 7.5 (DMSO was added immediately before use).
The cell suspension was filtered through a 100 µm Cell Strainer to purify the cell suspension and collected in a tube and stored on ice.

Sampling, fixation and storage of tissue for histotechnology and histopathology:
Part of stomach and duodenum from all animals was collected and fixed and stored in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No histotechnology and histopathology was performed.

Preparation of Slides:
To 20 µL of the cell suspension, 280 µL melted low melting point agarose (LMAgarose; Trevigen, Gaithersburg, USA) was added. The cells were mixed with the LMAgarose and
50 µL was layered on a pre-coated Comet slide (Trevigen) in duplicate. Three slides per tissue per animals were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for 15 - 17 minutes in the refrigerator in the dark until a clear ring appears at the edge of the Comet slide area.

Lysis, Electrophoresis and Staining of the Slides:
The cells on the slides were overnight (17-19 h) immersed in pre-chilled lysis solution (Trevigen) in the refrigerator. After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4, Sigma Aldrich). The slides were then placed in freshly prepared alkaline solution (Merck) for 20 minutes at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 0.7 Volt/cm, as requested by ECHA. The electrophoresis was performed for 20 minutes under constant cooling (actual temperature 4.5-5°C). After electrophoresis the slides were immersed/rinsed in neutralization buffer for 5-6 minutes. The slides were subsequently immersed for 5 minutes in Absolut ethanol (≥99.6%, Merck) and allowed to dry at room temperature. The slides were stained for approximately 5 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Bleiswijk, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark and fixed with a coverslip.

METHOD OF ANALYSIS:
To prevent bias, slides were randomly coded (per tissue) before examination of the Comets. An adhesive label with study identification number and code were placed over the marked slide. The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system (Perceptive instruments Ltd, Suffolk, United Kingdom). One hundred fifty Comets (50 comets of each replicate LMAgarose circle) were examined per sample. On a few slides, one of the agarose circles was damaged, therefore an agarose circle from the second backup slide was used for scoring.
The following criteria for scoring of Comets were used:
• Only horizontal orientated Comets were scored, with the head on the left and the tail on the right.
• Cells that showed overlap or were not sharp were not scored.
In addition the frequency of hedgehogs was determined and documented based on the visual scoring of at least 150 cells per tissue per animal in the repeat experiment. The occurrence of hedgehogs was scored in all treatment groups and the control. Since there was no effect of the test item Hedgehogs data was not reported and maintained in the raw data.

OTHER:
Evaluation criteria:
A test item is considered positive in the Comet assay if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity compared with the concurrent negative control
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative in the Comet assay if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.

Adopted acceptability criteria as requested by ECHA:
The in vivo comet is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent negative control data are considered acceptable when the means of the %DNA in tail are below 20%.
c) The positive control EMS should produce at least a statistically significant increase in the percentage Tail Intensity compared to the vehicle treated animals. The response should be compatible with the data in the historical control database. The positive control data will be analyzed by the Students t test (one-sided, p < 0.05) in case of homogeneous variances or by the Welch t test in case of inhomogeneous variances (one-sided, p < 0.05).
d) Adequate numbers of cells and doses have been analysed
e) The highest test dose is the MTD or 2000 mg/kg/day.
Statistics:
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
see section additional information on results
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Mortality and toxic signs:
The animals of the group treated with the positive control showed no treatment related clinical signs of toxicity or mortality.
Some minor effects were observed in the negative control group, low and mid dose group:
- All animals of the negative control group were lethargic and one animal showed ataxia within one hour after the first dosing. After approximately 21 hour the animals showed no abnormalities. Within one hour after the second and third treatment three animals showed no treatment related effects. The other two animals were lethargic and showed ataxia.
- All animals of the low (187.5 mg/kg bw) and mid dose group (375 mg/kg bw) were lethargic and one animal showed ataxia within one hour after the first dosing. After approximately 21 hour the animals showed no abnormalities. Within one hour after the second and third treatment the animals showed no treatment related effects with exception of one animal in the mid dose group and two animals in the low dose group which were lethargic after the second and third dosing. In addition one animal in the low dose group showed ataxia after the third dose.

- The following effects were observed in the high dose group: one animal was lethargic and showed ataxia after the first dosing. After approximately 21 hour the animal showed no abnormalities. This animals was lethargic and showed ataxia after the second and third dosing as well. The effects in a second animal were the same with the exception that the animal showed only lethargy after the first dosing. The remaining animals treated with 750 mg/kg bw died during the study (3/5), one after the first dosing and two after the second dosing.

DNA damage:
Glandular stomach:
No statistically significant increase in the mean Tail Intensity (%) was observed in stomach cells of Accelerator (PT 25E or PT 25E/2)-treated male animals at any of the dose levels tested compared to the vehicle treated animals.
The mean Tail Intensity (%) in stomach cells of vehicle treated male rats was 0.91 ± 0.63% (Mean ± SD). The mean vehicle control Tail Intensity was within the 95% control limits of the distribution of the historical negative control database and the mean Tail Intensity of the vehicle control was below 20%.
The positive control EMS was significantly increased and showed a mean Tail Intensity of 47.57% ± 3.32% (mean ± SD, 52-fold induction; p<0.001 Students t test). The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database.
Duodenum:
No statistically significant increase in the mean Tail Intensity (%) was observed in duodenum cells of Accelerator (PT 25E or PT 25E/2)-treated male animals at any of the dose levels tested compared to the vehicle treated animals.
The mean Tail Intensity (%) in duodenum cells of vehicle treated male rats was 2.47 ± 0.65% (Mean ± SD). The mean vehicle control Tail Intensity was within the 95% control limits of the distribution of the historical negative control database and the mean Tail Intensity of the vehicle control was below 20%.
The positive control EMS was significantly increased and showed a mean Tail Intensity of 45.39% ± 9.00% (mean ± SD, 18-fold induction; p<0.001 Students t test). The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database.
All negative and positive control values in glandular stomach and duodenum were within the acceptance criteria. Moreover, three dose levels were analyzed, 150 cells were scored per animal and the highest test item concentration was the MTD. Hence, all criteria for an acceptable Comet assay in glandular stomach and duodenum were met. The present study which was performed with a modified method resulted in low negative control tail intensities (<20%) as requested by ECHA.

Chemical analysis of dose preparations:
The concentrations analysed in the formulations were considered in agreement with target concentrations (i.e. mean accuracies between 93% and 98%). No test item was detected in the vehicle control samples.

The individual values for each animal, historical data and the results of the chemicals analysis are attached in the section "attached background material".


Dose formulation analysis:

The concentrations analyzed in the test item formulations were in agreement with target concentrations (i.e. mean accuracies between 93% and 98%). No test item was detected in the control formulation. The details are attached under "Attached background material".

Overview mean Tail Intensity in stomach Cells of Male Rats (individual data are attached under "Attached background material")

 

 

 

Tail Intensity (%)

S.D.

 

Vehicle Control

 

0.91

0.63

Accelerator (PT 25E or PT 25E/2)

187.5 mg/kg bw/day

0.91

 

0.22

 

375 mg/kg bw/day

0.71

0.39

 

750 mg/kg bw/day

0.69

0.20

EMS

200 mg/kg bw/day

47.57***

3.32

***= p < 0.001(Student’sttest); EMS = Ethyl Methanesulfonate; Vehicle Control = Propylene glycol

 

 

Overview mean Tail Intensity in duodenum Cells of Male Rats (individual data are attached under "Attached background material")

 

 

 

Tail Intensity (%)

S.D.

 

Vehicle Control

 

2.47

0.65

Accelerator (PT 25E or PT 25E/2)

187.5 mg/kg bw/day

1.23

0.69

 

375 mg/kg bw/day

1.16

0.52

 

750 mg/kg bw/day

2.41

0.08

EMS

200 mg/kg bw/day

45.39***

9.00

***= p < 0.001(Student’sttest); EMS = Ethyl Methanesulfonate; Vehicle Control = Propylene glycol

 

Conclusions:
In an alkaline Comet assay performed according to OECD guideline 489 and GLP principles, Accelerator (PT 25E or PT 25E/2) did not induce DNA damage in glandular stomach and duodenum cells when tested up to and including a maximum oral dose of 750 mg/kg bw in rats.
Executive summary:

An in vivo alkaline Comet assay was conducted according to OECD guideline 489 and GLP principles using Wistar WI (Han) male rats to assess the potential of Accelerator (PT 25E or PT 25E/2) to induce DNA damage. Based on on a previous Comet assay (CRL project 511920), the maximum tolerated dose was concluded to be 750 mg/kg bw/day, therefore the assay was performed with three dose levels 187.5, 375 and 750 mg/kg bw.

No mortality and no or minor treatment related clinical signs were noted in the animals treated with 187.5 or 375 mg/kg bw Accelerator (PT 25E or PT 25E/2) or control animals receiving vehicle or EMS. At the highest dose, 3/5 rats died, the two remaining animals showed ataxia and were lethargic.

No statistically significant increase in the mean Tail Intensity (%) was observed in stomach cells and in duodenum cells of Accelerator (PT 25E or PT 25E/2)-treated male animals at any of the dose levels tested compared to the vehicle treated animals. The mean Tail Intensity (%) in stomach cells of vehicle treated male rats was 0.91 ± 0.63%. The mean vehicle control Tail Intensity was within the 95% control limits of the distribution of the historical negative control database and the mean Tail Intensity of the vehicle control was below 20%. The positive control EMS was significantly increased and showed a mean Tail Intensity of 47.57%± 3.32%. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database. The mean Tail Intensity (%) in duodenum cells of vehicle treated male rats was 2.47 ± 0.65%. The mean vehicle control Tail Intensity was within the 95% control limits of the distribution of the historical negative control database and the mean Tail Intensity of the vehicle control was below 20%. The positive control EMS was significantly increased and showed a mean Tail Intensity of 45.39%± 9.00%. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database.

The present study was performed with a modified method compared to the previous Alkaline in vivo comet study (CRL project 511920) with the same test material and the same dose levels. This adjusted protocol resulted in low negative control tail intensities (<20%) as requested by ECHA. Lethargy and ataxia were observed in both the mid and high dose groups, confirming systemic exposure. At the highest dose, 3/5 rats died. Based on the previous study this dose was considered to be the MTD, and this high mortality rate was not expected. The present study gave negative results in the 2 surviving animals of the highest dose group and in the animals of the mid and low dose group, in glandular stomach and duodenum and, consequently, confirmed the result of the earlier study (CRL project 511920). Although the number in the high dose group was limited, all data taken together were considered sufficient to draw a conclusion on this endpoint. It was thus considered not justified both scientifically and from animal welfare perspective to perform further testing at an additional dose level.

In conclusion, the test is valid and it is concluded that Accelerator (PT 25E or PT 25E/2) does not cause DNA damage in the Comet assay in glandular stomach and duodenum cells.

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

Additional information

AMES test:

An Ames test was performed according to OECD guideline and GLP principles, with Salmonella typhimurium strains TA100, TA1535, TA 102, TA 98, TA 1537, without and with metabolic activation. No cytotoxicity or test substance precipitation was observed when tested up to 5000 µg/plate. In experiment 1, a significant increase in revertants seen at 5000 µg/plate in strains TA100 and TA1535 without S9-mix (p≤ 0.01) was recorded. This was confirmed in experiment 2 (p≤ 0.005) and a significant increase was also seen at 5000 µg/plate in strain TA100 with S9-mix (p≤ 0.05). In a third experiment without metabolic activation, a significant number of revertants was seen in strain TA100 at 2000, 3000 (both p≤ 0.05), 4000 and 5000 µg/plate (both p≤ 0.005). Therefore, based on these data it is concluded that Bisomer PTE is mutagenic under the conditions of this test.

Chromosome Aberration test:

A chromosome aberration study with Accelerator (PT 25E or PT 25E/2) was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. No precipitation of the substance was observed, but the test substance was tested up to and beyond cytotoxic concentrations. Both in the absence and presence of S9-mix Accelerator (PT 25E or PT 25E/2) did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. It can be concluded that Accelerator (PT 25E or PT 25E/2) does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

In vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells:

An in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells, performed according to OECD 476 and following GLP principles was performed. The test substance was tested up to cytotoxic concentrations. The positive control data and the solvent control data validated the study. In the presence of S9-mix, Accelerator (PT 25E or PT 25E/2) did not induce a significant increase in the mutation frequency in two experiments. In the absence of S9-mix, the test substance induced a 5.3-fold increase in the mutation frequency at the prolonged treatment period of 24 hours. This was verified in a further experiment, in which the substance induced an up to 3.3-fold increase in the mutation frequency. The mutation frequency of both the small and large colonies was significantly increased.

Based on these data, it is concluded that the test substance is positive in the in vitro mammalian cell gene mutation test.

First alkaline in vivo Comet assay in liver, stomach and duodenum of rats:

The alkaline in vivo Comet assay with Accelerator (PT 25E or PT 25E/2) was performed according to OECD guideline and GLP principles. One experiment was perfomed for liver and two experiments were performed for glandular stomach and duodenum since the first experiment did not pass the acceptance criteria for the control group. Groups of 5 male rats were dosed once daily via oral gavage with vehicle or with 187.5, 375 and 750 mg Accelerator (PT 25E or PT 25E/2) per kg body weight for three consecutive days. A positive control group was included.

Animals dosed with 750 mg/kg bw/day showed clinical signs including lethargy, tremors, hunched posture and ataxia. Approximately 3-4 hours after the second dose of EMS and third dose of the vehicle or Accelerator (PT 25E or PT 25E/2), liver, glandular stomach, and duodenum tissue were collected, and single cell suspensions were made followed by Comet slide preparation. No biologically relevant increase in the mean Tail Intensity (%) was observed in liver cells. No statistically significant increase in the mean Tail Intensity (%) was observed in glandular stomach and duodenum cells. As the acceptance criteria for the control group were not passed for glandular stomach and duodenum, no conclusion could be drawn about the potential DNA damaging properties of Accelerator (PT 25E or PT 25E/2) in these tissues. Therefore a repeat experiment was performed for these tissues, in which no statistically significant increase in the mean Tail Intensity (%) was observed in test substance treated animals. Overall the results in liver, glandular stomach and duodenum show that there is no statistically significant induction and thus also no trend of induction in the Tail Intensity (%) in Accelerator (PT 25E or PT 25E/2)-treated male animals.

It is concluded that the Comet assay in liver, duodenum and glandular stomach is valid and Accelerator (PT 25E or PT 25E/2) does not provoke DNA damage in the Comet assay up to and including a dose of 750 mg/kg bw/day (the maximum tolerated dose in accordance with current regulatory guidelines).

Second alkaline in vivo Comet assay in liver, stomach and duodenum of rats, requested by ECHA with a modified method and adopted acceptability criteria:

An in vivo alkaline Comet assay was conducted according to OECD guideline 489 and GLP principles using Wistar WI (Han) male rats to assess the potential of Accelerator (PT 25E or PT 25E/2) to induce DNA damage. Based on on a previous Comet assay (CRL project 511920), the maximum tolerated dose was concluded to be 750 mg/kg bw/day, therefore the assay was performed with three dose levels 187.5, 375 and 750 mg/kg bw.

No mortality and no or minor treatment related clinical signs were noted in the animals treated with 187.5 or 375 mg/kg bw Accelerator (PT 25E or PT 25E/2) or control animals receiving vehicle or EMS. At the highest dose, 3/5 rats died, the two remaining animals showed ataxia and were lethargic.

No statistically significant increase in the mean Tail Intensity (%) was observed in stomach cells and in duodenum cells of Accelerator (PT 25E or PT 25E/2)-treated male animals at any of the dose levels tested compared to the vehicle treated animals. The mean Tail Intensity (%) in stomach cells of vehicle treated male rats was 0.91 ± 0.63%. The mean vehicle control Tail Intensity was within the 95% control limits of the distribution of the historical negative control database and the mean Tail Intensity of the vehicle control was below 20%. The positive control EMS was significantly increased and showed a mean Tail Intensity of 47.57%± 3.32%. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database. The mean Tail Intensity (%) in duodenum cells of vehicle treated male rats was 2.47 ± 0.65%. The mean vehicle control Tail Intensity was within the 95% control limits of the distribution of the historical negative control database and the mean Tail Intensity of the vehicle control was below 20%. The positive control EMS was significantly increased and showed a mean Tail Intensity of 45.39%± 9.00%. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database.

The present study was performed with a modified method compared to the previous Alkaline in vivo comet study (CRL project 511920) with the same test material and the same dose levels. This adjusted protocol resulted in low negative control tail intensities (<20%) as requested by ECHA. Lethargy and ataxia were observed in both the mid and high dose groups, confirming systemic exposure. At the highest dose, 3/5 rats died. Based on the previous study this dose was considered to be the MTD, and this high mortality rate was not expected. The present study gave negative results in the 2 surviving animals of the highest dose group and in the animals of the mid and low dose group, in glandular stomach and duodenum and, consequently, confirmed the result of the earlier study (CRL project 511920).Although the number in the high dose group was limited, all data taken together were considered sufficient to draw a conclusion on this endpoint. It was thus considered not justified both scientifically and from animal welfare perspective to perform further testing at an additional dose level.

In conclusion, the test is valid and it is concluded that Accelerator (PT 25E or PT 25E/2) does not cause DNA damage in the Comet assay in glandular stomach and duodenum cells.

Justification for classification or non-classification

Based on the current data-set, Accelerator (PT 25E or PT 25E/2) does not need to be classified for genotoxicity in accordance with Regulation (EC) No 1272/2008 and GHS.