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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES test:

The study was performed to investigate the potential of Scentaurus clean (GR-86 -6599) to induce gene mutations according to OECD guideline No 471.

In conclusion, based on the results of this study it is concluded that GR-86 -6599 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Micronucleus test:

The study was performed to investigate the ability of Scentaurus clean (GR-86 -6599) to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity and aneugenicity of GR-86 -6599 was tested in two independent experiments according to OECD guideline No 487.

In conclusion, GR-86 -6599 is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Mouse Lymphoma test:

The test substance, Scentaurus Clean, was evaluated for its ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cellsin the presence and absence of an exogenous metabolic activation system according to OECD guideline No. 490.

The results indicate Scentaurus Clean was negative for the ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 to 18 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
conducted under GLP conditions
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
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:
Rat liver microsomal enzymes (S9 homogenate) was obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that had been injected intraperitoneal with Aroclor 1254 (500 mg/kg body weight).
Each S9 batch is characterised with the mutagens Benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.

S9-mix was prepared immediately before use and kept on ice. S9-mix contained per 10 ml:
30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 ml or 5.0 ml Milli-Q water (first or second experiment respectively) (Millipore Corp., Bedford, MA.,USA); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution (Merck); 1 ml 0.33 M KCl solution (Merck). The above solution was filter (0.22 μm)-sterilized. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 ml of S9-mix components 1.0 ml S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.
Test concentrations with justification for top dose:
First experiment
GR-86-6599 was tested in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA with concentrations of 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix.
Second experiment
To obtain more information about the possible mutagenicity of the test item, a second
mutation experiment was performed in the absence and presence of 10% (v/v) S9-mix. Based
on the results of the first mutation experiment, GR-86-6599 was tested up to concentrations
of 5000 μg/plate.
Vehicle / solvent:
dimethyl sulfoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191
Details on test system and experimental conditions:
Test system Salmonella typhimurium bacteria and Escherichia coli bacteria

Source Trinova Biochem GmbH, Germany (Master culture from Dr.
Bruce N. Ames) (TA1535: 2006, TA1537: 2016, TA98: 2015,
TA100: 2015) and (Master culture from The National
Collections of Industrial and Marine Bacteria, Aberdeen, UK)
(WP2uvrA, 2008)
The Salmonella typhimurium strains were regularly checked to confirm their
histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100),
UV-sensitivity and the number of spontaneous revertants.
The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an
excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a
wide variety of mutagens has been enhanced by permeabilization of the strain using
Tris-EDTA treatment (Ref.1). The strain was regularly checked to confirm the
tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.
Stock cultures of the five strains were stored in liquid nitrogen (-196°C).
Rationale for test conditions:
Rationale Recommended test system in international guidelines (e.g.
OECD, EC and MITI).
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.
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 specified
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.
The negative and strain-specific positive control values were within the laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that GR-86-6599 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Evaluation of the mutagenic activity of GR-86-6599 in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay.

GR-86-6599 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of

Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by induced by Aroclor 1254).

The study procedures described in this report were based on the most recent OECD, EC and MITI guidelines.

Batch 43 of GR-86-6599 was a yellow liquid. The test item was dissolved in dimethyl sulfoxide.

In the first mutation assay, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. The test item precipitated on the plates at dose levels of 1600 and 5000 μg/plate. The bacterial background lawn was not reduced at any of

the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation assay, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix. The test item precipitated on the plates at dose levels of 1600 and 5000 μg/plate. The bacterial background lawn was not reduced at any

of the concentrations tested and no decrease in the number of revertants was observed.

GR-86-6599 did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and

presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

In this study, the negative and 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.

Based on the results of this study it is concluded that GR-86-6599 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 December 2016 to 01 March 2017
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:
2014
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: stimulated human lymphocytes
Cytokinesis block (if used):
Cytochalasine B
Metabolic activation:
with and without
Metabolic activation system:
Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

Preparation of S9-mix:
S9-mix was prepared immediately before use and kept on ice. S9-mix components contained per ml physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox); 4 μmol HEPES (Life Technologies). The above solution was filter (0.22 m)-sterilized. To 0.5 ml S9-mix components 0.5 ml S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
Metabolic activation was achieved by adding 0.2 ml S9-mix to 5.3 ml of a lymphocyte culture (containing 4.8 ml culture medium, 0.4 ml blood and 0.1 ml (9 mg/ml)
phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).
Test concentrations with justification for top dose:
Dose range finding test / First cytogenetic assay:
At a concentration of 185 μg/ml GR-86-6599 precipitated in the culture medium. At the 3 h exposure time, blood cultures were treated in duplicate with 46, 93 and 185 μg test item/ml culture medium with and without S9-mix (first cytogenetic assay).
At the 24 hour exposure time single blood cultures were treated with 12, 23, 46, 93, 185 and 370 μg GR-86-6599/ml culture medium without S9-mix (dose range finding test).

Both in the absence and presence of S9-mix no appropriate dose levels could be selected for scoring of micronuclei since the highest tested concentration of 185 μg/ml was too toxic for scoring.
The experiment was repeated in cytogenetic assay 1A.
Without S9-mix : 5, 20, 40, 60, 80, 100 and 120 μg/ml culture medium
(3 hours exposure time, 27 hours harvest time).
With S9-mix : 20, 40, 80, 100, 120, 140, 160 and 180 μg/ml culture medium
(3 hours exposure time, 27 hours harvest time).
Both in the absence and presence of S9-mix no appropriate dose levels could be selected for scoring of micronuclei since at the concentrations of 40 and 80 μg/ml not enough cytotoxicity was observed (45% and 19%), whereas the next higher concentrations of 60 and 100 μg/ml were too toxic for scoring (69% and 79%).
The experiment was repeated in cytogenetic assay 1B.
Without S9-mix : 5, 20, 30, 40, 45, 50, 55, 60, 70, 80 and 100 μg/ml culture medium
With S9-mix : 20, 40, 80, 85, 90, 95 and 100 μg/ml culture medium
(3 hours exposure time, 27 hours harvest time for with and without S9 mix).

Second cytogenetic assay:
To obtain more information about the possible clastogenicity and aneugenicity of GR-86-
6599, a second cytogenetic assay was performed in which human lymphocytes were exposed
for 24 hours in the absence of S9-mix. The following dose levels were selected for the second
cytogenetic assay:
Without S9-mix : 10, 30, 40, 50, 60, 70 and 80 μg/ml culture medium
(24 hours exposure time, 24 hours harvest time).
Vehicle / solvent:
The vehicle for the test item was dimethyl sulfoxide.

Solvent for positive controls was Hanks’ Balanced Salt Solution (HBSS) (Life Technologies, Bleiswijk, The Netherlands), without calcium and magnesium.
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:
1) Dose range finding test / First cytogenetic assay
In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was obtained in a dose range finding test. GR-86-6599 was tested in the absence and presence of S9-mix.
Lymphocytes (0.4 ml blood of a healthy donor was added to 5 ml or 4.8 ml culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/ml)
Phytohaemagglutinin) were cultured for 46 ± 2 hours and thereafter exposed to selected doses of GR-86-6599 for 3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. Cytochalasine B (Sigma) was added to the cells simultaneously with the test item at the 24 hours exposure time. A vehicle control was included at each exposure time.
The highest tested concentration was determined by the solubility of GR-86-6599 in the culture medium.
The test item precipitated at concentrations of 185 μg/ml and upwards. The lymphocytes were cultured in duplicate at the 3 h exposure time and appropriate positive controls were included.
After 3 hours exposure to GR-86-6599 in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 ml culture medium with Cytochalasine B (5 μg/ml) and incubated for another 24 hours (1.5 times normal cell cycle). The cells that were exposed for 24 hours in the absence of S9-mix were not rinsed after exposure but were fixed immediately.
Cytotoxicity of GR-86-6599 in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index). Since the highest concentration was too toxic for scoring the experiment was repeated. To be able to select appropriate dose levels for scoring of micronuclei of the 3 h exposure time several repeat assays had to be performed.
Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the second cytogenetic assay considering the highest dose level was determined by the solubility.

2) Second cytogenetic assay
To confirm the results of the first cytogenetic assay a second cytogenetic assay was performed with an extended exposure time of the cells in the absence of S9-mix.
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of GR-86-6599 with cytochalasin B (5 μg/ml) for 24 hours in the absence of S9-mix.
Appropriate vehicle and positive controls were included in the second cytogenetic assay.

3) Preparation of slides
To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68 (Applichem, Darmstadt, Germany). After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride (Merck) solution. Immediately after, ethanol (Merck): acetic acid (Merck) fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiven, The Netherlands)/pertex (Histolab,
Gothenburg, Sweden) and mounted with a coverslip in an automated cover slipper (Leica Microsystems B.V., Rijswijk, The Netherlands).

4) Cytotoxicity assessment
A minimum of 500 cells (with a maximum deviation of 5%) per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells). The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI).
%Cytostasis = 100-100{(CBPIt – 1)/(CBPIc –1)}

CBPI = ((No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)/Total number of cells)
t= test item or control treatment culture
c= vehicle control culture

Three analysable concentrations were scored for micronuclei. The number of micronuclei per cell was not recorded. The highest dose level examined for micronuclei were the cultures that produced 55 ± 5% cytotoxicity. The lowest dose level had little or no cytotoxicity (approximately the same as solvent control). Also cultures treated with an intermediate dose level were examined.

5) Cytogenetic assessment/scoring of micronuclei
To prevent bias, all slides were randomly coded before examination of micronuclei and scored. An adhesive label with Charles River Den Bosch study identification number and code was stuck over the marked slide. At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were examined by light microscopy for micronuclei. In addition, at least 1000 (with a maximum deviation of 5%) mononucleated cells per culture were scored
for micronuclei separately. Since the lowest concentration of MMC-C resulted in a positive response the highest concentration was not examined for the presence of micronuclei. Due to cytotoxicity the number of examined bi- or mononucleated cells in the positive control groups might be <1000. However, when an expected statistical significant increase is observed, this has no effect on the study integrity.

The following criteria for scoring of binucleated cells were used (1 - 2, 6):
- Main nuclei that were separate and of approximately equal size.
- Main nuclei that touch and even overlap as long as nuclear boundaries are able to be
distinguished.
- Main nuclei that were linked by nucleoplasmic bridges.
The following cells were not scored:
- Trinucleated, quadranucleated, or multinucleated cells.
- Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone
already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996:
- The diameter of micronuclei should be less than one-third of the main nucleus.
- Micronuclei should be separate from or marginally overlap with the main nucleus as long
as there is clear identification of the nuclear boundary.
- Micronuclei should have similar staining as the main nucleus.
Rationale for test conditions:
Test conditions were based on OECD guideline.
Evaluation criteria:
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, onesided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.
Statistics:
REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA): temperature and humidity.
Key result
Species / strain:
lymphocytes: stimulated human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The ability of GR-86-6599 to induce micronuclei in human peripheral lymphocytes was investigated in two independent experiments. The highest concentration analysed was selected based on toxicity, cytokinesis-block proliferation index of 55 ± 5%.
The number of mono- and binucleated cells with micronuclei found in the solvent control was within the 95% control limits of the distribution of the historical negative control database.
The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. Although in the absence of S9-mix the response of Colchicine was above the upper control limits, these limits are 95% control limits and a slightly higher response is within the expected response ranges.
It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
GR-86-6599 did not induce a statistically significant and biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.
Conclusions:
GR-86-6599 did not induce a statistically significant and biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.
Finally, it is concluded that this test is valid and that GR-86-6599 is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.
Executive summary:

An in vitro micronucleus assay with GR-86-6599 in cultured peripheral human lymphocytes.

This report describes the effect of GR-86-6599 on the number of micronuclei formed in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible

clastogenicity and aneugenicity of GR-86-6599 was tested in two independent experiments.

The study procedures described in this report are in compliance with the most recent OECD guideline.

Batch 43 of GR-86-6599 was a yellow liquid with a purity of 96.5%. GR-86-6599 was dissolved in dimethyl sulfoxide.

In the first cytogenetic assay, GR-86-6599 was tested up to 50 and 85 μg/ml for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction, respectively. Appropriate toxicity was reached at these dose levels.

In the second cytogenetic assay, GR-86-6599 was tested up to 70 μg/ml for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C and cyclophosphamide both produced

a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. It was therefore concluded that the test

conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

GR-86-6599 did not induce a statistically significant and biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

Finally, it is concluded that this test is valid and that GR-86-6599 is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 August to 15 September 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
conducted uder 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
Target gene:
thymidine kinase locus of L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y/TK+/- mouse lymphoma cells are heterozygous at the normally diploid thymidine kinase (TK) locus. L5178Y/TK+/- cells, clone 3.7.2C were obtained from the American Type Culture Collection (repository number CRL-9518), Manassas, VA. Each batch of frozen cells was tested and found to be free of mycoplasma contamination. This test system has been demonstrated to be sensitive to the mutagenic activity of a variety of chemicals.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot No. 4141, Expiration Date: 05 Sep 2021) was purchased commercially from Moltox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at 60°C or colder until used. Each lot of S9 was assayed for sterility and its ability to metabolize at least two pro-mutagens to forms mutagenic to Salmonella typhimurium TA100.
The S9 mix was prepared on the day of use. The final concentrations of the components in the test system were as indicated below.
DL-isocitric acid: 17.4 mM in cultures
NADP (sodium salt): 3.0 mM in cultures
S9 homogenate: 10 microliters/mL in cultures
Test concentrations with justification for top dose:
In the preliminary toxicity assay, the concentrations tested were 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL. The maximum concentration evaluated the limit dose for this assay. Visible precipitate was observed at concentrations ≥250 µg/mL at the beginning of treatment and at concentrations ≥31.3 µg/mL(4-hour treatment with S9), ≥62.5 µg/mL(4-hour treatment without S9) and ≥15.6 µg/mL (24-hour treatment without S9) by the end of treatment (observed as possible cell debris). Relative suspension growth (RSG) was 43, 34 and 30% at concentrations of 15.6 µg/mL (4-hour treatment with S9), 15.6 µg/mL (4-hour treatment without S9) and 7.8 µg/mL (24-hour treatment without S9), respectively. RSG was or approximated 0% at higher concentration using all treatment conditions. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 6.56, 8.21, 10.3, 12.8, 16.0, 20.0, 25.0 and 31.3 µg/mL (4-hour treatment with S9), 6.56, 8.21, 10.3, 12.8, 16.0, 20.0, 25.0 and 31.3 µg/mL (4-hour treatment without S9) and 6.56, 8.21, 10.3, 12.8, 14.4, 16.0, 18.0 and 20.0 µg/mL (24-hour treatment without S9).
Vehicle / solvent:
DMSO, CAS 67-68-5, Supplier: Sigma-Aldrich, Lot number SHBM0878, Purity: 99.99%, Expiration date: Sep 2023

DMSO was the vehicle of choice based on information provided by the Sponsor and compatibility with the target cells. The test substance formed a clear solution in DMSO at a concentration of approximately 500 mg/mL in the solubility test conducted at BioReliance.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
methylmethanesulfonate
Details on test system and experimental conditions:
1) Preliminary Toxicity test for selection of dose levels
L5178Y/TK+/- cells was exposed to the vehicle alone in duplicate cultures and nine concentrations of test substance using single cultures. The maximum concentration evaluated the limit dose (2000 µg/mL) for this assay. The pH of the treatment medium was measured, and no pH adjustment was necessary to maintain neutral pH. Osmolality of the vehicle control, the highest concentration, the lowest precipitating concentration and the highest soluble concentration also was measured at the beginning of treatment. Precipitation was determined with the unaided eye at the beginning and end of treatment. Dose levels for the definitive assay were based upon post-treatment cytotoxicity (growth inhibition relative to the vehicle control).

2) Mouse Lymphoma assay
Eight concentrations were tested using duplicate cultures at appropriate dose intervals based on the toxicity profile of the test substance. The pH of the treatment medium was measured, and no pH adjustment was necessary to maintain neutral pH. Precipitation was determined with the unaided eye at the beginning and end of treatment.

3) Treatment of target cells
The preparation and addition of the test substance dose formulations was carried out under filtered lighting during the exposure period. Treatment was carried out by combining100 µL of test substance dose formulation, vehicle or positive control dose formulation and F0P medium or S9 mix (as appropriate) with 6 x 106 L5178Y/TK+/- cells in a total volume of 10 mL. All pH adjustments were performed prior to adding S9 or target cells to the treatment medium. Each S9 activated 10-mL culture contained 4 mL S9 mix (final S9 concentration of 1.0%). Cultures were capped tightly and incubated with mechanical mixing at 37 ± 1°C for 4 or 24 hours.
For the preliminary toxicity assay only, after a 4-hour treatment in the presence and absence of S9, cells were washed with culture medium and cultured in suspension for two days post treatment, with cell concentration adjustment on the first day. After a 24 hour treatment in the absence of S9, cells were washed with culture medium and immediately readjusted to 3 x 10^5 cells/mL. Cells were then cultured in suspension for an additional two days post-treatment with cell concentration adjustment on the first day.
For the definitive assay only, at the end of the exposure period, the cells were washed with culture medium and collected by centrifugation. The cells were resuspended in 20 mL F10P on Day 1 and in 10 mL F10P on Day 2, and incubated at 37 ± 1°C for two days following treatment. Cell population adjustments to 3 x 10^5 cells/mL were made as follows:
• 4 hour treatment – 1 and 2 days after treatment.
• 24 hour treatment – immediately after test substance removal, and 2 and 3 days after treatment.

4) Selection of mutant phenotype
Cells from selected dose levels were cultured in triplicate with 2-4 microg TFT/mL at a density of 1 x 10^6 cells/100 mm plate in cloning medium containing 0.22 to 0.24% agar. For estimation of cloning efficiency at the time of selection of those same cultures, 200 cells/100 mm plate were cultured in triplicate in cloning medium without TFT (viable cell (VC) plate). Cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 12 to 13 days.
The total number of colonies per culture was determined for the VC plates and the total relative growth calculated. The total number of colonies per TFT plate was then determined for those cultures with 10% total growth (including at least one concentration between 10 and 20% total growth, if possible). Colonies were counted and the diameter of the TFT colonies from the positive control and vehicle control cultures were determined over a range from 0.2 to 1.1 mm.

5) Extended treatmet and/or confirmatory assay
Verification of a clear positive response was not required. For negative results without activation, an extended treatment assay was performed in which cultures were continuously exposed to the test substance for 24 hours without S9 activation. The extended treatment assay was performed concurrently with the initial assay. For negative results with S9 activation, a confirmatory assay was not required unless the test substance was known to have specific requirements of metabolism.
Rationale for test conditions:
Test conditions were based on OECD guideline.
Evaluation criteria:
• A result was considered positive if a concentration-related increase in mutant frequency was observed in the treated cultures and one or more treatment conditions with 10% or greater total growth exhibited induced mutant frequencies of 90 mutants/106 clonable cells (based on the average mutant frequency of duplicate cultures). If the average vehicle control mutant frequency was >90 mutants/106 clonable cells, a doubling of mutant frequency over the vehicle would also be required (Mitchell et al., 1997).
• A result was considered negative if the treated cultures exhibited induced mutant frequencies of less than 90 mutants/106 clonable cells (based on the average mutant frequency of duplicate cultures) and there was no concentration-related increase in mutant frequency.

There are some situations in which a chemical would be considered negative when there was no culture showing between 10 to 20% survival (Office of Food Additive Safety, 2001).
• There was no evidence of mutagenicity (e.g. no dose response or increase in induced mutant frequencies between 45 and 89 mutants/106) in a series of data points within 100 to 20% survival and there was at least one negative data point between 20 and 25% survival.
• There was no evidence of mutagenicity (e.g. no dose response or increase in induced mutant frequencies between 45 and 89 mutants/106) in a series of data points between 100 to 25% survival and there was also a negative data point between 10 and 1% survival. In this case, it would be acceptable to count the TFT colonies of cultures exhibiting <10% total growth.
Statistics:
The primary computer or electronic systems used for the collection of data or analysis included, but were not limited to, the following:
- LIMS labware system for Test substance tracking
- Excel (Microsoft corporation) for Calculations
- Kaye Lab Watch Monitoring system (Kaye GE) for Environmental monitoring
- BRIQS for Deviation and audit reporting
- ProtoCOL Colony Counter for data collection
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
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Cultures treated at concentrations of 8.21, 10.3, 12.8, 16.0, 20.0 and 25.0 µg/mL (4-hour treatment with S9), 6.56, 8.21, 10.3, 12.8 and 16.0 µg/mL (4-hour treatment without S9) and 8.21, 10.3, 12.8, 14.4, 16.0, 18.0 and 20.0 µg/mL (24-hour treatment without S9) exhibited 13 to 94%, 9 to 96% and 7 to 95% RSG, respectively, and were cloned. Cultures treated at other concentrations were not selected for cloning due to sufficient number of concentrations were available for evaluation. Relative total growth of the cloned cultures ranged from 11 to 94% (4 hour treatment with S9), 7 to 94% (4-hour treatment without S9) and 9 to 139% (24 hour treatment without S9). At least one replicate had 10 to 20% or slightly lower RTG compared to the vehicle control in the highest concentration tested in all three conditions. No increases in induced mutant frequency ≥90 mutants/106 clonable cells were observed under any treatment condition. Although there was a positive dose dependent trend in the statistical analysis for 4-hour treatment without S9 condition, no increases in induced mutant frequency ≥90 mutants/106 clonable cells were observed. Therefore, this condition is not considered as biologically relevant. There was no positive statistical trend based in 4 hour treatment with S9 and 24 hour treatment without S9 conditions.
Trifluorothymidine-resistant colonies for the positive and vehicle control cultures, were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The colony sizing for the MMS and DMBA positive controls yielded the expected increase in small colonies (verifying the adequacy of the methods used to detect small colony mutants) and large colonies.
All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met.
Conclusions:
Under the conditions of the assay described in this report, Scentaurus Clean was concluded to be negative for the induction of forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro L5178Y/TK+/- mouse lymphoma assay.
Executive summary:

The test substance,Scentaurus Clean,was evaluated for itsability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cellsin the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the preliminary toxicity assay, the concentrations tested were 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL. The maximum concentration evaluated the limit dose for this assay. Visible precipitate was observed at concentrations ≥250 µg/mL at the beginning of treatment and at concentrations ≥31.3 µg/mL(4-hour treatment with S9), ≥62.5 µg/mL(4-hour treatment without S9) and ≥15.6 µg/mL (24-hour treatment without S9) by the end of treatment (observed as possible cell debris). Relative suspension growth (RSG) was 43, 34 and 30% at concentrations of 15.6 µg/mL (4-hour treatment with S9), 15.6 µg/mL (4-hour treatment without S9) and 7.8 µg/mL (24-hour treatment without S9), respectively. RSG was or approximated 0% at higher concentration using all treatment conditions. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 6.56, 8.21, 10.3, 12.8, 16.0, 20.0, 25.0 and 31.3 µg/mL (4-hour treatment with S9), 6.56, 8.21, 10.3, 12.8, 16.0, 20.0, 25.0 and 31.3 µg/mL (4-hour treatment without S9) and 6.56, 8.21, 10.3, 12.8, 14.4, 16.0, 18.0 and 20.0 µg/mL (24-hour treatment without S9).

In the definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. Cultures treated at concentrations of 8.21, 10.3, 12.8, 16.0, 20.0 and 25.0 µg/mL (4-hour treatment with S9), 6.56, 8.21, 10.3, 12.8 and 16.0 µg/mL (4-hour treatment without S9) and 8.21, 10.3, 12.8, 14.4, 16.0, 18.0 and 20.0 µg/mL (24-hour treatment without S9) exhibited 13 to 94%, 9 to 96% and 7 to 95% RSG, respectively, and were cloned. Cultures treated at other concentrations were not selected for cloning due to sufficient number of concentrations were available for evaluation. Relative total growth of the cloned cultures ranged from 11 to 94% (4‑hour treatment with S9), 7 to 94% (4-hour treatment without S9) and 9 to 139% (24‑hour treatment without S9). At least one replicate had 10 to 20% or slightly lower RTG compared to the vehicle control in the highest concentration tested in all three conditions. No increases in induced mutant frequency ≥90 mutants/106clonable cells were observed under any treatment condition. Although there was a positive dose dependent trend in the statistical analysis for 4-hour treatment without S9 condition, no increases in induced mutant frequency ≥90 mutants/106clonable cells were observed. Therefore, this condition is not considered as biologically relevant. There was no positive statistical trend based in 4‑hour treatment with S9 and 24‑hour treatment without S9 conditions.

These results indicateScentaurus Cleanwas negative for the ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system.

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

Additional information

Justification for classification or non-classification

Based on the study results which are negative, Scentaurus clean should not be classified as mutagenic nor clastogenic nor aneugenic according to the EU CLP regulation (No 1272/2008 and its adaption 286/2011).