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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An evaluation of the ability of the registration substance to induce chromosome aberrations in cultured peripheral human lymphocytes in line with OECD Guideline no. 473: In Vitro Mammalian Chromosome Aberration Test (adopted September 26, 2014) was carried out. Results showed that under the environmental conditions outlined in the study, the registration substance is not clastogenic in human lymphocytes.


Following OECD Guideline no. 471: "Genetic Toxicology: Bacterial Reverse Mutation Test" (Adopted July 21, 1997) a study to evaluate the registration substance for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains resulting in histidineindependent strains, and in a gene of tryptophan-requiring Escherichia coli bacterial strain resulting in a tryptophan-independent strain was carried out. Based on the results of this study it is concluded that the registration substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.


Following OECD 476 guideline (2016), An in-vitro gene mutation study in mammalian cells (HPRT) study concluded that tested substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
GR-50-1408 is the Givaudan identification code which was employed for ROSYFOLIA during the early, developmental and testing period.
Species / strain / cell type:
lymphocytes: Human
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
1st cytogenetic assay : 155 and 170 μg/ml , for 3 h exposure time exposure time with a 24 h fixation time with and without metabolic activation system
2nd cytogenetic assay : 100 μg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 75 μg/ml for a 48 h continuous exposure time with a 48 h fixation time without of S9-mix
Untreated negative controls:
yes
Remarks:
with vehicule
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulfoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without metabolic activation (-S9-mix)
Untreated negative controls:
yes
Remarks:
with vehicule
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulfoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation (+S9-mix)
Rationale for test conditions:
Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in international guidelines (OECD, EC).
Blood was collected from healthy adult, non-smoking, male volunteers. The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2014) are presented below:
Dose range finding study: age 23, AGT = 12.9 h
First cytogenetic assay: age 32, AGT = 12.8 h
Cytogenetic assay 1A: age 25, AGT = 12.8 h
Second cytogenetic assay: age 27, AGT = 12.6 h (24 h exposure time)
age 25, AGT = 12.8 h (48 h exposure time)
Key result
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Appropriate toxicity was reached at these dose levels
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

At a concentration of 512 µg/ml GR-50-1408 precipitated in the culture medium and was used as the highest concentration of GR-50-1408. In the dose range finding test blood cultures were treated with 5.4, 17, 52, 164 and 512 µg GR-50-1408/ml culture medium with and without S9-mix.

Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:

Without and with S9-mix: 5, 50, 100, 125, 150, 175 and 200 µg/ml culture medium

(3 h exposure time, 24 h fixation time).

Both in the absence and presence of S9-mix, GR-50-1408 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. Both in the absence and presence of S9-mix, GR-50-1408 did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

To obtain more information about the possible clastogenicity of GR-50-1408, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to GR-50-1408 in the absence of S9-mix for 24 or 48 hours. The following dose levels were selected for the second cytogenetic assay:

Without S9-mix : 5, 50, 75, 100, 125 and 150 µg/ml culture medium

(24 h and 48 h exposure time, 24 h and 48 h fixation time).

GR-50-1408 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. GR-50-1408 did not increase the number of polyploid cells and cells with endoreduplicated chromosomes

Conclusions:
Interpretation of results (migrated information): negative - it is concluded that this test is valid and that GR-50-1408 is not clastogenic in human lymphocytes.
Both in the absence and presence of S9-mix GR-50-1408 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.
No effects of GR-50-1408 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that GR-50-1408 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions describedt.
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:
2015
Reliability:
1 (reliable without restriction)
Qualifier:
according 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:
GR-50-1408 is the Givaudan identification code which was employed for ROSYFOLIA during the early, developmental and testing period.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
Untreated negative controls:
yes
Remarks:
The vehicle of the test substance
Negative solvent / vehicle controls:
yes
Remarks:
The vehicle of the test substance, which was DMSO.
True negative controls:
not specified
Positive control substance:
sodium azide
Remarks:
TA1535 , 5 μg , without S9
Untreated negative controls:
yes
Remarks:
The vehicle of the test substance
Negative solvent / vehicle controls:
yes
Remarks:
The vehicle of the test substance, which was DMSO.
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
TA1537, 2.5 μg, without S9
Untreated negative controls:
yes
Remarks:
The vehicle of the test substance
Negative solvent / vehicle controls:
yes
Remarks:
The vehicle of the test substance, which was DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA98, 10 μg, without S9
Untreated negative controls:
yes
Remarks:
The vehicle of the test substance
Negative solvent / vehicle controls:
yes
Remarks:
The vehicle of the test substance, which was DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA100 , 650 μg , without S9
Untreated negative controls:
yes
Remarks:
The vehicle of the test substance
Negative solvent / vehicle controls:
yes
Remarks:
The vehicle of the test substance, which was DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
WP2uvrA, 10 μg, without S9
Untreated negative controls:
yes
Remarks:
The vehicle of the test substance
Negative solvent / vehicle controls:
yes
Remarks:
The vehicle of the test substance, which was DMS
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA1535, TA1537 at 2.5 μg, TA1537 at 5 μg, TA98 an TA100 at 1 μg, TA100 at 1 μg, WP2uvrA at 15 μg with S9
Details on test system and experimental conditions:
Salmonella typhimurium bacteria and Escherichia coli bacteria.
The Salmonella typhimurium strains used in this study were TA1535, TA1537, TA98 and TA100. The Escherichia coli strain used was WP2uvrA. The strains TA1537 and TA98 are capable of detecting frameshift mutagens, strains TA1535, TA100 and WP2uvrA are capable of detecting base-pair substitution mutagens (1-5).
Rationale for test conditions:
The assay was conducted in the absence and presence of a metabolizing system (S9-mix).
Recommended test system in international guidelines (e.g. OECD, EC and MITI).
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No increase in the number of revertants
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
TA1535, TA1537, TA98 and TA100 at 512 μg/plate with and without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information): negative

All bacterial strains showed negative responses over the entire dose range, i.e. no significant doserelated increase in the number of revertants in two experiments.
The negative control values were within the laboratory historical control data ranges.
The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA98 in the first experiment, see protocol deviation 1.
Based on the results of this study it is concluded that GR-50-1408 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-2021
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Version / remarks:
OECD, 2016
Deviations:
no
Remarks:
no deviations that compromised the integrity of the study/study results
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from male Sprague Dawley rats induced with β-Naphthoflavone/Phenobarbital. The S-9 was supplied as lyophilized S-9 mix (MutazymeTM), stored frozen at <-10°C, and thawed and reconstituted with purified water to provide a 10% S-9 mix just prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).
Treatments were carried out both in the absence and presence of S-9 by addition of either 150 mM KCl or 10% S-9 mix respectively. The final S-9 volume in the test system was 1% (v/v).
Test concentrations with justification for top dose:
Test concentrations:
- without S9: 10, 20, 30, 40, 45, 50, 55, 60, 62.5, 65, 70, 80 μg/mL
- with S9: 20, 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120 μg/mL
The highest concentrations analysed were 50 μg/mL in the absence of S-9 and 70 μg/mL in the presence of S-9 due to the high cytotoxicity of the other highest doses.

Justification for top dose:
In the Mutation Experiment twelve concentrations, ranging from 10 to 80 µg/mL, were tested in the absence of S-9 and thirteen concentrations, ranging from 20 to 120 µg/mL, were tested in the presence of S-9. Upon addition of the test article to the cultures, precipitate was observed at the highest two concentrations tested in the presence of S-9 (100 and 120 µg/mL). Following the 3 hour treatment incubation period, no precipitation was observed in the absence or presence of S-9. At the end of the treatment incubation period, the highest two concentrations in the absence of S-9 (70 and 80 µg/mL) were not plated for survival due to extreme toxicity and concentrations of 85, 95, 100 and 120 µg/mL in the presence of S-9 were not plated for survival due to heterogeneity (85 µg/mL) or extreme toxicity (95, 100 and 120 µg/mL), measured by cell counts. Seven days after treatment, the highest four remaining concentrations in the absence of S-9 (55 to 65 µg/mL) and the highest three remaining concentrations in the presence of S-9 (75, 80 and 90 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 50 µg/mL in the absence of S-9 and 70 µg/mL in the presence of S-9, which gave 9% and 14% RS, respectively (see following table). In the absence of S-9, steep concentration-related toxicity was observed between two closely-spaced concentrations (45 and 50 µg/mL), which gave 27% and 9% RS, respectively, therefore both concentrations were analysed.
Vehicle / solvent:
The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).
Negative solvent / vehicle controls:
yes
Remarks:
DMSO diluted 100-fold
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Remarks:
100-fold dilution
Details on test system and experimental conditions:
Cytotoxicity Range-Finder Experiment
Treatment of cell cultures for the cytotoxicity Range-Finder Experiment was as described below for the Mutation Experiment. However, single cultures only were
used and positive controls were not included. The final treatment culture volume was 20 mL.
Following 3 hour treatment, cells were centrifuged (200 g) for 5 minutes, washed with tissue culture medium, centrifuged again (200 g) for 5 minutes and resuspended in 20 mL RPMI 10.
Cell concentrations were adjusted to 8 cells/mL and, for each concentration, 0.2 mL was plated into each well of a 96-well microtitre plate for determination of relative survival. The plates were placed in a humidified incubator, set to 37ºC and gassed with 5% v/v CO2 in air, for 8 days. Wells containing viable clones were identified by eye using background illumination and counted.

Mutation Experiment
Treatment of Cell Cultures
At least 107 cells in a volume of 17.8 mL of RPMI 5 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 0.2 mL vehicle, test article or positive control solution was added. S-9 mix or 150 mM KCl was added as described. Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL.
After 3 hours in an incubator set to 37°C with gentle agitation, cultures were centrifuged (200 g) for 5 minutes, washed with the appropriate tissue culture medium, centrifuged again (200 g) for 5 minutes and resuspended in 20 mL RPMI 10 medium.
Cell densities were determined using a Coulter counter and, where sufficient cells survived, the concentrations adjusted to 2 x 105 cells/mL. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival as described.
Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit may be responsible for an increase in mutant frequencies (Brusick, 1986; Scott et al., 1991). Osmolality and pH measurements on post-treatment media were taken in the cytotoxicity Range-Finder Experiment.

Plating for Survival
Following adjustment of the cultures to 2 x 105 cells/mL after treatment, samples from these were diluted to 8 cells/mL. Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells, averaging 1.6 cells/well). The plates were placed in a humidified incubator, set to 37ºC and gassed with 5% v/v CO2 in air, until scoreable (7 days). Wells containing viable clones were identified by eye using background illumination and counted.

Expression Period
Cultures were maintained in flasks for a period of 7 days during which the hprtmutation would be expressed. Sub-culturing was performed as required with the aim of retaining an appropriate concentration of cells/flask.

Plating for Viability
At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 105 cells/mL in readiness for plating for 6TG resistance. Samples from these were diluted to 8 cells/mL.
Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging
1.6 cells/well). The plates were placed in a humidified incubator, set to 37ºC and gassed with 5% v/v CO2 in air, until scoreable (8 days). Wells containing viable clones were identified by eye using background illumination and counted.

Plating for 6TG Resistance
At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 105 cells/mL. 6TG (1.5 mg/mL) was diluted 100-fold into these suspensions to give a final concentration of 15 μg/mL. Using a multichannel pipette, 0.2 mL of each suspension was placed into each well of 4 x 96-well microtitre plates (384 wells at 2 x 104 cells/well). Plates were placed in a humidified incubator, set to 37ºC and gassed with 5% v/v CO2 in air, until scoreable (12 days). Wells containing viable clones were identified by eye using background illumination and counted.
Rationale for test conditions:
The test conditions follow the requirements of OECD Guideline 476.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic in this assay if:
1. The Mutation Frequency (MF) at one or more concentrations was significantly greater than that of the vehicle control (p≤0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05)
3. If both of the above criteria were fulfilled, the results should exceed the upper limit of the last 20 studies in the historical vehicle control database (mean
MF +/- 2 standard deviations).
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
It is concluded that tested substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.
Executive summary:

Tested substance was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse

lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by a β-Naphthoflavone/Phenobarbitalinduced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3 hour treatment incubation period was used for each experiment. In the cytotoxicity Range-Finder Experiment, eight concentrations were tested in the absence and presence of S-9, ranging from 2.344 to 300 μg/mL (limited by solubility of the formulated test article in culture medium). The highest concentration to give >10% relative survival (RS) was 37.5 μg/mL, which gave 92% and 89% RS in the absence and presence of S-9, respectively.

In the Mutation Experiment twelve concentrations, ranging from 10 to 80 μg/mL, were tested in the absence of S-9 and thirteen concentrations, ranging from 20 to 120 μg/mL, were tested in the presence of S-9. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 50 μg/mL in the absence of S-9 and 70 μg/mL in the presence of S-9, which gave 9% and 14% RS, respectively. In the absence of S-9, steep concentration-related toxicity was observed between two closely-spaced concentrations (45 and 50 μg/mL), which gave 27% and 9% RS, respectively, therefore both concentrations were analysed.

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures were considered acceptable for addition to the laboratory historical vehicle control database and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore, the study was accepted as valid.

When tested up to toxic concentrations, no statistically significant increases in MF were observed following treatment with Rosyfolia at any concentration tested in the absence and presence of S-9 and there were no statistically significant linear trends.

A MF value of 7.08 mutants/106 viable cells, which marginally exceeded the historical vehicle control range of 1.39 to 6.80 mutants/106 viable cells, was observed at a concentration of 40 μg/mL in the absence of S-9. This was considered acceptable as the MF value was below the vehicle control MF in this experiment (7.95 mutants/

106 viable cells), which was acceptable for addition to the laboratory historical vehicle control database no statistically significant increase in MF was observed at this

concentration and there was no statistically significant linear trend over the concentration range analysed.

Although the highest concentration analysed in the absence of S-9 (50 μg/mL) gave 9% RS, the MF at this concentration was not statistically significant, compared to the

concurrent vehicle control and there was a clear negative result, taking all concentrations analysed into account. This did not prejudice the interpretation of the data or the outcome of the study.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification