4-Decenal, 9-hydroxy-5,9-dimethyl-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Bacterial Mutation Test (OECD 471, 2013, GLP, K, rel.1): not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 & TA100, and E.coli strain WP2uvrA

- Micronucleus test (OECD 487, 2018, GLP, K, rel. 1): non-clastogenic and non-aneugenic to human lymphocytes in vitro in either the absence or presence of metabolic activation.

- Mammalian Cell Gene Mutation Assay (OECD 476, HPRT): not mutagenic 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

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2020-10-05 to 2021-03-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 476 and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2016

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Date received: 02 September 2020

Target gene:

HPRT locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Cells: L5178Y tk+/- (3.7.2C) mouse lymphoma cells were obtained from Dr Donald Clive, Burroughs Wellcome Co. Cells. Cells are stored as frozen stocks in liquid nitrogen. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and placed in an incubator set to 37ºC. When the cells were growing well, subcultures were established in an appropriate number of flasks.
- Type and identity of media:
RPMI 1640 media, containing L-glutamine and HEPES were prepared. Resulting mediums are referred to as RPMI A (0% v/v), RPMI 10 (10% v/v) and RPMI 20 (20% v/v). RPMI 5 consisted of RPMI 10 diluted with RPMI A [prepared as RPMI 10 but with no serum added] to give a final concentration of 5% serum.
RPMI A medium supplemented with 10% horse serum (heat inactivated) referred to as RPMI 10, is used for general cell culture, e.g. when growing cells up from frozen stocks.
Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free.
All cell cultures are maintained in an incubator set to 37ºC.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction obtained from Molecular Toxicology Incorporated, USA, was prepared from male Sprague-Dawley rats dosed 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). The final content per mL of the 10% S9 mix is: sodium phosphate buffer pH 7.4 (100 µmol), glucose-6-phosphate (5 µmol), NADP (4 µmol), MgCl2 (8 µmol), KCl (33 µmol), S9 mix (100 µL) and water to volume.

Test concentrations with justification for top dose:

- Range-Finder (3 hours; -S9 and +S9): 1.548 to 198.2 mg/mL for a final concentration range of 15.48 to 1982 µg/mL.
- Mutation experiment (3 hours; -S9): 2.500 to 35.00 mg/mL for a final concentration range of 25.00 to 350.0 µg/mL.
- Mutation experiment (3 hours; +S9): 5.00 to 50.00 mg/mL for a final concentration range of 50.00 to 500.0 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO. Test article stock solutions were prepared by formulating Mahonial under subdued lighting in DMSO, with the aid of vortex mixing, to give the maximum required concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 2.5 hours of initial formulation.

- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Mahonial was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations up to at least 200.6 mg/mL. The solubility limit in culture medium was below 501.5 µg/mL, as indicated by the appearance of precipitate at this concentration approximately 3 hours after test article addition, with warming at 37°C

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Negative (vehicle) controls comprised treatments with the vehicle DMSO diluted 100-fold in the treatment medium.

True negative controls:

no

Positive controls:

yes

Remarks:

100-fold dilution

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

in the absence of S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Negative (vehicle) controls comprised treatments with the vehicle DMSO diluted 100-fold in the treatment medium.

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

in the presence of S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

CYTOTOXICITY RANGE-FINDER EXPERIMENT
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:
For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and placed in an incubator set to 37ºC. When the cells were growing well, subcultures were established in an appropriate number of flasks.
At least 10^7 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.
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 10^5 cells/mL. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival.
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 10^5 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 hprt mutation 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 10^5 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 (10 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 10^5 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 (13 days). Wells containing viable clones were identified by eye using background illumination and counted.

NUMBER OF REPLICATIONS: 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.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Rationale for test conditions:

A maximum concentration of 1982 µg/mL was selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to 10 mM and also a precipitating treatment concentration. Concentrations selected for the Mutation Experiment were based on the results of this cytotoxicity Range-Finder Experiment.
The data presented for Mutation Experiment were derived from a repeat experiment. In the initial Mutation Experiment, no colonies were observed on the vehicle control mutation plates, therefore scoring was aborted. The experiment was invalidated and data from this experiment are not further reported.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic in this assay if:
1. The 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.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No marked changes in pH were observed in the Range-Finder at the highest concentration tested (1982 µg/mL), compared to the concurrent vehicle controls.
- Effects of osmolality: No marked changes in osmolality were observed in the Range-Finder at the highest concentration tested (1982 µg/mL), compared to the concurrent vehicle controls.
Preliminary solubility data indicated that Mahonial was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations up to at least 200.6 mg/mL. The solubility limit in culture medium was below 501.5 µg/mL, as indicated by the appearance of precipitate at this concentration approximately 3 hours after test article addition, with warming at 37°C. A maximum concentration of 1982 µg/mL was therefore selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to 10 mM and also a precipitating treatment concentration.
- Evaporation from medium: not applicable
- Water solubility: not soluble in water
- Precipitation: yes
- Other confounding effects: none

PRELIMINARY TOXICITY TEST (see Table 7.6.1/1 below)
In the cytotoxicity Range-Finder Experiment, eight concentrations were tested in the absence and presence of S-9, ranging from 15.48 to 1982 µg/mL (equivalent to 10 mM at the highest concentration tested and also a precipitating concentration).
Upon addition of the test article to the cultures, precipitate was observed at the highest three concentrations tested in the absence and presence of S-9 (495.5 to 1982 µg/mL).
Following the 3 hour treatment incubation period, precipitate was observed at the highest concentration in the absence and presence of S-9 (1982 µg/mL). The highest concentrations to give >10% RS were 123.9 µg/mL in the absence of S-9 and 247.8 µg/mL in the presence of S-9, which gave 49% and 71% RS, respectively.

MAIN TEST (see Table 7.6.1/2 below)
The cell concentration was confirmed to be 1.2 x 10^6 cells/mL (i.e. 24 x 10^6 cells treated per cIn the Mutation Experiment twelve concentrations, ranging from 25 to 350 µg/mL in the absence of S-9 and from 50 to 500 µg/mL in the presence of S-9, were tested.
Upon addition of the test article to the cultures, precipitate was observed at the highest two concentrations tested in the presence of S-9 (450 and 500 µg/mL).
Following the 3 hour treatment incubation period, no precipitation was observed in the absence or presence of S-9. Seven days after treatment, the highest concentration in the absence of S-9 (350 µg/mL) and the highest three concentrations in the presence of S-9 (420 to 500 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition, the lowest concentration in the absence of S-9 (25 µg/mL) was not selected as there were sufficient concentrations to define the toxicity profile. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 280 µg/mL in the absence of S-9 and 390 µg/mL in the presence of S-9, which gave 15% and 14% RS, respectively.

MUTATION RESULTS
The MF could not be calculated for B[a]P at 4 µg/mL in the presence of S-9 because there were no colonies detected on the viability plates (due to probable technical error). However, the data were sufficiently robust for B[a]P at 6 µg/mL, therefore the acceptance criteria were met and the study was accepted as valid.

When tested up to toxic concentrations, no statistically significant increases in MF were observed following treatment with Mahonial at any concentration tested in the absence and presence of S-9. There was a statistically significant linear trend (p≤0.01) in the absence of S-9 but the MF values in all test article-treated cultures were not significantly higher than the concurrent vehicle control MF and were within the historical vehicle control range, therefore this observation was considered not biologically relevant. The result was considered negative under both treatment conditions.

Table 7.6.1/1: RS values - Range-Finder experiment - 3-hour treatments in the absence and presence of S9

cf. attached full study report

Concentration (µg/mL)	Percent relative survival (% RS)
	- S9 mix	+ S9 mix
0 15.48 30.97 61.94 123.9 247.8 495.5 P 991 P 1982 P, PP	100 81 76 55 49 9 0 0 0	100 81 102 78 84 71 0 0 0

P: precipitation noted at time of treatment

PP: precipitation noted at end of treatment incubation period

 

Table 7.6.1/2: Summary of Mutation Data - 3 Hour Treatments in the Absence and Presence of S-9

cf. attached full study report

 

3-hour Treatment -S9 mix			3-hour Treatment +S9 mix
Concentration µg/mL	% RS	MF*	Concentration µg/mL	% RS	MF*
0	100	2.83	0	100	3.35
50	80	1.76 NS	50	87	2.97 NS
100	68	2.35 NS	100	86	1.98 NS
125	52	3.48 NS	150	87	3.60 NS
150	57	4.36 NS	200	73	3.15 NS
175	55	3.12 NS	250	64	5.66 NS
200	42	2.25 NS	300	59	2.46 NS
220	39	5.29 NS	330	61	4.29 NS
240	27	4.88 NS	360	23	3.77 NS
260	25	5.86 NS	390	14	1.70 NS
280	15	5.35 NS
NQO 0.15	52	23.20
NQO 0.20	47	17.93	B[a]P 6	70	15.14

 

Test for Linear trend

- S9 mix		+ S9 mix
Slope Variance b²/Sb	1.06E-08 1.24E-17 8.998**	Slope Variance b²/Sb	-8.91E-10 5.29E-18 0.150

 

*6-TG resistant mutants/10^6 viable cells 7 days after treatment
%RS: Percent relative survival adjusted by post treatment cell counts

*, **, *** Test for linear trend: χ² (one-sided), significant at 5%, 1% and 0.1% level respectively
NS: Not significant

 

Table 7.6.1/3: Historical control ranges

The historical control ranges for the last 20 experiments performed in this laboratory are as follows:

 

Vehicle controls

S9 mix	Mean MF (mutants per 10^6 viable cells)	MF range* (mutants per 10^6 viable cells)
- S9 mix + S9 mix	4.40 4.53	1.09 to 7.70 0.77 to 8.30

*Range = Mean ± 2 x SD.

 

Positive controls

Control concentration	S9 mix	Mean MF (mutants per 10^6 viable cells)	MF range* (mutants per 10^6 viable cells)
NQO 0.15 µg/mL NQO 0.20 µg/mL	- S9 mix - S9 mix	20.55 30.60	5.89 to 35.22 16.37 to 44.82
B[a]P 2 µg/mL B[a]P 3 µg/mL	+ S9 mix + S9 mix	22.13 26.90	2.50 to 41.76 1.88 to 51.92

*Range = Mean ± 2 x SD.

 

The mean mutant frequency values described above differ from those stated in the historical control ranges attached which are based on the entire historical control ranges for this assay (updated after each experiment).

Conclusions:

Under the test conditions, it is concluded that Mahonial 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).

Executive summary:

In an in vitro mammalian cell mutation assay performed according to the OECD test guideline No. 476 and in compliance with GLP, mouse lymphoma L5178Y cells were exposed to the test item.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). Three-hour exposures were used both with and without activation (S9) in all tests.

 

In the cytotoxicity Range-Finder Experiment, eight concentrations were tested in the absence and presence of S-9, ranging from 15.48 to 1982 µg/mL (equivalent to 10 mM at the highest concentration tested and also a precipitating concentration). The highest concentrations to give >10% relative survival (RS) were 123.9 µg/mL in the absence of S-9 and 247.8 µg/mL in the presence of S-9, which gave 49% and 71% RS, respectively.

In the Mutation Experiment twelve concentrations, ranging from 25 to 350 µg/mL in the absence of S-9 and from 50 to 500 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 280 µg/mL in the absence of S-9 and 390 µg/mL in the presence of S-9, which gave 15% and 14% RS, respectively.

 

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 fell within acceptable ranges and clear increases in mutation were induced by one or both concentrations of 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 Mahonial at any concentration tested in the absence and presence of S-9. There was a statistically significant linear trend (p≤0.01) in the absence of S-9 but the MF values in all test article-treated cultures were not significantly higher than the concurrent vehicle control MF and were within the historical vehicle control range, therefore this observation was considered not biologically relevant. The result was considered negative under both treatment conditions.

 

It is concluded that Mahonial 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.

This study is considered as acceptable and satisfies the requirement for the mammalian cell gene mutation endpoint.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 04, 2013 to June 13, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 471 without any deviation.

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

Dated May 30, 2008

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

German GLP Certificate (signed on 11 April 2013)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Storage Conditions: in the refrigerator at +2-8°C, protected from light

Target gene:

Histidine and tryptophan for S. typhimurium and E. coli, respectively.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: in-house preparation by Harlan CRR
- method of preparation of S9 mix: Male Wistar rats were induced by administration of phenobarbital/beta-naphthoflavone. The S9 was prepared and stored according to the Harlan CCR SOP for rat liver S9 preparation and each batch was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test. The protein concentration of the S9 preparation was 44.9 mg/mL (Lot. No150213) in both experiments.
- concentration or volume of S9 mix and S9 in the final culture medium: An appropriate quantity of S9 supernatant is thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10 % v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix: 8 mM MgCl2; 33 mM KCl; 5 mM Glucose-6-phosphate; 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.During the experiment the S9 mix was stored in an ice bath.

Test concentrations with justification for top dose:

Pre-Experiment/Experiment 1 (plate incorporation test): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate both with and without liver microsomal activation.
The pre-experiment is reported as main experiment I, because of the following criteria is met:
Evaluable plates (>0 colonies) at five concentrations or more in all strains used.
Since toxic effects were observed eight concentrations were tested and 5000 µg/plate were chosen as maximal concentration.

Experiment 2 (pre-incubation test): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate both with and without liver microsomal activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: On the day of the experiment, the test item was dissolved in DMSO (purity > 99 %). The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
The test item precipitated in the overlay agar in the test tubes at 5000 µg/plate in experiment I.
No precipitation of the test item was observed in the overlay agar on the incubated agar plates. The
undissolved particles had no influence on the data recording.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD)

Remarks:

Without S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

With S9-mix

Details on test system and experimental conditions:

TEST SYSTEM: The bacteria used in the test were obtained from Trinova Biochem GmbH (35394 Gießen, Germany). The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.

METHOD OF APPLICATION:
- Plate incorporation (first assay): The following materials were mixed in a test tube and poured onto the selective agar plates: 100 µL Test solution at each dose level (solvent or reference mutagen solution (positive control)), 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), 100 µL Bacteria suspension (cf. test system, pre-culture of the strains) and 2000 µL Overlay agar.

- Pre-incubation (second assay): In the pre-incubation assay 100 µL test solution (solvent or reference mutagen solution (positive control)), 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

DURATION
- Preincubation period: 37°C for 60 minutes (with shaking)
- Exposure duration: After solidification plates were incubated at 37°C, in the dark, for at least 48 hours.

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

Rationale for test conditions:

The most widely used assays for detecting gene mutations are those using bacteria.
They are relatively simple and rapid to perform, and give reliable data on the ability of an agent to interact with DNA and produce mutations.

Evaluation criteria:

A test item is considered as a mutagen if :
- a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
- A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
- An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
- A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

A statistical analysis of the data is not mandatory.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The plates incubated with the test item showed reduced background growth in all strains.
Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in nearly all strains. See Tables 7.6.1/01 and 7.6.1/02 below.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix).
There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase
of induced revertant colonies.

Table 7.6.1/01: Reduced background growth during both assays at the following concentrations (µg/plate)

Strain	Experiment I		Experiment II
	Without S9-mix	With S9-mix	Without S9-mix	With S9-mix
TA 1535	2500-5000	2500-5000	2500-5000	1000-5000
TA 1537	2500-5000	2500-5000	1000-5000	1000-5000
TA 98	2500-5000	2500-5000	1000-5000	1000-5000
TA 100	2500-5000	2500-5000	1000-5000	1000-5000
WP2uvrA	2500-5000	2500-5000	5000	2500-5000

/ = no reduced background growth

Table 7.6.1/02: Toxic effects, evident as a reduction in the number of revertants occurred in both assays at the following concentrations (µg/plate)

Strain	Experiment I		Experiment II
	Without S9-mix	With S9-mix	Without S9-mix	With S9-mix
TA 1535	2500-5000	2500-5000	2500-5000	1000-5000
TA 1537	2500-5000	2500-5000	1000-5000	2500-5000
TA 98	2500-5000	2500-5000	2500-5000	1000-5000
TA 100	2500-5000	2500-5000	2500-5000	1000-5000
WP2uvrA	5000	2500-5000	5000	2500-5000

 

Conclusions:

Under the test condition, test substance is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA were exposed to test substance using the plate incorporation test (experiment I) and the pre-incubation test (experiment II) at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The test item was evaluated at the following concentrations in both experiments:
3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate. Negative, vehicle (DMSO) and positive control groups were also included in the mutagenicity tests.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in both experiments was selected as the maximum recommended dose level of 5000 μg/plate or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix. The test item induced a visible reduction in the growth of the bacterial background lawns of all strains in the absence and the presence of S9-mix between 2500 to 5000 µg/plate in the first experiment. Results from the second experiment showed a visible reduction in the growth of the bacterial background lawns in both the absence and presence of S9-mix, initially from 1000 μg/plate for the TA 1537, TA 98, TA 1535 (only with S9-mix) and TA100 strains, from 2500 µg/plate for TA 1535 without S9 mix and WP2 uvrA with S9 mix and at 5000 µg/plate for WP2 uvrA without S9 mix. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no substantial increases in revertant colony numbers among any of the five tester strains following treatment with test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency for higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. 

Under the test condition, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. It can be concluded that the test substance is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA according to the criteria of the Annex VI of the Regulation (EC) No.1272/2008 (CLP) and to the GHS. This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 April 2018 - 29 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2018

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Blood was collected from healthy adult, non-smoking volunteers
- Suitability of cells:
Recommended in the international OECD guideline.
- Age of volunteers and Average Geneteration Time:
Dose-range finding study: age 26, AGT = 15.8 h
Dose-range finding 2 study: age 30, AGT = 14.8 h
First cytogenetic assay: age 35, AGT = 13.7 h
Second cytogenetic assay: age 26, AGT = 14.2 h


- Blood sample collection: Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

MEDIA USED
- Culture medium: Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/ml respectively) and 30 U/mL heparin.
- Lymphocyte cultures: Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin was added.

Cytokinesis block (if used):

Cytochalasine B (5 μg/mL) for 24 hours (1.5 times normal cell cycle)

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).

Test concentrations with justification for top dose:

Dose range finding test (without and with S9): 39, 78, 156, 313, 625 and 1250 µg test item/mL

Experiment 1 (without and with S9): 50, 100, 250, 300, 350, 400 and 450 µg/mL culture medium
Doses selected for scoring (without S9): 100, 300 and 350 µg/mL culture medium
Doses selected for scoring (with S9): 100, 350 and 400 µg/mL culture medium

Experiment 2 (without S9): 20, 40, 50, 60, 70, 80, 90 and 100 µg/mL culture medium
Doses selected for scoring (without S9): 20, 60 and 90 µg/mL culture medium

Based on the results of the dose-range finding test, an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control. Initially, the 3 hours incubation in the presence of S9-mix was prematurely ceased since some of the reagents were not at hand at the time being. The repeat dose range finding was reported as part of the original dose range finding.

Vehicle / solvent:

- Vehicle used: dimethyl sulfoxide (DMSO)
- Justification for choice of vehicle: A solubility test was performed based on visual assessment. The test item formed a clear (colorless) solution in dimethyl sulfoxide (DMSO, SeccoSolv, Merck, Darmstadt, Germany).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Culture period of lymphocytes: 46 ± 2 hours
- Exposure duration: 3 hours (first assay) and 24 hours (second assay)
- Harvest time: 27 hours (first assay) and 24 hours (second assay)

ENVIRONMENTAL CONDITIONS:
- Humidity: set to maintain 80 - 100% (actual range 55 - 90%), containing 5.0 ± 0.5% CO2 in air
- Temperature: set to maintain 37.0 ± 1.0°C (actual range 34.8 - 37.2°C)

NUMBER OF REPLICATIONS:
2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
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. 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, ethano:acetic acid 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/ether 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 and mounted with a coverslip in an automated cover slipper.

NUMBER OF CELLS EVALUATED:
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 and CP 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 was observed, this has no effect on the study integrity.


METHOD OF SCORING: by light microscope

CRITERIA FOR SCORING: 
The following criteria for scoring of binucleated cells were used:
• 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.

DETERMINATION OF CYTOTOXICITY
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). Three analyzable 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.

- Method: cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI): %cytostasis = 100-100 [(CNPIt-1)/(CBPIc-1)]
where t=test item or control treatment culture and c=vehicle control culture
CBPI = [(no. mononucleate cells)+(2x no. binucleate cells)+(3x no. multinucleate cells)]/total no. of cells

Rationale for test conditions:

Test conditions were based on OECD guideline.

Evaluation criteria:

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:
- 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.
- The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
- 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:
- None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
- There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
- All results are inside the 95% control limits of the negative historical control data range.


ACCEPTABILITY CRITERIA:
- The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
- The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
- The positive control item colchicine induces a statistically significant increase in the number of mononucleated cells with micronuclei and the positive control items MMC-C and CP induces a statistically significant increase in the number of binucleated cells with micronuclei. The positive control data will be analyzed by the Chi-square test (one-sided, p < 0.05).

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) and ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) were used for statistical analysis of the data.

Key result

Species / strain:

lymphocytes: human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human peripheral lymphocytes

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Tables of results available on the section "Attached full study report".
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at a concentration of 625 µg/mL and upwards

RANGE-FINDING/SCREENING STUDIES: Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control.
- In all exposure conditions the highest concentration tested was 1250 μg/mL and a precipitate of the test item was observed at 1250 μg/mL in the 3-hour exposure groups in the absence and presence of metabolic activation (S9) and at and above 625 μg/mL for the 24-hour exposure group in the absence of S9.
- After 3-h treatment in the absence of S9-mix, a reduction in CBPI compared to vehicle control values, equivalent to 98% cytotoxicity, was obtained at 625 μg/mL. At higher tested concentrations overt toxicity was observed.
- After 3-h treatment in the presence of S9-mix, a reduction in CBPI compared to vehicle control values, equivalent to 96% cytotoxicity, was obtained at 625 μg/mL. At higher tested concentrations overt toxicity was observed.
- After 24-h treatment in the absence of S9-mix, a reduction in CBPI compared to vehicle control values, equivalent to 68% cytotoxicity, was obtained at 78 μg/mL. At higher tested concentrations overt toxicity was observed.
Binucleate cells were present at up to the maximum concentration (1250 μg/mL) in the 3-hour exposure in the absence of S9 and until 625 µg/mL in the 3-hour exposure in the presence of S9. The maximum dose with binucleate cells present in the 24-hour continuous exposure was 625 μg/mL. The test item induced some evidence of toxicity in all of the exposure groups.

CYTOKINESIS BLOCK: see tables in '"Attached full study report".

NUMBER OF CELLS WITH MICRONUCLEI: see tables in '"Attached full study report".

HISTORICAL CONTROL DATA
- 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 colchicine also showed a statistically significant increase in the number of binucleated 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. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

MAIN TEST:
FIRST ASSAY
- In the first assay, the following dose levels were selected for scoring of micronuclei:
Without S9-mix : 100, 300 and 350 µg/mL culture medium (3 hours exposure time, 27 hours harvest time). With S9-mix : 100, 350 and 400 µg/mL culture medium (3 hours exposure time, 27 hours harvest time).
In the rescoring 3-hour group in the absence of S9, toxicity was observed across the scorable dose levels where 3%, 31% and 49% cytostasis was achieved at 100, 300 and 350 μg/mL, respectively. Above this dose level, there were very few or no binucleate cells available for analysis. Therefore, the maximum dose level selected for analysis of binucleate cells was 350 μg/mL which approached the optimum toxicity range as stated in the OECD 487 test guideline (reduction in CBPI of 55±5%).
In the presence of S9, toxicity was observed across the scorable dose levels where 2%, 38% and 59% cytostasis was achieved at 100, 350 and 400 μg/mL, respectively. Above this dose level, there were very few or no binucleates available for analysis. Therefore, the maximum dose level selected for analysis of binucleate cells was 400 μg/mL, which slightly exceeded the optimum toxicity range as stated in the OECD 487 test guideline (55±5%), but this was considered to be acceptable.

SECOND ASSAY
- In the second assay, the following dose levels were selected for the second cytogenetic assay:
Without S9-mix : 20, 40, 50, 60, 70, 80, 90 and 100 µg/mL culture medium (24 hours exposure time, 24 hours harvest time).
A steep toxicity dose-response curve, compared to the preliminary toxicity test, was also observed in the 24-hour continuous exposure group, where -2%, 31% and 57% cytostasis was achieved at 20, 60 and 90 μg/mL. Above this dose level, there were very few or no binucleate cells available for analysis. Therefore, the maximum dose level selected for analysis of binucleate cells was 90 μg/mL whichslightly exceeded the optimum toxicity range as stated in the OECD 487 test guideline (55±5%), but this was considered to be acceptable.

MICRONUCLEUS ANALYSIS:
FIRST ASSAY
In the presence of S9-mix, test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei.
In the absence of S9-mix, test item induced a statistically significant increase in the number of binucleated with micronuclei at the mid dose level tested . However, since no dose relation was observed and the Cochran Armitage trend test was negative (p = 0.079), this increase was considered not biologically relevant. As quality control of the highest dose initially was not met, this part of the assay was rescored. In the end, this resulted in the same outcome with a negative Cochran Armitage trend test (p = 0.061).
In both original and rescoring, the number of micronucleated binucleate cells in the highest dose-group (7 and 17, respectively) was below the upper limit of the historical control data of the negative control (18) and furthermore the increase observed in the rescoring was caused by a high incidence in micronuclei in one single culture. All together, this increase in micronucleated cells was considered not biologically relevant.

SECOND ASSAY
Test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei.

Conclusions:

An in vitro micronucleus test, performed according to OECD guideline 487 and GLP principles, showed that the test item did not induce a statistically significant increase in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system under the experimental conditions described in the report. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Executive summary:

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to the test item using a 3-hour exposure in the presence and absence of a standard metabolizing system (S9) and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.
The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited by toxicity. The doses selected for the Main Experiment were as follows:
3-hour with and without S9: 50, 100, 250, 300, 350, 400 and 450 µg/mL culture medium; 24-hour without S9: 20, 40, 50, 60, 70, 80, 90 and 100 µg/mL culture medium   
Cytokinesis was blocked following mitosis using Cytochalasin B. Then the cells were harvested and slides prepared, so that binucleate cells could be examined for micronucleus induction.
 
All vehicle (DMSO) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
 
The results of the preliminary toxicity test indicated that the toxicity would coincide with the appearance of a precipitate of the test item and therefore, the main experiment should be limited by precipitate. In the 3-hour exposure groups and in the presence of S9-mix, the test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei. In the absence of S9-mix, the test item induced a statistically significant increase in the number of binucleated with micronuclei at the mid dose level tested. However, since no dose relation was observed and the Cochran Armitage trend test was negative (p = 0.079), this increase was considered not biologically relevant. As quality control of the highest dose initially was not met, this part of the assay was rescored. In the end, this resulted in the same outcome with a negative Cochran Armitage trend test (p = 0.061).
In both original and rescoring, the number of micronucleated binucleate cells in the highest dose-group (7 and 17, respectively) was below the upper limit of the historical control data of the negative control (18) and furthermore the increase observed in the rescoring was caused by a high incidence in micronuclei in one single culture. Altogether, this increase in micronucleated cells was considered not biologically relevant. In the 24-hour exposure group without metabolic activation, the test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei.
 
It was concluded that test item did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes in this in vitro test system, under the experimental conditions described. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Table 7.6/1: Summary of genotoxicity tests

Test No.	Test / GuidelineReliability	Endpoint	Strains/Cell Type	Metabolic Activation (S9)	Limit Concentration tested	Result and Comment
1	Ames Test(OECD 471)K, rel. 1, 2013	Gene mutation in bacteria	S. typhimurium strains TA1535, TA1537, TA98 & TA100, and E.coli strain WP2uvrA	With and without S9	Cytotoxicity	Negative with and without S9
2	Micronucleus test(OECD 487)K, rel. 1,2018	In vitro cytogenicity study in mammalian cells	Human lymphocytes	With and without S9	Cytotoxicity	Negative with and without S9
3	L5178Y cells/HPRT(OECD 476)	Gene mutation in mammalian cells	L5178Y mouse lymphoma cells	With and without S9	Up to toxic concentrations	Negative with and without S9

 

Gene mutation Assay (Test N° 1):

 In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP,Salmonella typhimuriumstrains TA 1535, TA 1537, TA 98 and TA 100 andEscherichia colistrain WP2 uvrA were exposed to test substance using the plate incorporation test (experiment I) and the pre-incubation test (experiment II) at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The test item was evaluated at the following concentrations in both experiments:
3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate. Negative, vehicle (DMSO) and positive control groups were also included in the mutagenicity tests.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in both experiments was selected as the maximum recommended dose level of 5000 μg/plate or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix. The test item induced a visible reduction in the growth of the bacterial background lawns of all strains in the absence and the presence of S9-mix between 2500 to 5000 µg/plate in the first experiment. Results from the second experiment showed a visible reduction in the growth of the bacterial background lawns in both the absence and presence of S9-mix, initially from 1000 μg/plate for the TA 1537, TA 98, TA 1535 (only with S9-mix) and TA100 strains, from 2500 µg/plate for TA 1535 without S9 mix and WP2 uvrA with S9 mix and at 5000 µg/plate for WP2 uvrA without S9 mix. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no substantial increases in revertant colony numbers among any of the five tester strains following treatment with test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency for higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. 

Under the test condition, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. It can be concluded that the test substance is not mutagenic with and without metabolic activation inS. typhimurium(strains TA1535, TA1537, TA98 and TA100) andE.coliWP2 uvrAaccording to the criteria of the Annex VI of the Regulation (EC) No.1272/2008 (CLP) and to the GHS. This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

 

Micronucleus test (Test N°2):

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to the test item using a 3-hour exposure in the presence and absence of a standard metabolizing system (S9) and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.
The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited by toxicity. The doses selected for the Main Experiment were as follows:
3-hour with and without S9: 50, 100, 250, 300, 350, 400 and 450 µg/mL culture medium; 24-hour without S9: 20, 40, 50, 60, 70, 80, 90 and 100 µg/mL culture medium 

 
Cytokinesis was blocked following mitosis using Cytochalasin B. Then the cells were harvested and slides prepared, so that binucleate cells could be examined for micronucleus induction.
 
All vehicle (DMSO) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
 
The results of the preliminary toxicity test indicated that the toxicity would coincide with the appearance of a precipitate of the test item and therefore, the main experiment should be limited by precipitate. In the 3-hour exposure groups and in the presence of S9-mix, the test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei. In the absence of S9-mix, the test item induced a statistically significant increase in the number of binucleated with micronuclei at the mid dose level tested. However, since no dose relation was observed and the Cochran Armitage trend test was negative (p = 0.079), this increase was considered not biologically relevant. As quality control of the highest dose initially was not met, this part of the assay was rescored. In the end, this resulted in the same outcome with a negative Cochran Armitage trend test (p = 0.061).
In both original and rescoring, the number of micronucleated binucleate cells in the highest dose-group (7 and 17, respectively) was below the upper limit of the historical control data of the negative control (18) and furthermore the increase observed in the rescoring was caused by a high incidence in micronuclei in one single culture. Altogether, this increase in micronucleated cells was considered not biologically relevant. In the 24-hour exposure group without metabolic activation, the test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei.
 
It was concluded that test item did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes in this in vitro test system, under the experimental conditions described. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

 

 

HPRT test (Test N°3):

In an in vitro mammalian cell mutation assay performed according to the OECD test guideline No. 476 and in compliance with GLP, mouse lymphoma L5178Y cells were exposed to the test item.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). Three-hour exposures were used both with and without activation (S9) in all tests.

In the cytotoxicity Range-Finder Experiment, eight concentrations were tested in the absence and presence of S-9, ranging from 15.48 to 1982 µg/mL (equivalent to 10 mM at the highest concentration tested and also a precipitating concentration). The highest concentrations to give >10% relative survival (RS) were 123.9 µg/mL in the absence of S-9 and 247.8 µg/mL in the presence of S-9, which gave 49% and 71% RS, respectively.

In the Mutation Experiment twelve concentrations, ranging from 25 to 350 µg/mL in the absence of S-9 and from 50 to 500 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 280 µg/mL in the absence of S-9 and 390 µg/mL in the presence of S-9, which gave 15% and 14% RS, respectively.

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 fell within acceptable ranges and clear increases in mutation were induced by one or both concentrations of 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 Mahonial at any concentration tested in the absence and presence of S-9. There was a statistically significant linear trend (p≤0.01) in the absence of S-9 but the MF values in all test article-treated cultures were not significantly higher than the concurrent vehicle control MF and were within the historical vehicle control range, therefore this observation was considered not biologically relevant. The result was considered negative under both treatment conditions.

It is concluded that Mahonial 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.

This study is considered as acceptable and satisfies the requirement for the mammalian cell gene mutation endpoint.

 

 

Conclusion on Genotoxicity

According to the ECHA integrated mutagenicity testing strategy, summarised in the flow chart shown on page 575 of Chapter R.7a, Version 6.0 – July 2017, if three of the Annex VIII in vitro tests are negative then the substance is considered not genotoxic and no further testing is required.

For this substance the Micronucleus mammalian cell assay and the in vitro gene mutation study in mammalian cells (HPRT) are clearly negative and evaluated as Klimisch grade 1 and are GLP compliant, fully robust and reliable as well as the bacterial mutagenicity assay performed on this substance which also showed clearly negative result. Therefore, it is concluded that the three in vitro studies that are required for the mutagenicity testing strategy are negative. To conclude on the genotoxicity potential of the substance, the substance is considered not genotoxic and no further testing is required.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

 

Self classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to the Annex I of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.