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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

3 GLP-compliant in vitro studies were carried out:

- OECD Guideline 471 - Bacterial reverse mutation assay (Ames test),

- OECD Guideline 490 - In vitro mammalian cell gene mutation tests using the thymidine kinase gene,

- OECD Guideline 473 - In vitro mammalian chromosome aberration test.

These studies allow investigating the 3 main genetic events leading to genotoxicity namely, gene mutation, structural and numerical chromosome aberrations.

The Ames test indicated the lack of mutagenic activity of the solutions of test item on this bacterial test system. In return, in the L5178Y mouse lymphoma assay using the thymidine kinase gene, a clear genotoxic activity of Solutions of test item was observed in the presence of metabolic activation. Results of the in vitro chromosomal aberrations test on human lymphocytes also indicated a clear clastogenic activity of solutions of test item with metabolic activation and after a 20-hour long-term treatment without metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 27, 2016 - October 14, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21 July 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed on 2 November 2016
Type of assay:
bacterial reverse mutation assay
Target gene:
Not applicable
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:
metabolic activation by a microsomal fraction of rat liver (S9-mix 10%v/v)
Test concentrations with justification for top dose:
- Concentrations for assay n°1: 50, 150, 500, 1500 and 5000 µg/plate
- Concentrations for assay n°2: 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
Solutions were prepared in ethanol.
Untreated negative controls:
yes
True negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
Remarks:
Test item was completely soluble in ethanol at all concentrations tested.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Stock solution was formulated in DMSO.
Negative solvent / vehicle controls:
yes
Remarks:
NaCl 0.15 M
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Stock solution was formulated in NaCl 0.15 M.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Stock solution was formulated in DMSO.
Negative solvent / vehicle controls:
yes
Remarks:
NaCl 0.15 M.
Positive controls:
yes
Positive control substance:
other: cis-Platinium (II) Diammine Dichloride: for E. Coli without S9
Remarks:
Stock solution was formulated in NaCl 0.15M.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene: for TA1535, TA1537, TA98 and TA100 with S9
Remarks:
Stock solution was formulated in DMSO.
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
Stock solution was formulated in acetone.
Details on test system and experimental conditions:
PREPARATION OF STOCK SOLUTION
-Test item is completely soluble in ethanol at all concentrations tested.
- Stock solution for each assay was prepared at 100 mg/mL in ethanol.
- Test item is tested at 5000, 1500, 500, 150 and 50 µg/plate.
- 0.1 mL volume additions of test item solution were used for all treatments.

BACTERIA
- Strains of Salmonella typhimurium and Eschericia coli are purchased from Moltox. They are maintained in LEMI laboratory.
- The genotype of bacterial strains was checked.

EXPERIMENTS
- Triplicate plates test material.
- Negative, positive and vehicle controls were included in triplicate with and without metabolic activation.
- S9 fraction, microsome fraction, prepared from Sprague Dawley rat liver homogenate, was provided by Moltox USA.
- For experiments without metabolic activiation: 0.1 mL bacterial culture, 0.1 mL test article solution or control and 0.5 mL of buffer solution were added to 2 mL overlay agar at 45°C containing 10%v/v of a L-Histidine-D-Biotine solution (0.5 mM) for Salmonella Typhimurium strains and 5%v/v of nutrient broth n°2 to which are added 5 µL of a L-Tryptophane solution at 2 mg/L for Escherichia coli strain. Plates are incubated at 37°C over 48-72 hour period.The pre-incubation phase is performed for the second assay if the first assay is negative.
- For experiments with metabolic activation: protocol similar to that described above, except that 500 µL of S9-mix fraction is quickly added before pouring the mixture onto the plates. Since the first assay was negative, the pre-incubation test was performed for the second assay.

COLONY COUNTING
- After a 48-72 hour incubation period at 37°C, revertant colonies are counted in each plate.
Evaluation criteria:
The result of the test is considered as negative if the revertant number is below three fold the number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions for TA 98, TA 100 and E. coli WP2 (uvrA) (pKM 101) strains without and with metabolic activation.
The result of the test is considered positive if a dependant relationship concentration is obtained in one, or several of the 5 strains, without and/or with metabolic activation, a mutagenic effect being taken into account for a given dilution of test item if the number of revertant colonies is at least two fold that of spontaneous revertant colonies for TA98, TA100 and E. coli WP2 (uvrA) (pKM 101) and three fold for TA1535 and TA1537.
All results must be confirmed in an independant experiment.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not valid
Additional information on results:
There is no significant difference between the number of spontaneous reversions, the number of reversions obtained for positive controls (with and without metabolic activation), and the mean of corresponding experimental historical values obtained in the laboratory.
There is no evidence of any increase in the number of revertant colonies in the presence of the test item stock solution and dilutions (5000, 1500, 500, 150 and 50 µg/plate) without and with metabolic activaiton in Salmonella typhimurium TA1535, TA1537, TA98, TA100 and Escherichia coli WP2(uvrA)(pKM101).
In presence of the highest dose 5000 µg/plate, we can observe for Salmonella typhirium TA1537 and TA100 strains a moderate thinning of the background bacterial lawn which is consistent with the toxicity measured at this dose and a decrease in the number of spontaneous revertant.
Results are confirmed in an independant experiment.
Conclusions:
Interpretation of results: negative
Doses (5000, 1500, 500, 150 and 50 µg/plate) performed from solutions of the test item do not induce any mutagenic change in Salmonella typhimurium TA1535, TA1537, TA98, TA100 and Eschirichia coli WP2(urA-)(pKM101) without, or with metabolic activation, according to the OECD guidelines n°471.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11.02.19 to 07.06.19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
version 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
dated 2 November 2016
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: from peropheral blood obtained from two healthy donors
Details on mammalian cell type (if applicable):
- Origin: peripheral blood collected in a heparinized tube.
- Culture medium: Complete culture medium is HAM F12 supplemented with 15 % (v/v) Fetal Calf Serum (FCS) (), 0.5 % (v/v) antibiotics () (penicillin 10 000 U/mL, streptomycin 10 000 μg/mL, amphotericin B 25 μg/mL) and 2 % (v/v) phytohemagglutinin A.
- Cell culture: Before exposure to solutions of the test item, lymphocytes are stimulated 48 hours in the presence of 2 % (v/v) phytohemagglutinin A (PHA) as follows: 0.35 mL of whole blood from two healthy non-smokers donors, taken on sterile heparin are added to 4.65 mL of complete culture medium in a 25 cm² culture flask. Cell cultures are incubated at 37 °C in a humid atmosphere containing 5 % (v/v) CO2.
Additional strain / cell type characteristics:
other: a stable karyotype with 46 chromosomes
Metabolic activation:
with and without
Metabolic activation system:
S9-mix 10% v/v
Test concentrations with justification for top dose:
1 000 μg/mL –700 μg/mL - 400 μg/mL - 280 μg/mL - 160 μg/mL - 112 μg/mL - 64 μg/mL – 44.8 μg/mL – 25.6 μg/mL for assay 1 (short-term treatment) and 2 (long-term treatment)
+ 17.9 μg/mL for assay 2 (long-term treatment)

A preliminary cytotoxicity test (using Balb/c 3T3 mouse embryo fibroblast) was performed to obtain a first estimate of the maximum concentration which should be tested in the chromosomal aberration test. Concentrations tested: 1000 - 500 - 250 - 125 – 62.5 and 31.25 μg/mL.
It is assessed by the determination of the Mitotic Index (% MI) with and without metabolic activation: this parameter evaluates the cytotoxicity of the five tested concentrations and allows the final selection of the concentrations to be tested in the chromosomal aberration test.
If cytotoxicity observed, analysable concentrations should cover a range from the maximum to little or no toxicity. The highest concentration used should induce cytotoxicity less than 50 %.
Vehicle / solvent:
Ethanol was chosen as vehicle:
- Test item is poorly soluble in water.
- A preliminary solubility test determined a maximum solubility in ethanol of 100 mg / mL.
- Solutions of the item are prepared at 100 mg/mL (ethanol) for assay 1 (short-term treatment) and assay 2 (long-term treatment), in sterile conditions.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
yes
Remarks:
Culture medium
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation; 0.25 µg/mL; 2 flasks per assay
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
yes
Remarks:
Culture medium
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation; 50 µg/mL; 2 flasks per assay
Details on test system and experimental conditions:
- EXPOSURE OF TEST ITEMS (SOLUTIONS)
Selected concentrations of test item solutions are placed in contact with the test system. Two independent tests are carried out.
- Assay 1: short-term treatment
Without metabolic activation: 4 hours exposure followed by 18 hours of expression.
With metabolic activation (S9-mix 10 % (v/v)): 3 hours exposure followed by 18 hours of expression.
- Assay 2: long-term treatment
Without metabolic activation: 20 hours of exposition.

Without metabolic activation (assay 1 and assay 2)
At the end of the period of stimulation mitotic cultures are centrifuged, the supernatant is removed and replaced by:
- Complete culture medium Complete culture medium*
- Vehicle HAM F12 + 1% Ethanol
- Test item: 1 000 μg/mL –700 μg/mL - 400 μg/mL - 280 μg/mL - 160 μg/mL - 112 μg/mL - 64 μg/mL – 44.8 μg/mL – 25.6 μg/mL for assay 1 and 2 + 17.9 μg/mL for assay 2
- Short-term treatment (Assay 1): After 4 hours at 37 °C in a humidified atmosphere containing 5 % (v/v) CO2, the culture medium is separated by centrifugation (200 g, 6 min) then discarded and the 3 OCDE 473: 24. If no precipitate or limiting cytotoxicity is observed, the highest test concentration should correspond to 10 mM, 2 mg/mL or 2 μL/mL, whichever is the lowest. When the test chemical is not of defined composition e.g. substance of unknown or variable
composition, complex reaction products or biological materials, environmental extracts etc., the top concentration may need to be higher (e.g. 5 mg/mL), in the absence of sufficient cytotoxicity, to increase the concentration of each of the components. It should be noted however that these requirements may differ for human pharmaceuticals.
- Long-term treatment (Assay 2): lymphocytes are incubated, 20 hours at 37 °C in a humidified atmosphere containing 5 % (v/v) CO2.

With metabolic activation (Assay 1)
- Preparation of S9 fraction
S9 (microsome fraction from the liver of Sprague Dawley rats treated with Aroclor 1254 (500 mg/kg over a 5-day period)) is prepared in compliance with Ames and al4, and provided by MOLTOXTM (POB Box 1189 – 157 Industrial Park Dr – Boone, NC 28607 – USA). The S9 fraction (Ref: 11-101.5 – Batch: 3919 – Expiry date: 07.02.2020) was previously validated on 06.07.2018 in the laboratory according to the LEMI SOP n° MB06/09.
- Preparation of S9-mix
S9 fraction 10 % (v/v)
MgCL2-6H2O 8 mM
KCl 33 mM
Glucose-6-Phosphate Na2 5 mM
NADP Na2 4 mM
Phosphate buffer pH 7.4 0.1 M
- Exposition
At the end of the period of stimulation mitotic cultures are centrifuged, cells washed with culture medium (HAM F12) and incubated with reaction mixture composed by culture medium supplemented with 10 % (v/v) S9-mix (final concentration: 1.5 %) and 2 % (v/v) phytohemagglutinin A.
Increasing quantities of solutions performed without FCS (Fetal Calf Serum) are added in the culture flasks (n = 2).
Vehicle: Mc Coy’s
Test item: 1 000 μg/mL –700 μg/mL - 400 μg/mL - 280 μg/mL - 160 μg/mL - 112 μg/mL - 64 μg/mL – 44.8 μg/mL – 25.6 μg/mL
- Short term treatment (Assay 1): After 3 hours at 37 °C in a humidified atmosphere containing 5 % (v/v) CO2, the culture medium is separated by centrifugation (200 g, 6 min) then discarded and the cells washed twice with culture medium.
5 mL of fresh complete culture medium are added and the lymphocytes incubated, 18 hours at 37 °C in a humidified atmosphere containing 5 % (v/v) CO2.

CELL HARVEST AND MICROSCOPE SLIDES PREPARATION
At the end of the incubation period (18 h for the short-term treatment and 20h for the long-term treatment), Colcemid® (SIGMA – D1925 – RNBG9164) (0.15 μg/mL) is added in each flask. Cells are incubated at 37° C for 2.5 hours in a humidified atmosphere containing 5 % (v/v) CO2 in order to block the cell division in the metaphase stage, then collected:
– centrifugation (200 g, 6 min)
– hypotonic shock (KCl 0.075 M) at 37° C for 10 minutes
– fixation (2 to 3 x 5 min) using the Carnoy mixture (methanol: acetic acid, 3:1)
– spread on coded microscope slides
– stained using Giemsa stain according to the LEMI SOP n° MB08/023.
Metaphases are analyzed under a microscope (Zeiss), magnification x 320 for the determination of the mitotic index and magnification x 1000 for the detection of chromosomal aberrations, polyploidy and endoreduplications.
Evaluation criteria:
The test item will be considered as clastogen in vitro with regards to human lymphocytes cells according to the following criteria:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent solvent control (Ethanol),
- the increase is dose-related when evaluated with an appropriate trend test,
- any of the results are outside the distribution of the historical negative control data.
The test item for which the results do not meet all the above criteria is considered non-clastogenic in this system.
Positive results indicate that the test item induces structural chromosome aberrations in human cell cultures.
The positive control shall largely fulfil to all three criteria.
The number of cells with chromosomal aberrations in the negative control shall be less than 5 %.
Equivocal or discutable results may not allow a clear positive response. Results shall be clarified by further testing using modification of experimental conditions (concentration spacing and metabolic activation conditions).
Statistics:
The number of cells presenting one, or more, aberration is considered as a direct response and evaluated statistically using the 2 trend test.
The results were considered significant if P < 0.05 comparing cultures treated with different solutions of the test substance with their corresponding negative control (solvent control), positive control and absolute negative control.
Species / strain:
lymphocytes: from humans
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
short-term treatment
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A slight to severe decrease in mitotic index was induced at dose-levels ≥ 280 μg/mL (10-100% decrease).
Vehicle controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
lymphocytes: from humans
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
short-term treatment
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
No decrease to slight decrease in mitotic index was induced at dose-levels ≥ 280 μg/mL (0-37% decrease).
Vehicle controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
lymphocytes: from humans
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
long-term treatment
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slight to severe decrease in mitotic index was induced at dose-levels ≥ 17.92 μg/mL (4-100% decrease). The solutions at 1000 and 700 µg/L caused complete cell destruction.
Vehicle controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
CYTOTOXICITY
- Preliminary study using Balb/c 3T3
The solution at 1000 μg/mL shows a 60 % inhibition of cell growth statistically significant (P < 0.01).
The solution at 500 μg/mL shows a 62 % inhibition of cell growth statistically significant (P < 0.001).
The solution at 250 μg/mL shows a 38 % inhibition of cell growth statistically significant (P < 0.01).
The solution at 125 μg/mL shows a 22 % inhibition of cell growth not statistically significant.
The solution at 62.5 μg/mL shows a 6 % inhibition of cell growth not statistically significant.
The solution at 31.25 μg/mL shows a 10 % inhibition of cell growth not statistically significant.
According to the evaluation criteria of the cytotoxicity, the solution prepared from the test item is cytotoxic at 1000 μg/mL, 500 μg/mL and 250 μg/mL for a 24 hour contact on the Balb A31 cells. The solution prepared from the test item is not cytotoxic at 125 μg/mL, 62.5 μg/mL and 31.25 μg/mL.
Positive control (phenol 0.64 mg/mL) induces a 57 % (P < 0.001) inhibition on cell growth which validates the study.
The maximum doses will be used in the chromosomal aberration test: 1000 μg/mL.

- Mitotic index
* Assay n°1 (short-term treatment) without metabolic activation
The solution at 1000 μg/mL of test item reduces the mitotic index by 100 %. This concentration is not compatible with the study (the reduction of human lymphocytes viability induced by the highest concentration chosen must not be greater than 45 % ± 5% when compared with the control).
The solution at 700 μg/mL of test item reduces the mitotic index by 39 %. This concentration is compatible with the study.
The solution at 400 μg/mL of test item reduces the mitotic index by 8 %. This concentration is compatible with the study.
The solution at 280 μg/mL of test item reduces the mitotic index by 10 %. This concentration is compatible with the study.
The positive control reduces the mitotic index by 9 %. This concentration is compatible with the study.
Solvent control (ethanol 1%) reduces the mitotic index by 13 %. This concentration is compatible with the study.
Therefore, the concentrations 700, 400 and 280 μg/mL will be used to determine the genotoxic effects.

* Assay n°1 (short-term treatment) with metabolic activation
The solution at 1000 μg/mL of the test item reduces the mitotic index by 37 %. This concentration is compatible with the study (the reduction of human lymphocytes viability induced by the highest concentration chosen must not be greater than 45 % +/- 5% when compared with the control).
The solution at 700 μg/mL of the test item does not reduce the mitotic index. This concentration is compatible with the study.
The solution at 400 μg/mL of the test item does not reduce the mitotic index. This concentration is compatible with the study.
The solution at 280 μg/mL of the test item does not reduce the mitotic index. This concentration is compatible with the study.
The positive control reduces the mitotic index by 16 %. This mitotic index reduction is compatible with the study.
Solvent control (ethanol 1%) reduces the mitotic index by 2 %. This concentration is compatible with the study.
Therefore, the concentrations 1000, 700 and 400 μg/mL will be used to determine the genotoxic effects.

* Assay n°2 (long-term treatment) without metabolic activation
The solutions at 1000 μg/mL and 700 μg/mL caused complete cell destruction (no metaphases and no interphase cells).
The solution at 400 μg/mL of test item reduces the mitotic index by 100 % without metabolic activation. This concentration is not compatible with the study. This concentration cannot be used to determine the genotoxic effect (see above).
The solution at 280 μg/mL of test item reduces the mitotic index by 100 % without metabolic activation. This concentration is not compatible with the study.
The solution at 160 μg/mL of test item reduces the mitotic index by 67 % without metabolic activation. This concentration is not compatible with the study.
The solution at 112 μg/mL of test item reduces the mitotic index by 42 % without metabolic activation. This concentration is compatible with the study.
The solution at 64 μg/mL of test item reduces the mitotic index by 17 % without metabolic activation. This concentration is compatible with the study.
The solution at 44.8 μg/mL of test item reduces the mitotic index by 12 % without metabolic activation. This concentration is compatible with the study.
The solution at 25.6 μg/mL of test item does not reduce the mitotic index without metabolic activation. This concentration is compatible with the study.
The solution at 17.92 μg/mL of test item reduces the mitotic index by 4 % without metabolic activation. This concentration is compatible with the study.
The positive control reduces the mitotic index by 38 %. This mitotic index reduction is compatible with the study.
Solvent control (ethanol 1%) reduces the mitotic index by 15 %. This concentration is compatible with the study.
Therefore, the concentrations 112, 64 and 44.8 μg/mL will be used to determine the genotoxic effects.

GENOTOXICITY
- Absolute negative control
* without metabolic activation: the percentage of cells with aberrations is 1.0 % for assay 1 (short-term treatment) and 2.0% for assay 2 (long-term treatment)* with metabolic activation: the percentage of cells with aberrations is 0.7 %

- Solvent control (Ethanol)
* without metabolic activation: the percentage of cells with aberrations is 1.0 % for assay 1 (short-term treatment) and 1.3 % for assay 2 (long-term treatment)
* with metabolic activation: the percentage of cells with aberrations is 1.7 %.

- Positive controls
* Without metabolic activation: Mitomycin C significantly increases the percentage of cells with aberrations compared to negative control (P < 0.001). This percentage is 22 % for assay 1 (short-term treatment) and 32 % for assay 2 (long-term treatment).
* With metabolic activation: Cyclophosphamide significantly increases the percentage of cells with aberrations compared to negative control (P < 0.001). This percentage is equal to 16 %.

- Solutions of test item
* Assay n°1 (short-term treatment) without metabolic activation
The solution at 700 μg/mL of test item do not significantly increase the percentage of cells with aberrations. This percentage is equal to 2.3 %.
The solution at 400 μg/mL of test item do not significantly increase the percentage of cells with aberrations. This percentage is equal to 1.7 %.
The solution at 280 μg/mL of test item do not significantly increase the percentage of cells with aberrations.This percentage is equal to 1.0 %.
No concentration exhibits a statistically significant increase compared with the concurrent solvent control.
The test item is considered not clastogenic in this test system (human lymphocytes) with a short-term treatment without metabolic activation.
* Assay n°1 (short-term treatment) with metabolic activation
The solution at 1000 μg/mL of test item significantly increase the percentage of cells with aberrations (P<0.001). This percentage is equal to 8.5 %.
The solution at 700 μg/mL of test item significantly increase the percentage of cells with aberrations (P<0.05). This percentage is equal to 4.7 %.
The solution at 400 μg/mL of test item do not significantly increase the percentage of cells with aberrations. This percentage is equal to 2.3 %.
Concentrations 1000 μg/mL and 700 μg/mL exhibits a statistically significant increase compared with the concurrent solvent control, the increase is dose-related and the result are outside the distribution of the historical negative control data.
The test item is considered clastogenic in this test system (human lymphocytes) with a short-term treatment with metabolic activation.
* Assay n°2 (long-term treatment) without metabolic activation
The solution at 112 μg/mL of test item significantly increase the percentage of cells with aberrations (P<0.001).
This percentage is equal to 6.3 %.
The solution at 64 μg/mL of test item do not significantly increase the percentage of cells with aberrations. This percentage is equal to 2.3 %.
The solution at 44.8 μg/mL of test item do not significantly increase the percentage of cells with aberrations. This percentage is equal to 1.3 %.
Concentration at 112 μg/mL exhibits a statistically significant increase compared with the concurrent solvent control, the increase is dose-related and the result are outside the distribution of the historical negative control data.
The test item is considered clastogenic in this test system (human lymphocytes) with a long-term treatment without metabolic activation.

POLYPLOIDY AND ENDOREDUPLICATION CASES
No increase in polyploidy and endoreduplication was observed compared to solvent control and absolute negative control.
Interpretation does not take into account the measurement uncertainties. These uncertainties are available and can be provided on request.
Conclusions:
According to the criteria of conclusion of the study protocol and OECD n°473, solutions obtanied from test item are considered clastogenic in the test system used (human lymphocytes) and in the conditions of the assay.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20-05-2019 to 08-10-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed on 11 July 2019
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
tk locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Mouse lymphoma L5178Y TK+/-cells (ATCC-CRL-9518) purchased from ATCC (American Type
Culture Collection-Rockeville, MD 20852 – USA)
Metabolic activation:
with and without
Metabolic activation system:
metabolic activation by a microsomal fraction of rat liver (S9-mix 2.5%v/v)
Test concentrations with justification for top dose:
Without S9-mix: 15.625 - 31.25 - 62.5 - 125 - 250 and 375 µg/mL
With S9-mix: 31.25 - 62.5 - 125 - 250 - 375 and 500 µg/mL
Vehicle / solvent:
Absolute Ethanol
Untreated negative controls:
yes
Remarks:
Culture medium
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Untreated negative controls:
yes
Remarks:
Culture medium
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
TEST SYSTEM:
- Test system description: Mouse lymphoma L5178Y TK+/-cells (ATCC-CRL-9518) have been used successfully in “in vitro” experiments for many years. These cells are characterized by their high proliferation rate (10 h – 12 h doubling time of the stock cultures) and their cloning efficiency, usually more than 50 %. They possess a nearly diploid karyotype (40 ± 2 chromosomes). They are heterozygous at the thymidine kinase (TK) locus which allows to detect mutation events at the TK-locus. Cells from the cell bank stored at- 80°C are systematically checked to be free from mycoplasma contamination.
- Test system purification: To prevent background arising from spontaneous mutation, cells lacking TK have to be eliminated by culturing them in a culture medium (Dulbecco’s modified Eagle’s medium (DMEM) GlutaMAXTM – I) supplemented with 10% (v/v) of inactivated horse serum containing HMTG (Cole et al10): 15 μg/mL hypoxanthine, 0.3 μg/mL methotrexate, 9 μg/mL thymidine, 22.5 μg/mL glycine. After 24 hours incubation at 37° C in a humidified atmosphere containing 5% (v/v) CO2, the culture is centrifuged (200 x G, 10 min) in order to eliminate methotrexate, and the cell pellet is suspended in medium, without methotrexate, containing HTG (hypoxanthine, thymidine and glycine) and incubated at 37° C in a humidified atmosphere containing 5% (v/v) CO2 for 1 day to 3 days. Cleaned cells are stored at -80°C. Each cell batch is checked free from mycoplasma contamination. Thawed cultures are maintained in complete culture medium (CCM).

CULTURE MEDIA:
- Complete culture medium: Dulbecco’s modified Eagle’s medium (DMEM) GlutaMAX– I (GIBCO - ref 31966-021 - batch 2026785 and batch 2026729 used only to test solubility) supplemented with 10 % (v/v) inactivated horse serum (10 %), (GIBCO – ref. 16050-130 - batch 1972988 used for the assyas and 1861235 used for pre-test viability) and 1 % (v/v) antibiotics (GIBCO – ref. 15240-096 - batch 2029640 and 1981203).
- Treatment medium (TM): DMEM GlutaMAXÔ – I supplemented with 5 % (v/v) inactivated horse serum and 1 % (v/v) antibiotics.
- Cloning medium (CM): DMEM GlutaMAXÔ supplemented with 20 % (v/v) inactivated horse serum and 1 % (v/v) antibiotics.
- Selective medium (SM): CCM supplemented with TFT (3 μg/mL) (Sigma - Ref T2255 - batch BCBW1167).

PRE-TEST FOR VIABILITY:
Prior to mutagenesis assay a pre-test is carried out in order to determine the viability of test item. This pre-test show a high toxicity of test item from 500 to 1 000μg/mL.
1 x 106 L5178Y cells /mL of TM are exposed to a range of concentrations of test item for 4 or 3 hours (without or with metabolic activation). Following treatment, cells are rinsed twice with complete culture medium (CCM) (10 mL) followed by centrifugation (200 g, 10 min). Subsequently, the cells are resuspended in 20 mL CCM for a 2 days growth period. Cell density is determined at days D1, D2 and adjusted to 2 x 105 cells/mL.
The relative suspension growth (RSG) and the relative total growth (RTG) of the treated cell cultures is calculated at the end of the growth period according to the method of Clive and Spector.

PREPARATION OF THE TEST ITEM:
Concentrations (n=2) of the test item are:
- without S9-mix: 15.625 - 31.25 - 625 - 125 - 250 - 375 μg/mL
- with S9-mix (2.5 %): 31.25 - 62.5 - 125 - 250 - 375 - 500 μg/mL
- The upper limit of cytotoxicity observed in experimental cultures should not be less than 10 % RTG, which is the case for the highest concentrations tested
without S9-mix in presence of 375 μg/mL (RTG value is 6%)
- the test item is soluble in Absolute Ethanol, the dissolution is performed in this solvent.
- The different solutions of the test item are prepared extemporaneously.
- Four different test concentrations were evaluated.
- without S9-mix: 31.25 – 62.5 – 125 and 250 μg/mL
- with S9-mix (2.5 %): 125 – 250 – 375 and 560 μg/mL

EXPERIMENTAL PROTOCOL:
Two short-term treatment assays without and with metabolic activiation are run independently using duplicate cultures. Only short-term treatments are planned.

Assay without metabolic activation (4 hours):
Viability: the methodology of the pre-test is applied in the main experiment.
Treatment: 1 x 106 cells/mL of TM supplemented with 5 % (v/v) are exposed to each concentration of test item at 37°C in a humidified atmosphere containing 5 % (v/v) CO2. After 4 hours, cells are rinsed twice by centrifugation (200g, 10 min). It is necessary to treat at least 6 x106 cells.
Plating for viability: Cell pellet is suspended in complete culture medium (CCM) and plated, at a statistical mean of 2 cells per well in 2 plates of 96 wells per concentration, in order to determine viability at T0 (colony counting 9 to 11 days later).
Expression period: Concurrent cells are suspended in CCM and incubated in order to allow TK locus phenotypic expression over 48 hours to 72 hours. The cell density is determined every day, and adjusted to 2 x 105 cells/mL, if necessary.
Mutagenesis test:
· Plating for survival: After the expression period, the relative cloning efficiency (RCE; percentage cloning efficiency of the test group in relation to the control) of the cells is determined according to Cole et al (1990) by seeding a statistical number of 2 cells/well in two 96-well-plates. Cells are incubated for 10 - 12 days at 37° C in a humidified atmosphere containing 5 % (v/v) CO2. Analysis of results is based on the number of cultures with cell growth (positive cultures) and/or those without cell growth (negative cultures) compared to the total number of cultures seeded. Relative total growth (RTG) of the treated cell cultures is calculated according to the method of Clive and Spector (10) as follows: RTG = % RSG x % RCE*
* RCE (Relative Cloning Efficiency) is determined by comparing plating efficiency PE in the test cultures and control cultures at day 2 and RSG (Relative Suspension Growth) is calculated from previous equation.
· Plating for 5-trifluorothymidine (TFT) resistance: Cultures are resuspended in selective medium with TFT at 3 μg/mL (see below). Cells from each experimental group are seeded in four 96-well plates at a density of approximately 2 000 cells/well in 200 μL selective medium. The plates are scored after an incubation period of 10 - 12 days at 37° C in a humidified atmosphere containing 5 % (v/v) CO2.
· Criteria for scoring mutation plates: The number of wells containing colonies are counted. A well without colonies is classified as negative. Colonies are characterized as follows:
- small colonies: colonies having a diameter less than 25 % of the diameter of the well. A small colony should also have a dense morphology and a clear contour;
- large colonies: colonies having a diameter greater than 25 % of the diameter of the well. Morphology is totally or partially diffuse.
- Any well which contains one or more than one small colony is scored as positive for small colony.
- Any well which contains one or more than one large colony is scored as positive for large colony.
- Any well which contains a combination of large and small colonies is scored as large colony and small colony.
The mutation frequencies are then calculated from the data obtained from cultures used for the cloning efficiency (cultures with non-selective medium) and those used for selection (cultures with selective medium).
A negative control (Complete Culture Medium DMEM glutamax) and a positive control (Methylmethanesulfonate at 10 μg/mL) are carried out in parallel under the same conditions.

Assay with metabolic activation (3 hours):
S9 fraction, microsome fraction prepared from Sprague Dawley rat liver homogenate, is provided by MOLTOXTM (POB Box 1189 - 157 Industrial Park Dr - Boone, NC 28607 – USA (S9 Moltox-11101-5-3919 validated on 06.07.2018 – expiry date: 07.02.2020).
Preparation of S9-mix
S9-mix is prepared at 4 °C on the day of use. The final concentration of cofactors and salts is as follow:
- S9 fraction 2.5 % (v/v)
- Mg Aspartate 8 mM
- KCl 33 mM
- Glucose-6-Phosphate Na2 5 mM
- NADP Na2 4 mM
- Phosphate buffer pH 7.4 0.1 M
The test is identical to the one described in the absence of metabolic activation (cf. §4.5.1.), except cells are treated for 3 hours in the presence of 2.5 % S9-mix.
A negative control (Complete Culture Medium DMEM glutamax), a solvent control (Absolute Ethanol) and a positive control, (Cyclophosphamide monohydrate at 2 μg/mL) are carried out in parallel under the same conditions.
Evaluation criteria:
Acceptability criteria for the assay:
A gene mutation assay is considered acceptable if it meets the following criteria:
- the test must be conducted under two experimental conditions (short treatment without and with metabolic activation) unless one resulted in positive results.
- adequate number of cells and concentrations should be analysable.

Acceptability criteria for negative and positive controls:
Every experiment should be evaluated as to whether the untreated control meets the IWGT MLA Workgroup acceptance criteria, below:
· Mutant Frequency 50 – 170 x 10-6
· Cloning Efficiency 65 – 120%
· Suspension Growth: 8 – 32 fold (3-4-hour treatment)- 32 – 180 fold (24-hour treatment, if conducted)
Every experiment should also be evaluated as to whether the positive controls meets at least one of the following two acceptance criteria:
- The positive control should demonstrate an absolute increase in total MF, that is, an increase above the spontaneous background MF [an induced MF (IMF)] of at least 300 x 10-6. At least 40 % of the IMF should be reflected in the small colony MF.
- The positive control has an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent untreated control (a small colony IMF of 150 x 10-6).
The upper limit of cytotoxicity observed in the positive control culture should be the same as of the experimental cultures. In other words, the RTG/RS should not be less than 10 %.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Results for untreatedand solvent negative controsl are in accordance with the acceptabiltiy criteria described in OECD 490 guideline.
Results for untreated positive controls are in accordance with the acceptability criteria described in OECD 490 guideline.

In the absence of metabolic activation – 4 hours treatment

a) An increase in the mutant frequency is observed.

Moreover, the GEF (Global Evaluating Factor) was calculated in these experimental conditions, since the GEF was recommended by the OECD 490 to help in evaluating the test results (Moore et al12 13 14). The GEF is applied as follows: if the negative control mutant frequency (MF) in a microwell experiment is 100 x 106, then one of the treatment groups must have a MF of at least [100+126 (the microwell GEF) = 226] x 10-6 in order to meet the GEF criterion for a positive call.

The above criteria, is not met any concentration tested, in the absence of metabolic activation for the short exposure time. The measured MF range from 124.2 to 214.9 x 10-6 falls below GEF criterion of [126+137.8]263.81 x 10-6.

b) Analysis of the size of colonies shows a light increase in induced small (0 to 18), and an increase in induced large colony (0 to 57).

In the presence of metabolic activation – 3 hours treatment (table 6b)

a) In the presence of 2.5 % S9-mix, an increase in the mutant frequency is observed.

The above criteria is met for the highest concentration tested 500 μg/mL, in the presence of metabolic activation. The measured MF range from 176.2 to 328.7 x 10-6. The value 328.7 x 10-6 measured in presence of 500 μg/mL is above GEF criterion of [126+157.4]283.4 10-6 (cf. table 6b).The increase in the MF is concentration related.

b) Analysis of the size of colonies shows an increase in induced small colonies (Number of induced mutants: from 15 to 157) and an increase in induced large colony (Number of induced mutants: from 1 to 43.

Conclusions:
In the framework of OECD 490 under the described experimental conditions, solutions obtained from test item induce a mutagenic effect in L5178Y TK+/-Mouse lymphoma cells in the presence of metabolic activation (2.5% S9-mix).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The search for potential genotoxic activity of the substance was investigated for in-vivo genotoxic potential by the means of the in vivo micronucleus test in bone marrow and the in vivo comet assay under alkaline conditions (SCGE) in the liver, glandular stomach and duodenum in male CD Sprague-Dawley rats, according to OECD Guidelines (Nos. 474 and 489, 2016). Under these experimental conditions, the test item does not present DNA strand breaks and/or alkalilabile sites inducer activities toward the liver, glandular stomach and duodenum from CD Sprague-Dawley male rats. Furthermore, in male CD Sprague-Dawley rats, the substance induced no genotoxic activity in bone marrow cells. As a conclusion, the substance induced no genotoxic activity under these experimental conditions.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 7 July 2021 to 20 January 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
CD Sprague Dawley rat
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Saint-Germain-surl’Arbresle, France
- Age at study initiation: young adult 8 weeks old
- Weight at study initiation: between 187 g and 226 g
- Not fasted at the treatment time
- Housing: animals were dispatched in polypropylene cages by random-distribution
- Diet: A04C-10 from SAFE
- Drinking water: Softened by reverse osmosis and filtered on 0.22 μm membrane, provided ad libitum
- Acclimatization period: 7 days, the animals received a clinical examination in order to retain only those which were healthy.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3°C
- Humidity: 30-70 %
- The cages were placed in a ventilated system in the animal room, which was also ventilated.
- Photoperiod (hrs dark / hrs light): 12h/12h
Route of administration:
oral: gavage
Vehicle:
corn oil (Sigma, batch MKCM3364 for the preliminary toxicity assays; batch MKCM9808 for the main assay)
Details on exposure:
In the main genotoxicity assay, three emulsions of the test item at the concentrations of 60 - 30 and 15 mg/mL were prepared, giving final doses of 600 - 300 and 150 mg/kg, respectively when administered at 10 mL/kg.
Duration of treatment / exposure:
Treatment took the form of 3 successive administrations at 24-hour intervals by oral route (gavage).
Samples were taken 2 to 6 hours after the last treatment.
Frequency of treatment:
3 successive administrations at 24-hour intervals
Post exposure period:
Samples were taken 2 to 6 hours after the last treatment.
Dose / conc.:
600 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males per dose, 7 males for highest dose
Control animals:
yes, concurrent vehicle
Positive control(s):
5 animals (males) received a single intraperitoneal injection 24 hours before sampling of the reference substance cyclophosphamide (CPA; Baxter, batch 9J125F) at a dose of 25 mg/kg under a volume of 10 mL/kg.
Tissues and cell types examined:
bone marrow tissue - polychromatic erythrocytes (PCE)
Details of tissue and slide preparation:
At the sampling time, the animals of the Cyclophosphamide control group were sacrificed by CO2 asphyxia; the femurs were removed, and the bone marrow was extracted with foetal calf serum (1 mL per animal).
The animals also used for the comet assay were anaesthetized with isoflurane and exsanguined.
Bone marrows from treated and vehicle control animals were sampled just before the sampling of liver, glandular stomach, duodenum and gonades for the comet assay.
The cell suspensions were centrifuged for 5 minutes at 1000 rpm. The supernatants were removed. After homogenisation, the centrifugate was spread on slides. The smears were stained using a technique, derived from the May Grunwald Giemsa technique (Schmid, 1975), which makes it possible to distinguish between immature polychromatic (PCE) and mature normochromatic erythrocytes (NCE): PCE are purple whereas NCE are red.
Evaluation criteria:
SLIDE COUNTING:
After blind coding of slides by a person not involved in the reading, two slides per animal were scored by two independent operators; for each animal, the number of polychromatic erythrocytes having one or more Howell-Jolly bodies (micronuclei) was determined from the microscopic examination of 4000 polychromatic erythrocytes.
The proportion of immature among total (immature and mature) erythrocytes was determined for each animal. The polychromatic/normochromatic erythrocyte ratio was determined from the microscopic examination of 1000 erythrocytes per animal. When analysing slides, the proportion of immature erythrocytes among total erythrocytes should not be less than 20% of the control value.

EXPRESSION OF RESULTS:
The results are reported in the form of tables giving the number of micronucleated cells per 4000 polychromatic erythrocytes for each animal, together with the total and the statistical analysis.
The ratio of polychromatic to normochromatic erythrocytes (PCE/NCE), calculated from 1000 polychromatic erythrocytes is also established for both treated and control animals.
Statistics:
The statistical comparison for the polychromatic/normochromatic erythrocyte ratio was performed using Student's t test.
Statistical analysis was performed for micronucleus number using a non-parametric test, the Mann Whitney U rank test, recommended by UKEMS (Lovell et al., 1989). Statistical analysis for micronucleus number was conducted.
The trend test was applied, using the statistical software GraphPad Version 3.10.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
clinical signs
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
CLINICAL SIGNS:
In the main experiment, the dose of 600 mg/kg elicited a slight or a moderate decrease in spontaneous motor activity in 2 and 3 animals, respectively, 15 minutes after the 1st treatment. Between 2 and 4 hours after the 1st treatment slight decrease in spontaneous motor activity was observed in 3 animals.
Fifteen minutes after both the 2nd and the 3rd treatment, a slight decrease in spontaneous motor activity was observed in 1 animal. Noteworthy, this animal was replaced by 1 of the 2 supplementary animals.
In return, no clinical sign was noted at the 2 inferior doses of 300 and 150 mg/kg b.w. /day (x 3).

PCE / NCE RATIO:
The ratio of polychromatic (PCE) to normochromatic erythrocytes (NCE) was established at each dose level. No statistically significant decrease in the ratio PCE to NCE was noted in the 3 treatment groups when compared to the vehicle control group. In consequence, no proof of systemic exposure was evidenced when using this parameter.
Nevertheless, the test item induced clinical signs including decrease in spontaneous motor activity when treated under 600 mg/kg indicating that a systemic exposure probably occurred.

FREQUENCY OF MICRONUCLEATED PCE:
Regarding the frequency of micronucleated polychromatic erythrocytes, no statistically significant increase in the frequencies of micronucleated polychromatic erythrocytes was found in the animals treated with the test item at any dose. Indeed, 0.75 to 0.85 micronucleated PCE/1000 PCE were obtained vs. 0.55 in the relative vehicle control.

Results:

 Sampling time

(2 - 6 H after

last treatment)*

 Test item

doses in

mg/kg bw/day (x3)

Sex

PCE/NCE ratio

Micronuclei for 1000 PCE

 Kruskall

Wallis

 

 

 

 Mean

Student's

t Test

(p) 

Mean

Mann

Whitney

(p) 

 

 Negative group

control

 Vehicle

10 mL/Kg

 M

 3.05

 

0.55 

 

 

 Positive group

control

 Cyclophosphamide

(10 mL/Kg - IP route)

25 mg/kg/day (x1)

 M

 1.14

 <0.05

 21.85

p<0.01 

 

 Test item

Treated groups

 600

3.85 

N.S. 

0.80 

N.S. 

N.S. 

 

 300

3.10 

  N.S.

0.75 

  N.S.

N.S. 

 

 150

M

 4.10

  N.S.

0.85 

  N.S.

N.S.

* 24H after the sinlge treatment for the positive group

No statistically significant increase in the number of micronuclei was noted at the doses of 600 - 300 - 150 mg/kg bw/day (x3) per os in male rats.

Conclusions:
Under the experimental conditions of this OECD 474 study, the test item induced no genotoxicity activity in bone marrow cells in male CD Sprague-Dawley rats.
Executive summary:

The search for potential genotoxic activity of test item was investigated for genotoxic potential by the means of the in vivo micronucleus test in bone marrow in male CD Sprague-Dawley rats, according to OECD Guideline No 474, 2016. Animals were treated orally once a day for 3 consecutive days, 24 hours apart, up to the dose of 600 mg/kg compatible with the toxic activity.

The control of concentrations of test item in treatment preparations was performed in a GLPcompliant laboratory using a validated method. The results were shown to be compliant with the recovery acceptance criteria (85-115% vs. theoretical concentrations). In addition, test item was not detected in the solvent control.

The acceptance criteria for the assay were considered as fulfilled. The current study was valid.

Under the experimental conditions, in male CD Sprague-Dawley rats, the test item induced no genotoxic activity in bone marrow cells.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 7 July 2021 to 20 January 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
CD Sprague Dawley rat
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Saint-Germain-surl’Arbresle, France
- Age at study initiation: young adult 8 weeks old
- Weight at study initiation: between 187 g and 226 g
- Not fasted at the treatment time
- Housing: animals were dispatched in polypropylene cages by random-distribution
- Diet: A04C-10 from SAFE
- Drinking water: Softened by reverse osmosis and filtered on 0.22 μm membrane, provided ad libitum
- Acclimatization period: 7 days, the animals received a clinical examination in order to retain only those which were healthy.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3°C
- Humidity: 30-70 %
- The cages were placed in a ventilated system in the animal room, which was also ventilated.
- Photoperiod (hrs dark / hrs light): 12h/12h
Route of administration:
oral: gavage
Vehicle:
corn oil (Sigma, batch MKCM3364 for the preliminary toxicity assays; batch MKCM9808 for the main assay)
Details on exposure:
In the main genotoxicity assay, three emulsions of the test item at the concentrations of 60 - 30 and 15 mg/mL were prepared, giving final doses of 600 - 300 and 150 mg/kg, respectively when administered at 10 mL/kg.
Duration of treatment / exposure:
Treatment took the form of 3 successive administrations at 24-hour intervals by oral route (gavage).
Samples were taken 2 to 6 hours after the last treatment.
Frequency of treatment:
3 successive administrations at 24-hour intervals
Post exposure period:
Samples were taken 2 to 6 hours after the last treatment.
Dose / conc.:
600 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males per dose, 7 males for highest dose
Control animals:
yes, concurrent vehicle
Positive control(s):
5 animals were treated orally with Methylmethane sulfonate (MMS; Aldrich, batch MKCL6261) at the doses of 100 mg/kg b.w. /day (x 2) and 70 mg/ kg b.w. /day PO (x1), 48, 24 hours and 2 to 6 hours before sacrifice, respectively.
Tissues and cell types examined:
liver, glandular stomach and duodenum cells
Details of tissue and slide preparation:
CELL ISOLATION:
A total 5 males of each group were assigned for cell isolation and assessed for DNA fragmentation.
Single cell preparation was done within one hour after animal sacrifice.
A 'V' shaped incision was made from the centre of the lower abdomen to the rib cage. The skin and muscles were removed to reveal the abdominal cavity.
A portion of the liver, glandular stomach, duodenum and gonad was removed and washed in the cold mincing buffer until as much blood as possible has been removed. The portion was minced with a pair of fine scissors to release the cells. The cell suspension was stored on ice for 15-30 seconds to allow large clumps to settle. The whole cell suspension was collected.
Cells were enumerated on a haemocytometer, and sufficient cells to obtain 25± 5 x 103 viable cells per slide were harvested from each cell suspension for proceeding to slides preparation.

DRIED SLIDES PREPARATION (PRE-LAYERING):
Conventional slides were dipped in hot 1.5 % normal melting point agarose in PBS. After gentle removal, the underside of the slides was wiped in order to remove excess agarose. The slides were then laid in a tray on a flat surface to dry.

SLIDE PREPARATION:
Before use, a volume of 85 μL of 0.8% of Normal Agarose (NA) was added on the microscope slide prelayered with 1.5% of NA and then covered with a glass coverslip. Slides were placed at +2-8°C until the agarose layer hardens. The cells of the different doses tested were mixed with 0.5% of Low Melting Point Agarose (LMPA) (75 μL/slide) kept at 37 °C and added on the microscope slide after gently sliding off the coverslip. The slides were then covered with a new glass coverslip, and were placed once again at +2-8°C.
Seven slides per animal were prepared for the Comet assay.
Evaluation criteria:
IMAGE ANALYSIS:
Slides were examined with a 200 x magnification, using a fluorescent microscope (Leica Microsystems SAS - DM 2000, Heerbrugg, Switzerland), equipped with an excitation filter of 515-560 nm and a barrier filter of 590 nm, connected through a gated monochrome CCD IEEE1394 FireWire video camera (Allied Vision Technologies), to the Comet Assay IV Image Analysis System, version 4.11 with Windows XP Pro Software (Perceptive Instruments Ltd, Suffolk, UK).
Only the slides from the set processed with a 20-minutes electrophoresis from liver, glandular stomach and duodenum were analysed. Indeed, in accordance with the final study plan, as no decrease in the percentage of DNA in tail was evidenced, the slides from the 40-minute electrophoresis were not analysed.
Moreover, as no primary DNA damage was noted in liver, glandular stomach and duodenum, the assessment of genotoxicity towards gonadal cells was not carried out.
For these groups, three slides were analysed with 50 nuclei per slide randomly scored. Five animals were retained per group, i.e. 15 slides per group, 750 analysed nuclei per group.

EXPRESSION OF THE RESULTS:
The results obtained in the different treatments are presented giving:
- the median per slide of the percentage of DNA in tail for at least 50 cells
- the mean of medians of the percentage of DNA in tail per animal (i.e. 3 slides, 150 cells)
- the mean of median per concentration (i.e. 5 animals, 750 cells)
In addition, each slide was also examined for presence of hedgehogs (possible indicator of toxicity and or apoptosis). Ghost cells were excluded from image analysis data collection. However, determining their frequency might be useful for data interpretation. Therefore, the percentage of hedgehogs was recorded for each slide per animal and per organ actually studied.
Statistics:
In order to quantify the test item effects on DNA, the following statistical analysis strategy was applied, using the statistical software GraphPad Version 3.10.
As the median of percentage of DNA in tail and other tail parameters do not follow a Gaussian distribution (E. Bauer et al., 1998), the non-parametric, one-way Kruskall-Wallis test was performed. This method is based on the analysis of variance by ranks for testing equality of population medians among groups.
The non-parametric Mann-Whitney U-test was applied to compare each of the doses tested with the vehicle control in order to determine statistical significance of differences in group median values between each group versus the vehicle control. This test was also used to compare vehicle control and positive control to
determine acceptable criteria of a valid test.
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
liver from rats
Toxicity:
yes
Remarks:
clinical signs
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
glandular stomach from rats
Toxicity:
yes
Remarks:
clinical signs
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
duodenum from rats
Toxicity:
yes
Remarks:
clinical signs
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
CLINICAL SIGNS:
In the main experiment, the dose of 600 mg/kg elicited a slight or a moderate decrease in spontaneous motor activity in 2 and 3 animals, respectively, 15 minutes after the 1st treatment. Between 2 and 4 hours after the 1st treatment slight decrease in spontaneous motor activity was observed in 3 animals.
Fifteen minutes after both the 2nd and the 3rd treatment, a slight decrease in spontaneous motor activity was observed in 1 animal. Noteworthy, this animal was replaced by 1 of the 2 supplementary animals.
In return, no clinical sign was noted at the 2 inferior doses of 300 and 150 mg/kg b.w. /day (x 3).

MAIN ASSAY FOR GENOTOXICITY IN RATS LIVER CELLS:
An increase in the mean of medians of percentage of tail DNA in was noted at the intermediary analysed dose of 300 mg/kg b.w. /day (x 3) with a value of 2.51 vs. 0.36% in the relative control. This value was over the intervals of historical data for the vehicle control (i.e. 1.10). However, the Kruskall-Wallis test was not significant, no statistically relevant effect was noted when using the Mann-Whitney test and no dose effect relationship was observed. Two animals out of 5 demonstrated a percentage of tail DNA over the intervals of historical data: animal No. 1451 and 1455 (Table 15a, Appendix No. 3a) with values of 7.91 and 3.68%.An out of range value of 17.17% for the slide No. 84A of the animal 1451 was noted, explaining the relative high a value for the percentage of DNA in tail for this animal. For the 3 other animals, percentage of DNA in tail were comparable to the mean of medians obtained in the vehicle control with values ranging from 0.25 to 0.38% vs. 0.36%.
From the overall data, the test item was thus considered as not genotoxic in liver from rats.

MAIN ASSAY FOR GENOTOXICITY IN RATS GLANDULAR STOMACH CELLS:
A statistically significant increase in the mean of medians of percentage of tail DNA was observed only at the intermediary analysed dose of 300 mg/kg b.w. /day (x 3), with a value of 3.96% vs. 0.90% for the vehicle control. Despite the Kruskall-Wallis test was significant, as this value was clearly within the intervals of historical data for vehicle control (i.e. 3.75-7.47% for the 95th confidence intervals) and as no clear dose effect relationship was noted, this effect was considered as not biologically relevant and the test item was considered as not genotoxic in glandular stomach from rats.

MAIN ASSAY FOR GENOTOXICITY IN RATS DUODENUM CELLS:
A statistically significant increase in the mean of medians of percentage of tail DNA was observed only at the lowest analysed dose of 150 mg/kg b.w. /day (x 3), vs. with a value of 3.21% vs. 1.42% for the vehicle control. However, the Kruskall-Wallis test was not significant, and this value was clearly within the intervals of historical data for vehicle control (i.e. 1.82-4.74% for the 95th confidence intervals) and no clear dose effect relationship was noted. This effect was considered as not biologically relevant The test item was thus considered as not genotoxic in duodenum from rats.

DETERMINATION OF SYSTEMIC EXPOSURE:

The determination of systemic exposure was not scheduled in a first intent. Indeed, clinical signs were noted at the top dose tested of 600 mg/kg b.w. /day (x 3), demonstrating the systemic exposure of the animals to the test item.

HISTOPATHOLOGICAL STUDY:

As no biologically significant effect was noticed on the 3 target organs, histopathological study was not performed.

Conclusions:
Under the experimental conditions of this OECD 489 study, the test item is considered as not genotoxic in liver, glandular stomach and duodenum from CD Sprague-Dawley rats.
Executive summary:

The search for potential genotoxic activity of test item was investigated for genotoxic potential by the means of the in vivo comet assay under alkaline conditions (SCGE) in the liver, glandular stomach and duodenum in male CD Sprague-Dawley rats, according to OECD Guideline No 489, 2016. Animals were treated orally once a day for 3 consecutive days, 24 hours apart, up to the dose of 600 mg/kg compatible with the toxic activity.

The control of concentrations of test item in treatment preparations was performed in a GLPcompliant laboratory using a validated method. The results were shown to be compliant with the recovery acceptance criteria (85-115% vs. theoretical concentrations). In addition, test item was not detected in the solvent control.

The acceptance criteria for the assay were considered as fulfilled. The current study was valid.

Under the experimental conditions, the test item does not present DNA breaks and/or alkalilabile sties inducer activities toward the liver, glandular stomach and duodenum from male CD Sprague-Dawley rats.

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

Additional information

3 GLP-compliant in vitro studies were carried out:

- OECD Guideline 471 - Bacterial reverse mutation assay (Ames test),

- OECD Guideline 490 - In vitro mammalian cell gene mutation tests using the thymidine kinase gene,

- OECD Guideline 473 - In vitro mammalian chromosome aberration test.

Regarding the results obtained in this standard battery of genotoxicity tests: the Ames test indicated the lack of mutagenic activity of the solutions of test item on this bacterial test system. In return, in the L5178Y Mouse Lymphoma Assay using the Thymidine Kinase gene, a clear genotoxic activity of solutions of test item was observed in the presence of metabolic activation. As both small and large colonies were increased, solutions of test item should be considered as possibly both mutagenic and clastogenic in vitro. Otherwise, due to a strong limitation of the assay without S9-mix, it is not possible to definitively conclude that solutions of test item are not genotoxic without metabolic activation. Results of the in vitro chromosomal aberrations test on human lymphocytes also indicated a clear clastogenic activity of solutions of test item with metabolic activation and after a 20-hour long-term treatment without metabolic activation, in particular, the presence of exchanges are real markers of clastogenesis which confirms the clastogenic activity of solutions of test item in the presence of metabolic activation.

In order to ensure that no such genotoxicity occurs in vivo, a follow-up in-vivo study was conducted. In-vivo potential genotoxic activity of the substance was investigated by the means of the in vivo micronucleus test in bone marrow and the in vivo comet assay under alkaline conditions (SCGE) in the liver, glandular stomach and duodenum in male CD Sprague-Dawley rats, according to OECD Guidelines (Nos. 474 and 489, 2016). Animals were treated orally once a day for 3 consecutive days, 24 hours apart, up to the dose of 600 mg/kg compatible with the toxic activity. The control of concentrations of the substance in treatment preparations was performed in a GLP compliant laboratory using a validated method. The results were shown to be compliant with the recovery acceptance criteria (85-115% vs. theoretical concentrations). In addition, the substance was not detected in the solvent control. The acceptance criteria for the assay were considered as fulfilled. The current study was valid. Under these experimental conditions, the test item does not present DNA strand breaks and/or alkalilabile sites inducer activities toward the liver, glandular stomach and duodenum from CD Sprague-Dawley male rats. Furthermore, in male CD Sprague-Dawley rats, test item induced no genotoxic activity in bone marrow cells. As a conclusion, the substance induced no in-vivo genotoxic activity under these experimental conditions.

Justification for classification or non-classification

According to the criteria for classification, packaging and labelling of dangerous substances and preparations in accordance with the Regulation EC n°1272/2008 and based on the results of in-vivo testing, the test item has not to be classified for genotoxic potential.