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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

3 genotoxicity studies were performed on E17 -194T.

1. Ames test (OECD 471) was conducted with E17 -194T at levels of 1.58, 5.0, 15.8, 50, 158, 500, 1580 and 5000 microgram/plate..No signs of precipitation or contamination were noticed in any of the strains at all dose levels. No signs of toxicity were seen in any of the strains in any dose levels.There was no contamination-related or substantial test substance related increases in the number of revertant colonies observed with all strains tested in both in the absence and presence of S9 using either plate incorporation or the pre incubation method. Based on these findings and on evaluation system used E17-194T did not elicit evidence of bacterial mutagenicity in the Ames assay.

2. In Vitro Mammalian Cell Micronucleus Test (OECD 487) was performed to assess the genotoxic potential of E17-194Tto induce micronuclei in human lymphocytes cultured in vitro in absence and presence of an exogenous metabolic activation system (liver S9 mix from male rats). Two valid and independent experiments were performed. No cytotoxicity was observed at any of the test item concentrations. Neither a statistically significant nor a biologically relevant increase in the number of binucleated cells containing micronuclei at the evaluated concentrations were observed. All positive control compounds caused large, statistically significant increases in the proportion of binucleate cells with micronuclei, demonstrating the sensitivity of the test system.

In conclusion, under the experimental conditions reported,E17-194Tdoes not induce the formation of micronuclei in human lymphocytes in vitro.

3. An in vitro mammalian gene mutation test using the Hprtgenes (OECD 476) was performed to investigate the potential of the test itemE17-194Tto induce mutations at the HPRT locuson chromosome X Chinese Hamster V79 cells. The assay was performed in two independent experiments, using two parallel cultures each (duplicates).In experiment I, 6 concentrations of the test item were tested with and without metabolic activation. The exposure time was 4 hours. The following nominal concentrations of the test item were investigated in experiment I: +S9 and -S9: 10.00 mM, 5.00 mM, 2.50 mM, 1.25 mM, 0.63 mM, 0.31 mM

Precipitation of the test item was not observed up to the highest tested concentration. EMS (750 µg/mL) and DMBA (5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies, without substantial reduction of the viability.

 No cytotoxic effects were observed in any of the test item concentrations in experiment I. Therefore, all concentrations were analysable for mutagenicity.

No dose dependent increase in mutant colony numbers was observed in experiment I.No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration.All values remained within the historical control range. Therefore, the result of experiment I is clearly negative. In conclusion it can be stated that under the experimental conditions of this studyE17-194Tdid not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation. Therefore, the test itemE17-194T is considered to be “non-mutagenic under the conditions of the HPRT assay”.


 

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan 9 2018 - Feb 29 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)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
composition 100%
Clear transparent liquid
The test substance was expected to be stable for the duration of the test
Expiration date: November 3, 2020
Target gene:
S. typhimurium: histidine
E. coli: tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: TA1535: rfa, uvrB; 1537: rfa, uvrB; 98: rfa, uvrB, R-factor; 100: rfa, uvrB, R-factor
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: uvrA
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (cofactor supplemented post-mitochondrial fraction) sourced from mate Sprague-Dawley rats induced with phenobarbital and benzoflavone.
Test concentrations with justification for top dose:
The test was conducted with E17-194T at levels of 1.58, 5, 15.8, 50, 158, 500, 1580 and 5000 microgram per plate, with the high level being the standard limit for this test.
Vehicle / solvent:
sterile water
Untreated negative controls:
yes
Remarks:
Test material not incorporated
Negative solvent / vehicle controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: Daunomycin ; 2-Aminoanthracene
Details on test system and experimental conditions:
The initial test followed the plate incorporation method, in which the following materials were mixed and poured over the surface of minimal agar plate:
10 microliter of prepared test substance, negative (vehicle) control, or prepared positive control substance.
500 microliter S9 mix or substitution buffer
100 microliter bacteria suspension (ST or EC)
2000 microliter overlay agar maintained at approx. 45 degrees C

Plates were prepared in triplicate at each experimental point and uniquely identified. after pouring, plates were placed on a level surface until the agar gelled then inverted and incubated at approx. 37 degrees C until growth was adequate for enumeration (approx 65 hours). appropriate sterility control check plates (treated with critical components in the absence of bacteria) were incubated as a standard procedural check. For the bacterial strains, plates were prepared for each treatment as follows (see table 1 below).
Conformatory test: this test employed the pre-incubation modification of the plate incorporation test. The test or control substances, bacteria suspension and S9/substitution buffer were incubated under agitation for approximately 30 minutes at approx. 37 degrees C prior to mixing with the overlay agar and pouring onto the minimal agar plates before proceeding as described for the initial test. The atudy design for the confirmatory test, including strains, dose levels ect. was as described above for the initial test.
A supplemental test wes performed to clarify results obtained with TA1537 in the initial phase of testing. The study utilized the same methodolgies and dose levels described above in the original phase of testing. Appropriate vehicle and positive controls were included.


Rationale for test conditions:
The background lawn for vehicle control plates should appear normal. The mean revertant colony counts for each strain treated with the vehicle should lie close to or within the expected range taking into account the laboratory historical control range / or published values. The positive controls (with S9 where required) should produce substantial increases in revertant colony numbers with the appropriate bacterial strain as specified below.
Evaluation criteria:
Evalation of Toxicity: Toxic effects of the test substance are indicated by the partial or complete absence of a background lawn of the non-revertant bacteria or a substantial dose-related reduction in revertant colony counts compared with lower dose levels and concurrent vehicle control taking into account the laboratory historical control range. Where precipitation obscures observations on the condition of the background lawn, the lawn can be considered normal and intact if the revertant colony counts are within the expected range based on results for lower dose levels and historical counts for that strain.
Evaluation of Mutagenicity: For each experiment point, the mutation factor (MF) was calculated by dividing the mean revertant colony count by the mean revertant colony count for the corresponding concurrent vehicle control group. The mutagenic activity of the test item was assessed by applying the following criteria:

Positive results (mutagenic potential): The results for the test item showed a substantial increase in revertant colony counts, i.e., response MF ≥ 2 for strains TA98, TA 100 and WP2 uvrA or MF ≥ 3 for strains TA1535 and TA 1537, with mean value(s) outside the laboratory historical control range. Otherwise, results were considered negative.
The above increae must be dose related and/or reproducible, i.e., increses must be obtained at more than one experimental point. if this criteria is not met, the results may be classified as equivocal and further testing may be appropriate.
A test substance that produces neither a concentration related increase in the number of revertant colonies nor a reproducible substantial increase in revertant colonies to be non-mutagenic in this test system.
Statistics:
means and standard deviations for all quantitative data were collected by the lab.
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
pre incorporation method-main test
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
pre-incubation method- confirmatory test
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
plate incorporation method-supplemental main test 1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
pre-incubation method-supplemental confirmatory test 1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
plate incorportaion method - main test
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
pre-incubation method - confirmatory test
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
plate incorporation method - main test
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
pre-incubation method-confirmatory test
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
plate incorporation method - main test
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
pre-incubation method - confirmatory test
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The mean revertant colony counts for each strain treated with the vehicle were close to or within the expected range, considering the laboratory historical control range and/or published values. The positive control substances caused the expected substantial increses in revertant colony counts in both absence and presence of S9 mix.
No signs of precipitation or contamination were noticed in any of the strains at all dose levels. No signs of toxicity were seen in any of the strains in any dose levels.
There was no contamination-related or substantial test substance related increases in the number of revertant colonies observed with all strains tested in both in the absence and presence of S9 using either plate incorporation or the pre incubation method
Remarks on result:
other: not mutagenic
Conclusions:
Based on these findings and on evaluation system used E17-194T did not elicit evidence of bacterial mutagenicity in the Ames assay.
Executive summary:

Ames test was conducted with E17 -194T at levels of 1.58, 5.0, 15.8, 50, 158, 500, 1580 and 5000 microgram/plate. The main test was conducted using the plate incorporation method in both the absence and presence of metabolic activation. The results of the test were confirmed using a similar study design but employing the pre-incubation modification of the Ames test.

The mean revertant colony counts for each strain treated with the vehicle were close to or within the expected range, considering the laboratory historical control range and/or published values. The positive control substances caused the expected substantial increses in revertant colony counts in both absence and presence of S9 mix.

No signs of precipitation or contamination were noticed in any of the strains at all dose levels. No signs of toxicity were seen in any of the strains in any dose levels.

There was no contamination-related or substantial test substance related increases in the number of revertant colonies observed with all strains tested in both in the absence and presence of S9 using either plate incorporation or the pre incubation method.

Based on these findings and on evaluation system used E17-194T did not elicit evidence of bacterial mutagenicity in the Ames assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 October 2019- 14 July 2020
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)
Version / remarks:
adopted 29. Jul. 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Adopted 30. May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
Name E17-194T
Batch no. 300567-78-7
Appearance Clear, colorless, transparent liquid
Composition Phosphinic acid, P,P diethyl-, mono ester with 1,2 propandiol
Purity 92.9 %
Homogeneity homogeneous
Expiry date 30. Aug. 2022
Storage Room Temperature (20 ± 5 °C)
Source of test item: Bromine Compounds Ltd., Beer-Sheva, Israel

Molecular formula C7 H17 O3 P1     
Molecular weight 180 g/mol
Vapour pressure 0.056 hPa at 20 °C
Stability H2O: not stated; EtOH: not stated; acetone: not stated; CH3CN: not stated; DMSO: not stated
Solubility H2O: > 1 g/L; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown

See also Copy of certificate of analysis- attached
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
The Chinese hamster cells V79 (V79-4 clone) are lung fibroblasts, growing adherent. They have been used successfully in in vitro experiments for many years because of its sensitivity to chemical mutagens. Especially the high proliferation rate (doubling time 12 – 14 h in stock cultures) and a high cloning efficiency (80 %) of untreated cells, both nec-essary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22 (Bradley et al., 1981). The cells were purchased by ATCC and were sold under the name ATCC® CCL-93™.
Metabolic activation:
with and without
Metabolic activation system:
In vitro methods are valuable when it is desired to accurately control the concentration and exposure time of cells to the test item under study. However, due to the limited ca-pacity for metabolic activation of potential mutagens an exogenous metabolic activation system is necessary. For this purpose, the cells were exposed to the test item with (+S9) and without (-S9) exogenous metabolic activation for 4 hrs (experiment I). Following an expression time the descendants of the treated cell population were monitored for the loss of functional HPRT enzyme by culturing in selective medium.
Test concentrations with justification for top dose:
nominal concentrations of the test solution (mM) 100 50 25 12.5 6.25 3.13 1.57
resulting concentrations in the experiment (mM) 10 5 2.5 1.25 0.63 0.32 0.16
Vehicle / solvent:
DMEM
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:

Before starting the experiments an initial test (pre-test) was performed to better define the concentrations to be used in the main experiment (experiment I). In this pre-test only the cytotoxicity expressed as Cloning Efficiency CErel (corresponds to the CE I values of the main experiments, see chapter 7.5, page 20) was determined.
HPRT catalyses the conversion of the nontoxic 6-thioguanine (6-TG) to its toxic ribophos-phorylated derivative. For this purpose, cells deficient in HPRT due to a forward gene mu-tation are resistant to 6-TG. These cells are able to proliferate in the presence of 6-TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 - 9 days.
The mutant frequency was determined by seeding known numbers of cells in DMEM cul-ture medium containing the selective agent to detect mutant cells, and in DMEM culture medium without selective agent to determine the surviving cells. After a suitable period of time the colonies were counted. Mutation frequencies were calculated from the number of mutant colonies corrected for cell survival.
In order to establish a concentration response effect of the test item at least 4 concentra-tions were tested that should yield a concentration related effect. To reduce the risk of false positive or false negative results caused by outliers a minimum of 2 parallel cultures (culture A and culture B) were made. For the analysis of the survival and the viability du-plicates of each culture were made. For the analysis of the mutagenicity 5 replicates of each culture were analysed.
Reference mutagens were tested in parallel to the test item in order to demonstrate the sensitivity of the test system.

6.4.2 Cell Cultures
Prior to use in the experiments, a Master Cell Bank (MCB) and a Working Cell Bank (WCB) were prepared. Stocks of cells were stored in liquid nitrogen in the cell bank of LAUS GmbH. The cells of the MCB were screened for mycoplasma contamination, the doubling time was determined (14h) and the cells were cleansed of pre-existing mutant cells by culturing in HAT medium (medium containing Hypoxanthine, Aminopterin and Thymidine). Cleansed cells build the WCB. Directly after Cleansing the modal chromo-some number was analysed and the cells were expanded (max. 2 passages), stocks were frozen in liquid nitrogen.
MCB and WCB are routinely checked regarding modal chromosome number, doubling time and mycoplasma contamination to allow a continuous working stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells. The data are stored in the archive of LAUS GmbH.
The cells were thawed 9 d prior treatment and cultivated in DMEM complete culture medi-um with 10 % FBS in cell culture flasks at 37.0 ± 1.5 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.
Rationale for test conditions:
Ethylmethane sulfonate (EMS) and 7,12-Dimethylbenzanthracene (DMBA)
Evaluation criteria:
7.6 Acceptability
The gene mutation assay is considered acceptable if it meets the following criteria:
1. the mutant frequency found in the solvent controls falls within the laboratory historical data and should ideally be within the 95% control limit.
2. the positive control substances must produce a significant increase (p < 0.05) in mu-tant frequency compared with the concurrent solvent controls and should induce re-sponses that are compatible with those, generated in the historical data base (within the 95% control limit).
3. two experimental conditions (+S9 and -S9) are tested unless one resulted in positive results.
4. adequate number of cells (treatment: at least 20*106) and never less than 2*106 cells must be cultured during the expression period and plated for mutant selection. At least 4 concentrations, that meet the acceptance criteria are analysable.
5. If the maximum concentration is based on cytotoxicity, the RS of the highest concen-tration is between 20 % and 10 %. If no precipitate or limiting cytotoxicity is observed, the highest test item concentration should be 10 mM, 2 mg/mL or 2 µL/mL and for poor-ly soluble test items paragraph 21 of the OECD 476 has to be fullfilled.

If these criteria are not completely fulfilled, the acceptability of the study should be evalu-ated by the study director. In case of an invalid experiment, the experiment should be re-peated.

Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
It can be stated that under the experimental conditions of this study E17-194T did not in-duce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation.
Therefore, the test item E17-194T is considered to be “non-mutagenic under the condi-tions of the HPRT assay”.
Executive summary:

Summary

This study was performed to investigate the potential of E17-194T to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster cells (V79).

The assay comprised a pre-test and one experiment (experiment I). An experiment II was conducted, to confirm the results of experiment I, but was invalid. Therefor this experiment is not reported, all documentation is kept with the raw data and will be archived at the GLP test facility. The invalid experiment II was not repeated, since experiment I was clearly negative and according to the Guideline OECD476 there is no requirement for verification of a clearly negative response.[CP1] 

The pre-test was done to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the main experiment were determined.

Experiment I was performed with and without metabolic activation (liver S9 mix from male rats, treated with Aroclor 1254) and a treatment period of 4 h.

The highest nominal concentration (10 mM) applied, was chosen with regard to the solubility of the test item in organic solvents and aqueous media as well as the results of the pre-test.

Precipitation or turbidity of the test item was not visible in all experimental parts at the maximum concentration of the test item.

Ethylmethane sulfonate (EMS)and7,12-Dimethylbenzanthracene (DMBA)as appropriate reference mutagens were used as positive controls. Both induced a distinct increase in mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity of the metabolic activation system.

The evaluated experimental points and the results are summarized in chapter8, page23.

 No dose dependent increase in mutant colony numbers was observed in experiment I. No statistically significant increase in mutant colony number in comparison to the solvent control was detected up to the maximal concentration of the test item. All values remained within the historical control range. Therefore, the result of experiment I is clearly negative.

 Conclusion:

It can be stated that under the experimental conditions of this studyE17-194Tdid not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation.

Therefore, the test itemE17-194Tis considered to be “non-mutagenic under the conditions of the HPRT assay”.

 [CP1]Due to GLP reasons, we have to state all performed experiments and the addition, what is part of the Final report and which data are reported or kept at LAUS archive.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 October 2019 - 12 December 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
OECD Guidelines for the Testing of Chemicals, Part 487, adopted 29. Jul. 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EU B.49
Version / remarks:
Commission Regulation (EU) 2017/735 amending Regulation (EC) No. 440/2008, EU Method B.49: “In Vitro Mammalian Cell Micronucleus Test”, adopted 14. Feb. 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Name E17-194T
Batch no. 300567-78-7
Appearance Clear, colorless, transparent liquid
Composition Phosphinic acid, P,P diethyl-, mono ester with 1,2 propandiol
Purity 92.9%
Homogeneity homogeneous
Expiry date 30. Aug. 2022
Storage Room Temperature (20 ± 5 °C)
Source of test item: Bromine Compounds Ltd., Beer-Sheva, Israel

Molecular formula C7 H17 O3 P1
Molecular weight 180 g/mol
Density 1063 g/L at 20°C
Stability H2O: not stated; EtOH: not stated; acetone: not stated; CH3CN: not stated; DMSO: not stated
Solubility H2O: > 1 g/L; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown

See also Copy of Certificate of analysis
Target gene:
micronuclei
Species / strain / cell type:
lymphocytes: Human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
Human whole blood treated with anti-coagulant (heparin).
Cytokinesis block (if used):
Cytotoxicity Test
Cytotoxicity is characterised by the cytokinesis-block proliferation index in comparison with the controls by counting 500 cells per culture in duplicate.
5 concentrations of the test item were used in the pre-experiment that was evaluated as experiment I and tested without and with metabolic activation.
In experiment I,
Nominal concentrations of the test solutions [mM] 2000 1000 500 250 125
Resulting concentrations in the experiment [mM] 10 5 2.5 1.25 0.63
Exposure time was 4 h, exposure date was 28. Oct. 2019. The culture harvest time was 22 h after start of exposure. On the basis of the cytokinesis-block proliferation index the concentrations were selected for micronuclei scoring.

In experiment II, 5 concentrations of the test item were used and tested without metabolic activation.

Nominal concentrations of the test solutions [mM] 2000 1000 500 250 125
Resulting concentrations in the experiment [mM] 10 5 2.5 1.25 0.63

Exposure time was 24 h and directly afterwards, preparation started. The exposure date of the test was 25. Nov. 2019. On the basis of the cytokinesis-block proliferation index the concentrations were selected for micronuclei scoring.
Metabolic activation:
with and without
Metabolic activation system:
S9 (liver enzyme mixture used for the test with metabolic activation) was obtained from Trinova Biochem GmbH, Gießen, and stored at – 80 ± 5°C.
Batch nos.: 3941
Specification: produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intra-peritoneall

Test concentrations with justification for top dose:
Dose Selection
According to OECD 487, the maximum concentration of the test item should be 2 µL/mL, 2 mg/mL or 10 mM, whichever is the lowest. When cytotoxicity occurs, the highest con-centration should aim to produce 55 ± 5 % cytotoxicity. When the test item is a substance of unknown or variable composition, a complex reaction product or of biological origin (UVCB), testing may be started at a higher concentration to increase the amount of each of the test item components. Generally, concentration intervals of approximately 2 to 3-fold are used. When solubility is a limiting factor, the maximum concentration, if not lim-ited by cytotoxicity, should be the lowest concentration at which turbidity or minimal pre-cipitate is visible in the cultures.
For the test item E17-194T, the maximum concentration according to these criteria was 10 mM, corresponding to 1.69 µL/mL.
Vehicle / solvent:
DMSO was used as solvent control for the test item.
0.9% NaCl was used as solvent control for the positive controls Cyclophosphamide mon-ohydrate (CPA), Mitomycin C (MMC) and Colchicine.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
colchicine
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Specification: Human whole blood treated with anti-coagulant (heparin).
Blood Collection: Blood samples were obtained from healthy donors who neither smoke nor receive medication.
Rationale for test conditions:
As per testing the appropriate guidleine
Evaluation criteria:
Acceptability
The genotoxicity assay is considered acceptable if it meets the following criteria:
• All experimental conditions are tested (short exposure with and without metabolic acti-vation, extended exposure without metabolic activation) unless a positive result is achieved in any experiment.
• In each experiment, an adequate number of cells is analysable both in the controls and in at least 3 test item concentrations.
• The micronucleus induction of the solvent and positive controls is compatible with the historical laboratory control data or the literature data.
• The positive control shows a statistically significant increase of binucleated cells with micronuclei compared with the concurrent solvent control.
• The criteria for cell proliferation and for the selection of concentrations are fulfilled.
Statistics:
Statistical Evaluation
The number of binucleated cells with micronuclei in each treatment group was compared with the solvent control. Statistical significance was tested using Fisher’s exact test at the five per cent level (p 0.05) with the following equation:

a number of binucleated cells with micronuclei of the solvent control
b number of binucleated cells with micronuclei of the test item of the respective concentration
c number of binucleated cells without micronuclei of the solvent control
d number of binucleated cells without micronuclei of the test item of the respective concentration
n total number of cells

For positive controls with high values of binucleated cells with micronuclei, the chi-square-test with the following equation was used:

a number of binucleated cells with micronuclei of the solvent control
b number of binucleated cells with micronuclei of the test item of the respective concentration
c number of binucleated cells without micronuclei of the solvent control
d number of binucleated cells without micronuclei of the test item of the respective concentration
n total number of cells
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In experiment I without and with metabolic activation, no cytotoxicity was observed at any of the evaluated test item concentrations.
No relevant increase of the number of binucleated cells with micronuclei was detected at the evaluated concentrations and a 2nd experiment (experiment II without metabolic acti-vation, extended exposure) was performed.

In experiment II, no cytotoxicity was observed at any of the evaluated test item concentra-tions.
No relevant increase of the number of binucleated cells with micronuclei was detected at the evaluated concentrations.

Tabulated data are attached.
Remarks on result:
other: under the experimental conditions reported, E17-194T does not induce the formation of micronuclei in human lymphocytes in vitro.
Conclusions:
In conclusion, under the experimental conditions reported, E17-194T does not induce the formation of micronuclei in human lymphocytes in vitro.
The result of the micronucleus test with the test item E17-194T is considered as “negative” under the conditions of the test.

Executive summary:

Summary

This study was performed to assess the genotoxic potential ofE17-194Tto induce micronuclei in human lymphocytes culturedin vitroin absence and presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254).

The test item was dissolved in DMSO to prepare a stock solution with a concentration of 338.7 µL/mL (± 10%) of test item in DMSO, corresponding to a maximum concentration of 10 mM in the test. In addition, a geometric series of dilutions was prepared from the stock solution (see also chapter8.2, page21for exp. I and chapter8.3, page22for exp. II).

Two valid and independent experiments were performed.

Human peripheral blood lymphocytes in whole blood culture were stimulated to divide by addition of phytohaemagglutinin and exposed to solvent control, test item and positive control. All cell cultures were set up in duplicates. After exposure and harvesting, slides were prepared and stained. In order to assess the toxicity of the test item to the cultivated human lymphocytes, the cytokinesis-block proliferation index (CBPI) was calculated for all evaluable cultures. On the basis of these data, the appropriate concentrations were selected (see chapter8.2.2, page21for experiment I and chapter8.3.2, page22for experiment II) to determine the proportion of binucleated cells containing micronuclei.

No cytotoxicity was observed at any of the test item concentrations.

Neither a statistically significant nor a biologically relevant increase in the number of binucleated cells containing micronuclei at the evaluated concentrations were observed.

All positive control compounds caused large, statistically significant increases in the proportion of binucleate cells with micronuclei, demonstrating the sensitivity of the test system.

In conclusion, under the experimental conditions reported,E17-194Tdoes not induce the formation of micronuclei in human lymphocytesin vitro.

The result of the micronucleus test with the test itemE17-194Tis considered as “negative” under the conditions of the test.

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

Additional information

Justification for classification or non-classification

Not genotoxic in any of the sytudies performed. Not subject to calssification