Reaction mass of 3,7-dimethyloct-7-en-1-yl 3-methylbut-2-enoate and 3,7-dimethyloctyl 3-methyl-2-butenoate and citronellyl 3-methylcrotonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not mutagenic (similar to OECD 471, QSAR prediction for the 5th strain TA102)

Not mutagenic (OECD 490, GLP)

Not aneugenic or clastogenic (OECD 487, GLP)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 21, 1982 to January 1983

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

A sample of the test product, a colourless liquid with a mild flavour, designated Sinodor (X-09648, 13.12.82), was received from the sponsor on December 21, 1982. The test material was dissolved in ethanol just prior to use.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9
S9 mix is prepared by mixing the thawed S9 with a NADPH generating system. The final concentrations of the various ingredients in the S9 mix are:
MgCl2 8 mM
KCl 33 mM
G6P 5 mM
NADP 4 mM
Sodium phosphate pH 7.4 100 mM
S9 10%
The S9 mix is prepared just prior to use with cold sterile solutions and is kept in ice water before and during use

Test concentrations with justification for top dose:

A preliminary test was carried out to assess the chemical toxicity of the test substance for the bacteria. The results show that 10 mg of the test substance per plate was slightly toxic whereas 1 mg per plate did not show a growth-inhibiting effect. However, at a dose of 1 mg per plate, the compound precipitated in the plate. Therefore 500 micrograms per plate was chosen as the highest dose level for the mutagenicity study.
Appropriate test solutions in ethanol containing 0, 0.06, 0.19, 0.56, 1.67 and 5.00 mg/ml were prepared immediately before use.

Vehicle / solvent:

Ethanol

Positive controls:

yes

Positive control substance:

sodium azide

Positive controls:

yes

Positive control substance:

other: hycanthone methanesulphonate (12.5 micrograms per 0.1 ml water per plate) for strains TA 1537, TA 1538 and TA 98 without S9 mix

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (0.5 micrograms per 0.1 ml DMSO per plate) for TA 1535, TA 1538, TA 98, TA 100 in the presence of S9 mix. For TA 1537 2.0 micrograms per plate was used in the presence of S9 mix.

Details on test system and experimental conditions:

Preparation and storage of stock cultures:
A fresh culture of each strain is prepared by inoculating nutrient broth with bacteria of the strain in question and incubating the broth for 16h (overnight) at 37°C while shaking. The cultures are then mixed with DMSO to a final DMSO concentration of 8.75%. Subsequently 0.5 ml portions are pipetted into sterile polypropylene vials which are then quickly frozen on dry ice and stored at -80°C.
Part of each culture is retained and examined for the number of spontaneous revertants, histidine requirement and sensitivity to ampicilline, crystal violet and UV radiation. In addition the reversion induced by reference mutagens is determined.

Preparation of cultures for the tests:
To obtain a culture for the mutagenicity test, nutrient broth is inoculated with a thawed aliquot of the stock culture in question (0.1 ml per 10 ml nutrient bouillon). The broth is incubated for 16h (overnight) at 37°C while shaking. The viable count of each culture is determined by plating appropriate dilutions of the culture on nutrient broth agar plates. The cultures are subsequently stored in a refrigerator at 5°C until use, but no longer than 5 days.

Evaluation criteria:

A positive response in the assay system is taken to be a two-fold or greater increase in the mean number of revertant colonies appearing in the test plates over and above the background spontaneous reversion rate observed with the solvent, together with evidence of a dose response.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: High concentration based on precipitation.

Conclusions:

It is concluded that SINODOR does not show any mutagenic activity in the Salmonella/mammalian microsome mutagenicity test under the conditions employed in this evaluation.

Executive summary:

Sinodor was examined for mutagenic activity in the Ames test using the histidine requiring S.typhimurium mutants TA 1535, TA 1537, TA 1538, TA98 and TA100 as indicator strains and a liver microsome fraction of Aroclor-induced rats for metabolic activation.

It is concluded that SINODOR does not show any mutagenic activity in the Salmonella/mammalian microsome mutagenicity test under the conditions employed in this evaluation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
OECD [Q]SAR Toolbox
2. MODEL (incl. version number)
QSAR Toolbox 3.4.0.17
Database version: 3.8.8/3.1.2
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CAS: 20770-40-5
SMILES: CC(CCC=C(C)C)CCOC(=O)C=C(C)C
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
Please see the attached OECD [Q]SAR Toolbox report.
5. APPLICABILITY DOMAIN
The prediction is based on 5 neighbours' values, 5 of them equal to prediction.
Prediction confidence is measured by the p-value = 4.12E-03 (strong confidence)
6. ADEQUACY OF THE RESULT
Negative Ames in the absence of S9 with strain TA102.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
OECD [Q]SAR Toolbox
2. MODEL (incl. version number)
QSAR Toolbox 3.4.0.17
Database version: 3.8.8/3.1.2
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CAS: 20770-40-5
SMILES: CC(CCC=C(C)C)CCOC(=O)C=C(C)C
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
Please see the attached OECD [Q]SAR Toolbox report.
5. APPLICABILITY DOMAIN
The prediction is based on 5 neighbours' values, 5 of them equal to prediction.
Prediction confidence is measured by the p-value = 4.12E-03 (strong confidence)
6. ADEQUACY OF THE RESULT
Negative Ames in the presence of S9 with strain TA102.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 January 2020 to 7 May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Identity SINODOR CQ
Batch no. VE00609758
Sponsor Reference 6099932
EC 946-248-1
Purity 99,5%
Expiry date 09 December 2020
Appearance yellowish-colorless to yellow liquid
Storage conditions room temperature, protected from light
ERBC no. 16728

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y TK+/− (Clone 3.7.2C) mouse lymphoma cells were obtained from American Type
Culture Collection, Rockville,Maryland (ATCC code: CRL 9518). The generation time and
mutation rates (spontaneous and induced) have been checked in this laboratory. The cells
are checked at regular intervals for the absence of mycoplasmal contamination.
Permanent stocks of the L5178Y TK+/− cells are stored in liquid nitrogen, and subcultures
are prepared from the frozen stocks for experimental use. Prior to use, cells were cleansed
of pre-existing mutants.

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue homogenate
One batch of S9 tissue fraction, provided by Trinova Biochem GmbH, was used in this study and had the following characteristics:
Species Rat
Strain Sprague Dawley
Tissue Liver
Inducing Agents Phenobarbital – 5,6-Benzoflavone
Producer MOLTOX,Molecular Toxicology, Inc.
Batch Number 3971

The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each 10 ml):
S9 tissue fraction 0.408mL
NADP (30 mM) 0.204mL
G-6-P (590 mM) 0.204mL
KCl (150 mM) 0.204mL
Complete medium (5%) 8.98mL
Total 10.0 mL

Test concentrations with justification for top dose:

A preliminary solubility trial indicated that the maximum practicable concentration of the test item in the final treatment medium was 2.00 µL/mL (the upper limit to testing as indicated in the Study Protocol) using dimethylsulfoxide (DMSO) as solvent. On the basis of this result, a cytotoxicity assay was performed. Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 2.00 µL/mL and at a wide range of lower dose levels: 1.00, 0.500, 0.250, 0.125, 0.0625, 0.0313, 0.0156 and 0.00781 µL/mL.

Based on the results obtained in the preliminary trial, two independent assays for mutation at the TK locus were performed using the dose levels described in the following table:

Assay No.: S9 Treatment time (hours) Dose level (µl/mL)
1 − 3 0.0417, 0.0333, 0.0267, 0.0213, 0.0171, 0.00853, 0.00427 and 0.00213
1 + 3 0.120, 0.100, 0.0833, 0.0694, 0.0347, 0.0174, 0.00868 and 0.00434
2 − 24 0.0417, 0.0333, 0.0267, 0.0213, 0.0171, 0.00853, 0.00427 and 0.00213

Vehicle / solvent:

The solvent used in this study was dimethylsulfoxide (DMSO), batch no.: H079S obtained from Honeywell.
Solubility of the test item was evaluated in a preliminary trial using DMSO and ethanol. These solvents were selected since they are compatible with the survival of the cells and
the S9 metabolic activity. The test item was found to be soluble in all the solvents tested at 200 µL/mL. However when added to the culture medium (1% v/v) precipitation was observed and some particle in suspension were noticed up to 0.5 µL/mL final concentration.
Since no relevant differences were observed using the different solvents, DMSO was selected as it is the most widely used and with the largest number of historical controls available.
The concentration 200 µL/mL, when added to culture medium in the ratio of 1:100, gave a maximum dose level of 2.00 µL/mL corresponding to the upper limit to be tested as
indicated in the study protocol.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

Fresh solutions of methylmethanesulphonate(MMS) (batch no.: MKBL6789V obtained from Sigma) were prepared in sterile injectable water (batch no.: 19E3002 obtained from Baxter and batch no.:19J2501 obtained from Eurospital) and served as positive controls in the absence of S9 metabolism. Fresh solutions of benzo(a)pyrene (B(a)P) (batch no.: 129K1892 obtained fromSigma) were prepared in DMSO and served as positive controls in the presence of S9 metabolism.

Solutions of the test item were prepared immediately before use in dimethylsulfoxide (DMSO) on a volume/volume basis. Concentrations were expressed in terms of test item as received. All test item solutions were used within 14 minutes from the initial preparation. All dose levels in this report are expressed to three significant figures.

In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained, a second experiment in the absence of S9 metabolism was performed, using a longer treatment time (24 hours).
Results of the first mutation assay were obtained in a repeated assay since in the original experiment there was not adequate number of analysable concentrations.
After washing in Phosphate Buffered Saline (PBS), cells were resuspended in fresh complete medium (10%) and cell densities were determined. The number of cells was adjusted to give 2×105 cells/mL. The cultures were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.
During the expression period (two days after treatment), the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period, the cell densities of each culture were determined and adjusted to give 2×105 cells/mL.

Plating for 5-trifluorothymidine resistance:
After dilution, the cell suspensions in complete medium B (20%) were supplemented with trifluorothymidine (final concentration 3.0 µg/mL) and an estimated 2 × 103 cells were plated in each well of four 96-well plates.
Plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified by eye using background illumination and counted. In addition, the number of wells containing large colonies as well as the number of those containing small colonies were scored.

Plating for viability:
After dilution, in complete medium A (20%), an estimated 1.6 cells/well were plated in each well of two 96-well plates. These plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified as above and counted.

Rationale for test conditions:

Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 2.00 µL/mL and at a wide range of lower dose levels: 1.00, 0.500, 0.250, 0.125, 0.0625, 0.0313, 0.0156 and 0.00781 µL/mL. No precipitate was noted upon addition of the test item to the cultures and at the end of the treatment incubation periods at any concentration tested.
In the absence of S9 metabolic activation, using the 3 hour treatment time, no cells survived treatment from 0.0625 µL/mL onwards. At the next two lower dose levels of 0.0313 and 0.0156 µL/mL, slight toxicity was observed, reducing relative survival (RS) to 74% of the concurrent negative control value. No relevant toxicity was noted at 0.00781 µL/mL. Using the 24 hour treatment time, no cells survived treatment from 0.125 µL/mL onwards, slight toxicity was observed at 0.0625 µL/mL, reducing relative survival to 67% of the concurrent negative control. No relevant toxicity was observed over the remaining dose levels tested.
Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), severe toxicity was observed between 2.00 and 0.250 µL/mL. Test item treatment at 0.125 µL/mL yielded slight toxicity reducing RS to 75%, while no relevant toxicity was observed over the remaining dose levels tested.

Evaluation criteria:

For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF)
suggested for the microwell method (126×10−6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis.
Results which only partially satisfy the above criteria will be dealt with on a case-by-case
basis. Similarly, positive responses seen only at high levels of cytotoxicity will require
careful interpretation when assessing their biological significance. Any increase in mutant
frequency should lie outside the historical control range to have biological relevance.

Statistics:

Statistical analysis was performed according to UKEMS guidelines (RobinsonW.D., 1990).

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In the first Assay, in the absence of S9 metabolic activation, dose related cytotoxic effects were observed reducing the relative total growth (RTG) to 4% of the concurrent negative control at 0.0417 µL/mL and to 18% at the next lower dose level (0.0333 µL/mL).
In the presence of S9 metabolism, severe toxicity reducing RTG to 1% of the concurrent negative control was noted at 0.120 µL/mL, marked toxicity reducing RTG to 12% of the concurrent negative control was noted at 0.100 µL/mL, mild toxicity was observed at 0.0833 and 0.0694 µL/mL, while no relevant toxicity was noted over the remaining dose levels tested.
In the second Assay, in the absence of S9 metabolic activation using a long treatment time, the highest dose level selected (0.0417 µL/mL) yielded moderate toxicity reducing RTG to 37% of the concurrent negative control value. The next two lower dose levels of 0.0333 and 0.0267 µL/mL yielded slight toxicity reducing RTG to 70-68%, respectively. No relevant toxicity was noted over the remaining dose levels tested.
At low survival levels, the mutation data are prone to a variety of artefacts (selection effects, sampling error, founder effects). Mechanisms other than direct genotoxicity per se can lead to positive results that are related to cytotoxicity and not genotoxicity (e.g. events associated with apoptosis, endonuclease release fromlysosomes, etc.). For this reason it is generally recommended that such data are treated with caution or excluded from consideration.
Accordingly, we have excluded from the statistical analyses, mutation data obtained in the first experiment in the absence and presence of S9 metabolism at 0.0417 µL/mL and 0.120 µL/mL, respectively.

In all experimental conditions examined there is no concentration related response as indicated by the linear trend analysis. The induced mutant frequency (IMF) was lower than the global evaluation factor at all dose levels tested. No statistically significant or biologically relevant increase in mutant frequency values was observed at any concentration tested, in any treatment series in the absence or presence of S9 metabolic activation.

The positive control items induced an increase in the small colony MF of at least 150×10−6 above that seen in the concurrent solvent control, demonstrating that one criterion of acceptance for the small colony mutant frequency was achieved.

The study was accepted as valid.

The addition of the test item solution did not have any obvious effect on the osmolality or pH of the treatment medium.

Conclusions:

It is concluded that SINODOR CQ does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

The test item SINODOR CQ was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.

A preliminary solubility trial indicated that the maximum practicable concentration of the test item in the final treatment medium was 2.00 µL/mL (the upper limit to testing as indicated in the Study Protocol) using dimethylsulfoxide (DMSO) as solvent.

On the basis of this result, a cytotoxicity assay was performed. Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 2.00 µL/mL and at a wide range of lower dose levels: 1.00, 0.500, 0.250, 0.125, 0.0625, 0.0313, 0.0156 and 0.00781 µL/mL.

In the absence of S9 metabolic activation, using the 3 hour treatment time, no cells survived treatment from 0.0625 µL/mL onwards. At the next two lower dose levels of 0.0313 and 0.0156 µL/mL, slight toxicity was observed, reducing relative survival (RS) to 74% of the concurrent negative control value. No relevant toxicity was noted at 0.00781 µL/mL. Using the 24 hour treatment time, no cells survived to treatment from 0.125 µL/mL onwards, slight toxicity was observed at 0.0625 µL/mL, reducing relative survival to 67% of the concurrent negative control value. No relevant toxicity was observed over the remaining dose levels tested. Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), severe toxicity was observed from 2.00 up to 0.250 µL/mL. Test item treatment at 0.125 µL/mL yielded slight toxicity reducing RS to 75%, while no relevant toxicity was observed over the remaining dose levels tested.

Based on the results obtained in the preliminary trial, two independent assays for mutation at the TK locus were performed using the dose levels described in the following table:

Assay No.:	S9	Treatment time (hours)	Dose level (microliter/mL)
1	-	3	0.0417, 0.0333, 0.0267, 0.0213, 0.0171, 0.00853, 0.00427 and 0.00213
1	+	3	0.120, 0.100, 0.0833, 0.0694, 0.0347, 0.0174, 0.00868 and 0.00434
2	-	24	0.0417, 0.0333, 0.0267, 0.0213, 0.0171, 0.00853, 0.00427 and 0.00213

Negative and positive control treatments were included in each mutation experiment both in the absence and presence of S9 metabolism. The cloning efficiencies at Day 2, the suspension growth over 2 days and the mutant frequencies in the solvent control cultures fell within the normal range. Both positive controls, methylmethanesulphonate and benzo(a)pyrene, induced a clear increase above the spontaneous background in the small colony mutation frequency, higher than 150×10=6. The study was accepted as valid.

Adequate levels of cytotoxicity, covering a range from the maximum to slight or no toxicity, were observed in all treatment series. No relevant increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism.

It is concluded that SINODOR CQ does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Reference 5

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 January 2020 to 09 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Identity SINODOR CQ
Batch no. VE00609758
Sponsor Reference 6099932
EC No. 946-248-1
Purity 99.5%
Expiry date 09 December 2020
Appearance yellowish - colorless to yellow liquid
Storage conditions room temperature, protected from light
ERBC number 16728

Target gene:

Not applicable - In vitro Micronucleus in Mammalian cells

Species / strain / cell type:

lymphocytes: peripheral human lymphocytes

Details on mammalian cell type (if applicable):

For Main Assay 1, human whole blood was provided by Biopredic International (France).
The following two batches were pooled and had the following characteristics:

Sex Male
Age 33 years old
Donor information healthy, no smoker without any recent exposure to drugs or radiation
Anticoagulant Sodium heparin, 556 IU/mL of whole blood
Collection date 16 January 2020
ERBC Code Number 2020/3

Sex Female
Age 30 years old
Donor information healthy, no smoker without any recent exposure to drugs or radiation
Anticoagulant Sodium heparin, 556 IU/mL of whole blood
Collection date 16 January 2020
ERBC Code Number 2020/4

For Main Assay 2, human whole blood was provided by BioIVT (UK). The following two batches were pooled and had the following characteristics:

Sex Female
Age Less than 35 years old
Donor information healthy, no smoker without any recent exposure to drugs or radiation
Anticoagulant Sodium heparin, 12 to 30 IU/mL of whole blood
Collection date 06May 2020
ERBC Code Number 2020/17

Sex Female
Age Less than 35 years old
Donor information healthy, no smoker without any recent exposure to drugs or radiation
Anticoagulant Sodium heparin, 12 to 30 IU/mL of whole blood
Collection date 06May 2020
ERBC Code Number 2020/18

Cytokinesis block (if used):

Cytochalasin-B

Metabolic activation:

with and without

Metabolic activation system:

One batch of S9 tissue fraction, provided by Trinova Biochem GmbH, was used in this study and had the following characteristics:

Species Rat
Strain Sprague Dawley
Tissue Liver
Inducing Agents Phenobarbital – 5,6-Benzoflavone
Producer MOLTOX,Molecular Toxicology, Inc.
Batch Number 3971

The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each
10 mL):
S9 tissue fraction 1.0mL
NADP (100mM) 0.4mL
G-6-P (100mM) 0.5mL
MgCl2 (100mM) 0.2mL
Phosphate buffer (pH 7.4, 200mM) 5.0mL
Distilled Water 2.9mL

Test concentrations with justification for top dose:

The CBPI was calculated for each of the treatment series.
Following the 3 hour treatment in the presence of S9 metabolism, marked cytotoxicity was seen from 0.444 µL/mL onwards, while mild or no remarkable cytotoxicity was noticed over the remaining dose range.
Following the continuous treatment in the absence of S9 metabolism severe cytotoxicity was observed from 0.148 µL/mL onwards. Moderate to no remarkable cytotoxicity was seen over the remaining dose range.
For the second Main Assay, following the short treatment in the absence of S9 metabolism, marked cytotoxicity was seen from 0.0756 µL/mL onwards, while moderate to no cytotoxicity was seen over the remaining dose range.

On the basis of the above results, the dose levels selected for scoring of micronuclei were as follows: see table in "any other information on materials and methods including tables" section below

Vehicle / solvent:

The solvent used for the test item was dimethylsulfoxide (DMSO, batch nos.H079S and J128BIL obtained from Honeywell).

In a preliminary solubility test performed for ERBC Study No. A3803 the test item was found soluble in DMSO and ethanol at the concentration of 200 µL/mL.
For the present study DMSO was selected as solvent/vehicle since it is the most widely used and with the largest number of historical controls available. Based on the preliminary toxicity results obtained in ERBC Study No. A3803, the maximum dose level selected for the short termtreatment was 1.00 µL/mL, while 0.500 µL/mL was selected for the continuous treatment.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Solutions of the test item, as received, were prepared immediately before use in dimethylsulfoxide (DMSO). Solutionswere prepared on a weight/volume basis without correction for the displacement due to the volume occupied by the test item. Concentrations of solutions were expressed in terms of test item as received. All test item solutions were used within 15 minutes from the initial preparation.

For Main Assay 1, three treatment series were performed including solvent/negative and positive controls. ForMain Assay 2, one treatment series was performed including solvent/negative and positive control. Two replicate cultures were prepared at each test point. Lymphocyte cultures were treated approximately forty-eight hours after they were initiated. Before treatment, cultures were centrifuged at 1000 rpm for 10 minutes and the culture medium was decanted and replaced with treatment medium.

For the short treatment series, the composition of the treatment media was as follows:

Presence of S9 metabolism

Test item solution or solvent/vehicle 0.05mL
S9 mix 1.00mL
Culture medium (without PHA) 3.95mL

Absence of S9 metabolism

Test item solution or solvent/vehicle 0.05mL
Culture medium (without PHA) 4.95mL


For the continuous treatment, due to the concurrent addition of Cytochalasin B dissolved in DMSO, it was necessary to reduce the volume of test item solutions (25 µL/tube instead of 50 µL/tube), to maintain the final concentration of organic solvents to 1%.

The composition of the treatment medium was as follows:

Absence of S9 metabolism

Test item solution or solvent/vehicle 0.025mL
Culture medium (without PHA) 4.975mL

Treatment medium for positive controls was prepared in the following proportions:

Presence of S9 metabolism

Positive control solution 0.05mL
S9 mix 1.00mL
Culture medium (without PHA) 3.95mL

Absence of S9 metabolism

Positive control solution 0.05mL
Culture medium (without PHA) 4.95mL

For the short treatment series, the treatment mediawere added to the tubes and the cultures were incubated for 3 hours at 37°C. At the end of treatment time, the cell cultures were centrifuged and washed twice with Phosphate Buffered Saline Solution. Fresh medium was added and the cultures were incubated for a further 28 hours (Recovery Period) before harvesting. At the same time, Cytochalasin-B was added to achieve a final concentration of 6 µg/mL.
For the continuous treatment series, 3 hours after beginning of treatment, Cytochalasin-B was also added and the cultures were incubated for a further 28 hours before harvesting.

Rationale for test conditions:

For the first Main Assay, following the 3 hour treatment in the absence of S9 metabolism, severe cytotoxicity was observed over the whole dose range tested excluding the low dose levels of 0.0585 and 0.0390 µL/mL.
Following the 3 hour treatment in the presence of S9 metabolism, marked cytotoxicity was seen from 0.444 µL/mL onwards, while mild or no remarkable cytotoxicity was noticed over the remaining dose range.
Following the continuous treatment in the absence of S9 metabolism severe cytotoxicity was observed from 0.148 µL/mL onwards. Moderate to no remarkable cytotoxicity was seen over the remaining dose range.
For the second Main Assay, following the short treatment in the absence of S9 metabolism, marked cytotoxicity was seen from 0.0756 µL/mL onwards, while moderate to no cytotoxicity was seen over the remaining dose range.
On the basis of the above results, the dose levels selected for scoring of micronucleis were indicated in the table in "any other information on materials and methods including tables" section below

Evaluation criteria:

In this assay, the test item is considered as clearly positive if the following criteria are met:
– Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations.
– The proportion of micronucleated cells at such data points exceeds the normal range based on historical control values (95% control limits).
– There is a significant dose effect relationship.

The test item is considered clearly negative if the following criteria are met:
– None of the dose levels shows a statistically significant increase in the incidence of micronucleated cells.
– There is no concentration related increasewhen evaluated with the Cochran-Armitage trend test.
– All the results are inside the distribution of the historical control data (95% control limits).

Statistics:

For the statistical analysis, a modified χ2 test was used to compare the number of cells with micronuclei in control and treated cultures.
Cochran-Armitage Trend Test (one-sided) was performed to aid determination of concentration response relationship.

Species / strain:

lymphocytes: peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In the first Main Assay following treatment with the test item in the presence of S9 metabolism, no statistically significant increase in the incidence of micronucleated cells over the concurrent solvent control value was observed. All incidences were within the normal distribution of historical control values (95% confidence limits). Besides a statistically significant dose dependent decrease in the incidence of micronucleated cells was indicated by the linear trend analysis.
Following the continuous treatment in the absence of S9 metabolism, a concentration related effect was seen (p<5%). However, no statistically significant increase in the incidence of micronucleated cells was observed at any dose level and all results were inside the distribution of the historical negative control data. Hence, the linear trend was considered to be attributable to a chance event, not related to the action of the test item and of no biological relevance.
For the second Main Assay, following treatment with the test item in the absence of S9 metabolism, a statistically significant increase in the incidence of micronucleated cells was seen at the highest dose level selected for scoring. All incidences however were within the normal distribution range of the historical negative control data (95% confidence limits). An extended analysis to increase the sample size was performed by an additional scoring of two thousand binucleated cells for the highest dose level and for the solvent control. No statistically significant increase in the incidence of micronucleated cells was seen at the dose level examined nor a dose effect relationship was present. All incidences were within the normal distribution range of our historical negative control values (95% confidence limits).

Results are summarised in the table in "Any other information on results incl. tables" section below (results per assay).

Controls results:

Adequate cell proliferation was observed in negative control cultures and the appropriate number of doses and cells was analysed.

Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide, Colchicine and Mitomycin-C and the responseswere compatible with those generated in our historical control database, indicating the correct functioning of the test system.

The study was accepted as valid.

Results per assay:

Main assay	S9	Treatment time (hours)	Harvest time (hours)	Dose level (microL/mL)	Incidence of micronucleated cells (%)	Statistical significance
1	+	3	32.5	0.00	1.15	-
1	+	3	32.5	0.444	0.50	L
1	+	3	32.5	0.198	0.90	NS
1	+	3	32.5	0.0878	0.65	NS
1	-	31	31	0.00	0.30	-
1	-	31	31	0.0988	0.75	NS
1	-	31	31	0.0658	0.40	NS
1	-	31	31	0.0439	0.50	NS
2	-	3	32	0.00	0.23	-
2	-	3	32	0.0658	0.35	NS
2	-	3	32	0.0497	0.45	NS
2	-	3	32	0.0376	0.20	NS

NS: Not significant

L: Significantly lower or significant decrease

Conclusions:

On the basis of the results obtained, it is concluded that SINODOR CQ does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Executive summary:

The test item SINODOR CQ was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation.

Three treatment conditions were performed. A short termtreatment, where the cells were treated for 3 hours, was performed in the absence and presence of S9 metabolism. The harvest time of approximately 32.5 hours, corresponding to approximately two cell cycle lengths, was used. A long term (continuous) treatment was also performed only in the absence of S9 metabolism, until harvest at 31 hours. Solutions of the test item were prepared in dimethylsulfoxide (DMSO). Based on the preliminary toxicity results obtained in ERBC Study No. A3803, the maximum dose level selected for the short term treatment was 1.00 µL/mL. The dose level of 0.500 µL/mL was selected for the continuous treatment.

Treatments were performed as follows and for the short termexposure in the absence of S9 metabolism, a steep increase of cytotoxicity was observed and no concentration tested showed an adequate cytotoxicity (55 ± 5%) to assess the frequency of micronucleated cells, therefore a Main Assay 2 was performed where treatments were as follows:

Main assay	S9	Treatment time (hours)	Harvest time (hours)	Dose level (microL/mL)
1	-	3	32.5	1.00, 0.667, 0.444, 0.296, 0.198, 0.132, 0.0878, 0.0585 and 0.0390
1	+	3	32.5	1.00, 0.667, 0.444, 0.296, 0.198, 0.132, 0.0878, 0.0585 and 0.0390
1	-	31	31	0.500, 0.333, 0.222, 0.148, 0.0988, 0.0658, 0.0439, 0.0293 and 0.0195
2	-	3	32	0.100, 0.0870, 0.0756, 0.0658, 0.0572, 0.0497, 0.0432 and 0.037

Each experiment included appropriate negative and positive controls. Two replicate cell cultures were prepared at each test point. The actin polymerisation inhibitor Cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. The cytokinesisblock proliferation index CBPI was calculated in order to evaluate cytotoxicity.

Dose levels for the scoring of micronuclei were selected with the aim to evaluate the test item concentrations at adequate levels of cytotoxicity, covering a range from the maximum (approximately 55±5%) to slight or no toxicity.

Based on the results obtained, the following concentrations were selected for the scoring of micronuclei:

Main assay	S9	Treatment time (hours)	Harvest time (hours)	Dose level (microL/mL)	Cytotoxicity (%)	Presence of precipitation/opacity
1	+	3	32.5	0.00	0	-
1	+	3	32.5	0.444	61	No
1	+	3	32.5	0.198	39	No
1	+	3	32.5	0.0878	2	No
1	-	31	31	0.00	0	-
1	-	31	31	0.0988	55	No
1	-	31	31	0.0658	37	No
1	-	31	31	0.0439	16	No
2	-	3	32	0.00	0	-
2	-	3	32	0.0658	51	No
2	-	3	32	0.0497	24	No
2	-	3	32	0.0376	-4	No

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells. Adequate cell proliferation was observed in negative control cultures and the appropriate number of doses and cells was analysed. Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide, Colchicine and Mitomycin-C and the responseswere compatible with those generated in our historical control database, indicating the correct functioning of the test system.

The study was accepted as valid.

In Main Assay 1, following treatment with the test item in the presence of S9 metabolism, no statistically significant increase in the incidence of micronucleated cells of the solvent control was observed. All incidences were within the normal distribution of historical control values (95% confidence limits) and no dose related increase in the incidence of micronucleated cells was indicated by the linear trend analysis.

Following the continuous treatment in the absence of S9 metabolism, a statistically significant dose effect relationship was seen (p<5%). However, no statistically significant increase in the incidence of micronucleated cells was observed at any dose level and all results were inside the distribution of the historical negative control data. Hence, the linear trend was considered to be attributable to a chance event, not related to the action of the test item and of no biological relevance.

In Main Assay 2, a statistically significant increase in the incidence of micronucleated cells was seen at the highest dose level selected for scoring. All incidences however were within the range of distribution of the historical negative control data (95% confidence limits).

An additional scoring of two thousand binucleated cells was performed for the highest dose level and for the solvent control cultures to increase the sample size. No statistically significant increase in the incidence of micronucleated cells was seen nor a dose effect relationship was present. All incidences were within the distribution range of our historical negative control values (95% confidence limits).

Results are summarised in the following table:

Main assay	S9	Treatment time (hours)	Harvest time (hours)	Dose level (microL/mL)	Incidence of micronucleated cells (%)	Statistical significance
1	+	3	32.5	0.00	1.15	-
1	+	3	32.5	0.444	0.50	L
1	+	3	32.5	0.198	0.90	NS
1	+	3	32.5	0.0878	0.65	NS
1	-	31	31	0.00	0.30	-
1	-	31	31	0.0988	0.75	NS
1	-	31	31	0.0658	0.40	NS
1	-	31	31	0.0439	0.50	NS
2	-	3	32	0.00	0.23	-
2	-	3	32	0.0658	0.35	NS
2	-	3	32	0.0497	0.45	NS
2	-	3	32	0.0376	0.20	NS

NS: Not significant

L: Significantly lower or significant decrease

It is concluded that SINODOR CQ does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

There is no genotoxic potential observed in any of the in vitro tests performed on the substance Sinodor.

The battery of tests includes QSAR predictions (OECD QSAR Toolbox), an Ames test, an MLA (OECD 490) and a MNT (OECD 487).

Additional information

Mutagenicity in bacteria:

Sinodor was examined for mutagenic activity in the Ames test using the histidine requiring S.typhimurium mutants TA 1535, TA 1537, TA 1538, TA98 and TA100 as indicator strains and a liver microsome fraction of Aroclor-induced rats for metabolic activation.

It is concluded that SINODOR does not show any mutagenic activity in the Salmonella/mammalian microsome mutagenicity test under the conditions employed in this evaluation.

As the Ames test was performed before the latest update requiring the testing of a fifth strain (TA 102 or E.Coli WP2), a QSAR approach was used to predict the result on TA 102 in the presence and absence of metabolic activation. The outcome of the QSAR was negative (see complete report attached in the corresponding Endpoint Study Record).

Mutagenicity in Mammalian cells (MLA OECD 490, GLP):

The test item SINODOR CQ was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.

A preliminary solubility trial indicated that the maximum practicable concentration of the test item in the final treatment medium was 2.00 µL/mL (the upper limit to testing as indicated in the Study Protocol) using dimethylsulfoxide (DMSO) as solvent.

On the basis of this result, a cytotoxicity assay was performed. Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 2.00 µL/mL and at a wide range of lower dose levels: 1.00, 0.500, 0.250, 0.125, 0.0625, 0.0313, 0.0156 and 0.00781 µL/mL.

In the absence of S9 metabolic activation, using the 3 hour treatment time, no cells survived treatment from 0.0625 µL/mL onwards. At the next two lower dose levels of 0.0313 and 0.0156 µL/mL, slight toxicity was observed, reducing relative survival (RS) to 74% of the concurrent negative control value. No relevant toxicity was noted at 0.00781 µL/mL. Using the 24 hour treatment time, no cells survived to treatment from 0.125 µL/mL onwards, slight toxicity was observed at 0.0625 µL/mL, reducing relative survival to 67% of the concurrent negative control value. No relevant toxicity was observed over the remaining dose levels tested. Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), severe toxicity was observed from 2.00 up to 0.250 µL/mL. Test item treatment at 0.125 µL/mL yielded slight toxicity reducing RS to 75%, while no relevant toxicity was observed over the remaining dose levels tested.

Based on the results obtained in the preliminary trial, two independent assays for mutation at the TK locus were performed using the dose levels described in the following table:

Assay No.:	S9	Treatment time (hours)	Dose level (microliter/mL)
1	-	3	0.0417, 0.0333, 0.0267, 0.0213, 0.0171, 0.00853, 0.00427 and 0.00213
1	+	3	0.120, 0.100, 0.0833, 0.0694, 0.0347, 0.0174, 0.00868 and 0.00434
2	-	24	0.0417, 0.0333, 0.0267, 0.0213, 0.0171, 0.00853, 0.00427 and 0.00213

Negative and positive control treatments were included in each mutation experiment both in the absence and presence of S9 metabolism. The cloning efficiencies at Day 2, the suspension growth over 2 days and the mutant frequencies in the solvent control cultures fell within the normal range. Both positive controls, methylmethanesulphonate and benzo(a)pyrene, induced a clear increase above the spontaneous background in the small colony mutation frequency, higher than 150×10=6. The study was accepted as valid.

Adequate levels of cytotoxicity, covering a range from the maximum to slight or no toxicity, were observed in all treatment series. No relevant increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism.

It is concluded that SINODOR CQ does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Cytogenicity in vitro (MNT OECD 487, GLP):

The test item SINODOR CQ was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation.

Three treatment conditions were performed. A short termtreatment, where the cells were treated for 3 hours, was performed in the absence and presence of S9 metabolism. The harvest time of approximately 32.5 hours, corresponding to approximately two cell cycle lengths, was used. A long term (continuous) treatment was also performed only in the absence of S9 metabolism, until harvest at 31 hours. Solutions of the test item were prepared in dimethylsulfoxide (DMSO). Based on the preliminary toxicity results obtained in ERBC Study No. A3803, the maximum dose level selected for the short term treatment was 1.00 µL/mL. The dose level of 0.500 µL/mL was selected for the continuous treatment.

Treatments were performed as follows and for the short termexposure in the absence of S9 metabolism, a steep increase of cytotoxicity was observed and no concentration tested showed an adequate cytotoxicity (55 ± 5%) to assess the frequency of micronucleated cells, therefore a Main Assay 2 was performed where treatments were as follows:

Main assay	S9	Treatment time (hours)	Harvest time (hours)	Dose level (microL/mL)
1	-	3	32.5	1.00, 0.667, 0.444, 0.296, 0.198, 0.132, 0.0878, 0.0585 and 0.0390
1	+	3	32.5	1.00, 0.667, 0.444, 0.296, 0.198, 0.132, 0.0878, 0.0585 and 0.0390
1	-	31	31	0.500, 0.333, 0.222, 0.148, 0.0988, 0.0658, 0.0439, 0.0293 and 0.0195
2	-	3	32	0.100, 0.0870, 0.0756, 0.0658, 0.0572, 0.0497, 0.0432 and 0.037

Each experiment included appropriate negative and positive controls. Two replicate cell cultures were prepared at each test point. The actin polymerisation inhibitor Cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. The cytokinesisblock proliferation index CBPI was calculated in order to evaluate cytotoxicity.

Dose levels for the scoring of micronuclei were selected with the aim to evaluate the test item concentrations at adequate levels of cytotoxicity, covering a range from the maximum (approximately 55±5%) to slight or no toxicity.

Based on the results obtained, the following concentrations were selected for the scoring of micronuclei:

Main assay	S9	Treatment time (hours)	Harvest time (hours)	Dose level (microL/mL)	Cytotoxicity (%)	Presence of precipitation/opacity
1	+	3	32.5	0.00	0	-
1	+	3	32.5	0.444	61	No
1	+	3	32.5	0.198	39	No
1	+	3	32.5	0.0878	2	No
1	-	31	31	0.00	0	-
1	-	31	31	0.0988	55	No
1	-	31	31	0.0658	37	No
1	-	31	31	0.0439	16	No
2	-	3	32	0.00	0	-
2	-	3	32	0.0658	51	No
2	-	3	32	0.0497	24	No
2	-	3	32	0.0376	-4	No

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells. Adequate cell proliferation was observed in negative control cultures and the appropriate number of doses and cells was analysed. Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide, Colchicine and Mitomycin-C and the responseswere compatible with those generated in our historical control database, indicating the correct functioning of the test system.

The study was accepted as valid.

In Main Assay 1, following treatment with the test item in the presence of S9 metabolism, no statistically significant increase in the incidence of micronucleated cells of the solvent control was observed. All incidences were within the normal distribution of historical control values (95% confidence limits) and no dose related increase in the incidence of micronucleated cells was indicated by the linear trend analysis.

Following the continuous treatment in the absence of S9 metabolism, a statistically significant dose effect relationship was seen (p<5%). However, no statistically significant increase in the incidence of micronucleated cells was observed at any dose level and all results were inside the distribution of the historical negative control data. Hence, the linear trend was considered to be attributable to a chance event, not related to the action of the test item and of no biological relevance.

In Main Assay 2, a statistically significant increase in the incidence of micronucleated cells was seen at the highest dose level selected for scoring. All incidences however were within the range of distribution of the historical negative control data (95% confidence limits).

An additional scoring of two thousand binucleated cells was performed for the highest dose level and for the solvent control cultures to increase the sample size. No statistically significant increase in the incidence of micronucleated cells was seen nor a dose effect relationship was present. All incidences were within the distribution range of our historical negative control values (95% confidence limits).

Results are summarised in the following table:

Main assay	S9	Treatment time (hours)	Harvest time (hours)	Dose level (microL/mL)	Incidence of micronucleated cells (%)	Statistical significance
1	+	3	32.5	0.00	1.15	-
1	+	3	32.5	0.444	0.50	L
1	+	3	32.5	0.198	0.90	NS
1	+	3	32.5	0.0878	0.65	NS
1	-	31	31	0.00	0.30	-
1	-	31	31	0.0988	0.75	NS
1	-	31	31	0.0658	0.40	NS
1	-	31	31	0.0439	0.50	NS
2	-	3	32	0.00	0.23	-
2	-	3	32	0.0658	0.35	NS
2	-	3	32	0.0497	0.45	NS
2	-	3	32	0.0376	0.20	NS

NS: Not significant

L: Significantly lower or significant decrease

It is concluded that SINODOR CQ does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Justification for classification or non-classification

Based on the data available and key results described in this summary, the substance has shown no genotoxicity potential and should therefore not be classified for genotoxicity according to the (EC) No 1272/2008 Regulation (CLP).