2,4-dimethyl-4-phenyltetrahydrofuran

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two in vitro gene mutation test in bacteria were performed:

- Ames Test on S. typhimurium TA98, TA100, TA1535, TA1537 and E.coli WP2 uvrA (OECD 471, rel.1, K): not mutagenic with and without metabolic activation.

- Ames Test on S. typhimurium TA98, TA100, TA1535, TA1537, TA1538 (eq. to OECD 471, rel.2, S): not mutagenic with and without metabolic activation.

 

- Micronucleus test (OECD 487, Rel.1, K): non-clastogenic and non-aneugenic to human lymphocytes in vitro.

 

- Mammalian Cell Gene Mutation Assay (OECD 476, HPRT, Rel.1, K): 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).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 18 September 2020 to 02 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test guideline No. 471 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

updated and adopted 21 July 1997

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan

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):

Salmonella tester strains were derived from Dr. Bruce Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

Metabolic activation:

with and without

Metabolic activation system:

10 % (v/v) S9: S9-mix from the livers of male Sprague-Dawley rats induced with a single injection intraperitoneal of Aroclor 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice.
The S9 (Lot No. 4287, Exp. Date: 11 Aug 2022) was purchased commercially from MolTox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at 60°C or colder until used.
Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and 2aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.

The S9 mix was prepared on the day of use as indicated below:

- beta-nicotinamideadenine dinucleotide phosphate (4 mM)
- Glucose-6-phosphate (5 mM)
- Potassium chloride (33 mM)
- Magnesium chloride (8 mM)
- Phosphate Buffer at pH 7.4 (100 mM)
- S9 homogenate (10% v/v)

Test concentrations with justification for top dose:

- initial toxicity-mutation assay: 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate in DMSO in TA1535, TA1537, TA98, TA100 and WP2 uvrA, with and without S9-mix via the plate incorporation method.
- confirmatory mutagenicity assay: 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate in DMSO in TA1535, TA1537, TA98, TA100 and WP2 uvrA, with and without S9-mix via the plate incorporation method.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO), Lot number SHBL2820 supplied by Sigma-Aldrich.
- Justification for choice of solvent/vehicle: DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance in DMSO formed a clear solution at a concentration of approximately 500 mg/mL in the solubility test conducted at BioReliance.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

Remarks:

Without S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene at 2.0 µg/plate for TA98 and TA1535, 5.0 µg/plate for TA100 and TA1537 and 15 µg/plate for WP2 uvrA

Remarks:

With S9-mix

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976).
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).
Salmonella tester strains were derived from Dr. Bruce Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

PREPARATION OF TESTER STRAIN: On the day of use in each assay, all tester strain cultures were checked for the appropriate genetic markers.
Overnight cultures were prepared by inoculating from the appropriate frozen permanent stock into a vessel, containing 45 to 65  mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a shaker/incubator programmed to begin shaking at 125 to 175  rpm and incubating at 37±2°C for approximately 10 +/-2.5 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x10^9 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

STERILITY CHECKS: To confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective agar. To confirm the sterility of the test substance and the vehicle, all test substance dose levels and the vehicle used in each assay were plated on selective agar with an aliquot volume equal to that used in the assay. These plates were incubated under the same conditions as the assay.

METHOD OF APPLICATION: in agar (plate incorporation)
Media used in the treatment of the test system were indicated in the table 7.6/01 below.

MUTAGENICITY ASSAY: Onehalf (0.5)  milliliter of S9 or Sham mix, 100 µL of tester strain (cells seeded) and 50.0 µL of vehicle or test substance dilution were added to 2.0 mL of molten selective top agar at 45±2°C. When plating the positive controls, the test substance aliquot was replaced by a 50.0 µL aliquot of appropriate positive control. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2°C.

DURATION
- Exposure duration: 48 to 72 h at 37 ± 2 °C

NUMBER OF REPLICATIONS:
- initial toxicity-mutation assay: 2 plates/dose
- Mutagenicity assay: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: The condition of the bacterial background lawn was evaluated for evidence of test material toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate. See Table 7.6/02.

Rationale for test conditions:

The initial toxicity-mutation assay was used to establish the doserange for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation.
The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test substance.

Evaluation criteria:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated. For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:

- For TA1535 and TA1537 strains, data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.

- For TA98, TA100 and WP2 uvrA strains, data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.

Statistics:

None

Key result

Species / strain:

other: S. typhimurium TA1535, TA1537, TA98, TA100 and E.coli WP2 uvrA.

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

Additional information on results:

No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: None
- Precipitation: Not observed.
- Other confounding effects: None

INITIAL TOXICITY-MUTATION ASSAY: No precipitate was observed, but toxicity was generally observed at 5000 µg/plate.
Several increases were observed with WP2 uvrA in the absence of S9 activation.  However, the values were within the 95% control limits.  Since, the vehicle control mean revertant value was towards the lower end of the range, the treated plates appear to have greater fold increase.  There was no dose dependent trend.  These increases were not considered to be biologically significant.

No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

CONFIRMATORY MUTAGENICITY ASSAY: No precipitate was observed, but toxicity was observed from 1500 µg/plate for TA98 and from 5000 µg/plate for TA100 and TA 1535 in the presence of metabolic activation and from 1500 µg/plate for TA100 and WP2 uvrA and from 5000 µg/plate for TA98, TA1535 and TA1537 in the absence of metabolic activation.
Several increases were observed with TA1537 in the absence of S9 activation.  However, the values were within the 95% control limits.  Since, the vehicle control mean revertant value was towards the lower end of the range, the treated plates appear to have greater fold increase.  There was no dose dependent trend.  These increases were not considered to be biologically significant.

No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

COMPARISON WITH HISTORICAL CONTROL DATA: The comparison was made with the historical negative and positive control values of 2018.

See table of results in the full study report attached.

All criteria for a valid study were met as described in the protocol.

Conclusions:

The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, Rhubafuran did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA1535, TA1537, TA98, TA100 and E.coli strain WP2 uvrA were exposed to test material both in the presence and absence of metabolic activation system (10% liver S9-mix) using the plate incorporation method. The first phase, the initial toxicity-mutation assay was used to establish the dose range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone, positive controls and eight dose levels of the test substance (1.50 to 5000 µg/plate), in duplicate, in the presence and absence of Aroclo-rinduced rat liver S9. Dose levels for the confirmatory mutagenicity assay were based upon post-treatment toxicity.

The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test substance.  TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone, positive controls and six dose levels of the test substance (15.0 to 5000 µg/plate), in triplicate, in the presence and absence of Aroclor-induced rat liver S9.  Vehicle (dimethyl sulphoxide) and positive control groups were also included in mutagenicity assay.

 

In the initial toxicity-mutation assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. No precipitate was observed. Toxicity was observed at 5000 µg per plate with most conditions. Several increases in revertant frequencies were observed with WP2 uvrA in the absence of S9 activation.  However, the values were within the 95% control limits.  Since, the vehicle control mean revertant value was towards the lower end of the range, the treated plates only appear to have greater fold increase.  Moreover there was no dose dependent trend.  As such these increases were not considered to be biologically significant. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Based upon these results, the maximum dose tested in the confirmatory mutagenicity assay was 5000 µg per plate.

In the confirmatory mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. No precipitate was observed. Toxicity was observed beginning at 1500 or at 5000 µg per plate with most conditions. Several increases in revertant frequencies were observed with TA1537 in the absence of S9 activation.  However, the values were within the 95% control limits.  Since, the vehicle control mean revertant value was towards the lower end of the range, the treated plates only appear to have greater fold increase.  Moreover there was no dose dependent trend.  As such these increases were not considered to be biologically significant. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

 

These results indicate that Rhubafuran was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

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

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2020-10-09 to 2021-06-06

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

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.
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.
- 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.

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): 1.172 to 150.0 mg/mL for a final concentration range of 11.72 to 1500 µg/mL.
- Mutation experiment (3 hours; -S9): 10.00 to 40.00 mg/mL for a final concentration range of 100.0 to 400.0 µg/mL.
- Mutation experiment (3 hours; +S9): 5.000 to 45.00 mg/mL for a final concentration range of 50.00 to 450.0 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO. Test article stock solutions were prepared by formulating Rhubafuran 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 Rhubafuran was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 206.4 mg/mL. The solubility limit in culture medium was in the range of 258.0 to 516.1 µg/mL, as indicated by precipitation at the higher concentration which persisted for an unspecified period after test article addition, with warming at 37°C. A maximum concentration of 1500 µg/mL was selected for the cytotoxicity Range Finder Experiment in order that treatments were performed up to a precipitating concentration. Concentrations selected for the first Mutation Experiment were based on the results of this cytotoxicity Range-Finder Experiment.

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 10 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. From observations on recovery and growth of the cultures during the expression period, cultures were selected to be plated for viability and 6TG resistance .

- 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 (8 days). Wells containing viable clones were identified by eye using background illumination and counted.

- Plating for 6TG resistance:
At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 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 10^4 cells/well). Plates were placed in a humidified incubator, set to 37ºC and gassed with 5% v/v CO2 in air, until scoreable (13 to 14 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 1500 µg/mL was selected for the cytotoxicity Range Finder Experiment in order that treatments were performed up to a precipitating concentration. Concentrations selected for the first Mutation Experiment were based on the results of this cytotoxicity Range-Finder Experiment.
The data presented for the Mutation Experiment were derived from the first Mutation Experiment performed in the presence of S-9 and the third Mutation Experiment performed in the absence of S-9. In the first Mutation Experiment, steep concentration-related toxicity was observed in the absence of S-9 and no concentration giving approximately 10-20% relative survival (RS) could be identified. In the repeat experiment, the experiment was terminated as the survival data did not meet the acceptance criteria. The data for the first Mutation Experiment in the absence of S-9 and the second Mutation Experiment in presence and absence of S-9 were invalidated and the data 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 (375, 750 and 1500 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 (375, 750 and 1500 µg/mL), compared to the concurrent vehicle controls.
Preliminary solubility data indicated that Rhubafuran was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 206.4 mg/mL. The solubility limit in culture medium was in the range of 258.0 to 516.1 µg/mL, as indicated by precipitation at the higher concentration which persisted for an unspecified period after test article addition, with warming at 37°C. A maximum concentration of 1500 µg/mL was selected for the cytotoxicity Range Finder Experiment in order that treatments were performed up to a precipitating 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 11.72 to 1500 µg/mL (limited by precipitation of the formulated test article in culture medium). Upon addition of the test article to the cultures, precipitate was observed at the highest four concentrations tested in the absence and presence of S-9 (187.5 to 1500 µg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the highest concentration in the presence of S-9 only (1500 µg/mL). At the end of the treatment incubation period, the highest two concentrations tested in the absence of S-9 (750 and 1500 µg/mL) and the highest concentration tested in the presence of S-9 (1500 µg/mL) were not plated for survival due to extreme toxicity (measured by cell counts). The highest concentration to give >10% RS was 187.5 µg/mL, which gave 83% and 75% RS in the absence and presence of S-9, respectively.

MAIN TEST (see Table 7.6.1/2 below)
In the Mutation Experiment thirteen concentrations, ranging from 100 to 400 µg/mL, were tested in the absence of S-9 and twelve concentrations, ranging from 50 to 450 µg/mL, were tested in the presence of S-9. Upon addition of the test article to the cultures, precipitate was observed at the highest eleven concentrations tested in the absence of S-9 (150 to 400 µg/mL) and the highest nine concentrations in the presence of S-9 (170 to 450 µg/mL). Following the 3 hour treatment incubation period, no precipitation was observed in the absence or presence of S-9. At the end of the treatment incubation period, the highest two concentrations in the absence of S9 (370 and 400 µg/mL) were not plated for survival due to extreme toxicity (measured by cell counts). Seven days after treatment, the highest three remaining concentrations in the absence of S-9 (330 to 350 µg/mL) and the highest two concentrations in the presence of S-9 (380 and 450 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 320 µg/mL in the absence of S-9 and 350 µg/mL in the presence of S-9, which gave 14% and 18% RS, respectively.

MUTATION RESULTS
The acceptance criteria were met and the study was accepted as valid.

When tested up to toxic concentrations in the absence and presence of S-9, no statistically significant increases in MF were observed following treatment with Rhubafuran at any concentration tested and there were no statistically significant linear trends. This was indicative of a negative result under both treatment conditions.
The MF values for several test article concentrations (and the vehicle controls) in the absence of S-9 in the Mutation Experiment exceeded the historical vehicle control ranges generated by the last 20 experiments performed in this laboratory (0.73 to 7.71 mutants per 106 viable cells). However, the MF values were consistent with the requirements of OECD Guideline 476, which states that the spontaneous mutant frequency is generally between 5 and 20 mutants per 106 viable cells, therefore the vehicle control MF value in the absence of S-9 was considered acceptable for addition to the laboratory historical vehicle control database in accordance with the guidelines and the assay acceptance criteria. The guidelines also describe that where concurrent vehicle control data fall outside the control limit they are acceptable for inclusion in the historical control distribution if the data are not extreme outliers and there is evidence that the test system is “under control”, with no evidence of technical or human failure. In the absence of S-9, the vehicle control data were not considered to be extreme outliers, based on previous data generated in this laboratory, and the test system was considered to be under control, therefore the vehicle control data were acceptable .

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 11.72 23.44 46.88 93.75 187.5 P 375 P 750 P	100 102 98 113 111 83 0 NP	100 128 139 117 87 75 1 0

P: precipitation noted at time of treatment

NP: Not plated due to toxicity

 

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	11.35	0	100	4.72
100	101	6.77 NS	50	124	4.23 NS
150 P	98	7.24 NS	100	110	6.45 NS
200 P	84	8.38 NS	140	113	4.62 NS
250 P	73	6.48 NS	170 P	87	2.58 NS
280 P	50	7.32 NS	200 P	76	5.65 NS
300 P	39	6.36 NS	230 P	76	2.99 NS
310 P	17	3.41 NS	260 P	59	5.95 NS
320 P	14	10.93 NS	290 P	65	5.98 NS
			320 P	58	2.79 NS
			350 P	18	4.78 NS
NQO 0.15	49	31.27	B[a]P 4	52	47.20
NQO 0.20	30	40.75	B[a]P 6	37	41.11

 

Test for Linear trend

- S9 mix		+ S9 mix
Slope Variance b²/Sb	-1.33E-08 3.43E-17 5.156	Slope Variance b²/Sb	-2.02E-09 1.29E-17 0.317

 

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

NS: Not significant

P: Precipitation noted at time of treatment

 

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	4.22 4.26	0.73 to 7.71 0.46 to 8.05

*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	20.54 30.12	5.89 to 35.19 15.15 to 45.09
B[a]P 2/4 µg/mL(1) B[a]P 3/6 µg/mL(2)	+ S9 mix + S9 mix	20.07 24.63	3.31 to 36.83 0.33 to 48.94

*Range = Mean ± 2 x SD.

(1) “Low” B[a]P concentration (2 or 4 µg/mL), dependent on use of Aroclor 1254 or β‑Naphthoflavone/Phenobarbital S-9 fraction, respectively, for metabolic activation.

(2) “High” B[a]P concentration (3 or 6 µg/mL), dependent on use of Aroclor 1254 or β‑Naphthoflavone/Phenobarbital S-9 fraction, respectively, for metabolic activation.

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 Rhubafuran 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 11.72 to 1500 µg/mL (limited by precipitation of the formulated test article in culture medium). The highest concentration to give >10% relative survival (RS) was 187.5 µg/mL, which gave 83% and 75% RS in the absence and presence of S-9, respectively.

In the Mutation Experiment thirteen concentrations, ranging from 100 to 400 µg/mL, were tested in the absence of S-9 and twelve concentrations, ranging from 50 to 450 µg/mL, were tested in the presence of S‑9. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 320 µg/mL in the absence of S-9 and 350 µg/mL in the presence of S-9, which gave 14% and 18% 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 were considered acceptable for addition to the laboratory historical vehicle control database and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) without S-9 and benzo(a)pyrene (B[a]P) with S-9. Therefore, the study was accepted as valid.

When tested up to toxic concentrations in the absence and presence of S-9, no statistically significant increases in MF were observed following treatment with Rhubafuran at any concentration tested and there were no statistically significant linear trends. This was indicative of a negative result under both treatment conditions

 

It is concluded that Rhubafuran 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 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 06 to September 03, 2014

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:

2010

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on March 12 to 14, 2014/ signed on May 15, 2014)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

20% (v/v) S9 fraction; S9 fraction was obtained from the liver homogenates of male rats induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4.

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 6.65, 13.30, 26.59, 53.19, 106.38, 212.75, 425.5, 851 and 1702μg/mL; 4 h exposure with and without S9-mix; 24 h continuous exposure to the test item without S9-mix followed by a 28 h incubation period in treatment-free media

Experiment 1:
4 h exposure to the test item formulations without S9-mix, followed by a 28 h incubation period in treatment-free media: 0*, 52.5, 105, 210*, 280*, 350* and 420 μg/mL (Mitomycin C 0.2*)
4 h exposure to the test item formulations with S9-mix (2%), followed by a 28 h incubation period in treatment-free media: 0*, 52.5, 105, 210, 280*, 350* and 420* μg/mL (Cyclophosphamide 5*)

Experiment 2:
24 h continuous exposure to the test item without S9-mix, followed by a 28 h incubation period in treatment-free media: 0*, 26.25, 52.5*, 105*, 210*, 245*, 280 and 350 μg/mL (Demecolcine 0.075*)

*Dose levels selected for analysis of micronucleus frequency in binucleate cells

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test tem was insoluble in minimal essential medium (MEM) at 17.02 mg/mL but was soluble in DMSO at 170.2 mg/mL in solubility checks performed in-house. The test item was accurately weighed, dissolved in DMSO and serial dilutions prepared.
The molecular weight of the test item was initially incorrectly calculated as 170.21, therefore, the maximum dose level tested was 1702 µg/mL, which was calculated to be equivalent to 10 mM, the maximum recommended dose level. It was subsequently realized that the molecular weight had been underestimated and the correct value was 176.26, giving a maximum recommended dose level of 1763 µg/mL. This error was considered to have no impact on the study outcome since the test item was tested to toxic dose levels and dose selection for the main experiments was limited by toxicity. The purity of the test item was 98.8% and was accounted for in the test item formulations.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: demecolcine

Remarks:

mitomycin C: 0.2 μg/mL; demecolcine: 0.075 μg/mL without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

5 μg/mL with S9 mix

Details on test system and experimental conditions:

TEST SYSTEM: For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The mean value of the AGT for the pool of regular donors used in this laboratory has been determined to be approximately 16 h under typical experimental exposure conditions.
The details of the donors used are:
Preliminary Toxicity Test: male, aged 29 years;
Experiment 1: male, aged 38 years
Experiment 2: male, aged 26 years
The age of the donor used for Experiment 1 was marginally above the range stated in the Study plan and the OECD 487 Guideline, approximately 18-35 years, however, since the age range is approximate and the micronucleus data generated from this donor was within the historical range, it is considered that there is no impact on the study outcome.

CELL CULTURE: Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 °C with 5 % CO2 in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

DURATION
- Exposure duration: 4 h (± S9) and 24 h continuous exposure (-S9) in preliminary toxicity test; 4 h (± S9) in Experiment 1, 24 h continuous exposure (-S9) in Experiment 2
- At the end of the exposure period, the cell cultures were washed and then incubated for a further 28 h in the presence of Cytochalasin B.

SPINDLE INHIBITOR (cytogenetic assays): Prior to the mitosis (after exposure of the test substance) the chemical cytochalasin B (4.5 μg/mL) was added to the cultures.
STAIN (for cytogenetic assays): 5 % Giemsa for 5 minutes

NUMBER OF REPLICATIONS:
- Duplicate cultures per dose for test item, vehicle and positive controls

NUMBER OF CELLS EVALUATED:
- Cytotoxicity: A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the cytokinesis block proliferation index (CBPI) value expressed as a percentage of the vehicle controls.
- Scoring of Micronuclei: The micronucleus frequency in 2000 binucleated cells was analysed per concentration (1000 binucleated cells per culture, two cultures per concentration). Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data. Experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity of test item in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
Cytotoxicity = 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 substance treatment culture
C = vehicle control culture

OTHER:
The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Bi-nucleate cell were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

Evaluation criteria:

The following criteria was used to determine a valid assay:
Negative Control: The frequency of binucleate cells with micronuclei in the vehicle control cultures will normally be within the laboratory historical control data range. The level of spontaneous background micronuclei may be slightly elevated above the normal range and the experiment still considered to be valid.
Positive Control Values: All the positive control chemicals must induce positive responses (p≤0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9 mix.

Statistics:

The frequency of cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analysis may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of cells with aberrations which was reproducible.

Key result

Species / strain:

lymphocytes: human

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).

PRELIMINARY TOXICITY TEST:
- A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 425.5 µg/mL and 851 µg/mL, in the 4-hour exposure groups in the absence and presence of S9 respectively, and at and above 425.5 µg/mL in the 24-hour continuous exposure group. A reduced cell pellet was also noted at the end of exposure at and above 425.5 µg/mL in the 4-hour exposure in the absence of S9, at and above 212.75 µg/mL in the 4-hour exposure in the presence of S9, and at and above 851 µg/mL, in the 24-hour exposure in the absence of S9.
- Haemolysis was observed following exposure to the test item at and above 106.38 μg/mL in all three exposure groups. Haemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
- Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 212.75 μg/mL in all three exposure groups. The test item induced marked evidence of toxicity in all three exposure groups.
- The selection of the maximum dose level was based on toxicity and was 420 μg/mL for the 4-hour exposure groups and was 350 µg/mL for the 24-hour exposure group used in Experiment 2.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected for both exposure groups.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1:
- The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were binucleate cells suitable for scoring at the 420 μg/mL in the presence of S9. In the absence of S9 the maximum dose level of the test item with binucleate cells suitable for scoring was 350 μg/mL.
- No precipitate of test item was noted at the end of the exposure period in either exposure group. Haemolysis was observed at and above 210 μg/mL and a reduced cell pellet was noted at and above 350 µg/mL at the end of exposure in both exposure groups.
- CBPI data confirm the qualitative observations in that a dose-related inhibition of CBPI was observed, and that 54% inhibition of cell proliferation was achieved at 420 μg/mL in the presence of S9. In the absence of S9 the toxicity curve was very steep with 16% inhibition of CBPI at 350 µg/mL and no binucleate cells available for scoring at 420 µg/mL. The maximum dose level selected for analysis of binucleate cells was 350 µg/mL in the absence of S9 and 420 µg/mL in the presence of S9.
- The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, either in the absence or presence of metabolic activation.

Experiment 2:
- The qualitative assessment of the slides determined that there were binucleate cells suitable for scoring at the 245 μg/mL in the 24-hour exposure group in the absence of S9.
No precipitate of the test item was observed at the end of the exposure period. Haemolysis was observed at and above 105 μg/mL and a reduced cell pellet was observed at and above 280 µg/mL at the end of the exposure period.
- CBPI data confirm the qualitative observations in that a dose-related inhibition of CBPI was observed, and that 44% and 65% inhibition of cell proliferation was achieved at 210 μg/mL and 245 µg/mL respectively. Therefore, the maximum dose level selected for binucleate cell analysis was 245 μg/mL where marginally greater than optimum toxicity was achieved.
- The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, in the 24-hour exposure in the absence of metabolic activation.

Table 7.6.1/1: Cytokinesis block proliferation index (CBPI) and Micronucleus Data

 

Dose Level (μg/mL)	Replicate	Nucleate cells /500 cells			CBPI	% Control CBPI	Micronuclei (MN) per 1000 Binucleate cells			%Cells with MN	Mean % Cells with MN
		Mono	Bi	Multi			1 MN	2 MN	>2 MN
Experiment 1 – 4 h Exposure Without Metabolic Activation (S9)
0	A	283	172	45	1.52	100	1	0	0	0.10	0.20
	B	254	205	41	1.57		3	0	0	0.30
210	A	250	239	11	1.52	92	4	0	0	0.40	0.25
	B	279	200	21	1.48		1	0	0	0.10
280	A	294	194	12	1.44	87	2	0	0	0.20	0.50
	B	274	195	31	1.51		7	1	0	0.80
350	A	346	136	18	1.34	84	5	0	0	0.50	0.55
	B	216	278	6	1.58		6	0	0	0.60
MMC 0.2	A	214	274	12	1.60	96	65	2	0	6.70	4.30***
	B	286	202	12	1.45		18	1	0	1.90
Experiment 1 - 4 h Exposure With Metabolic Activation (S9)
0	A	138	342	20	1.76	100	4	0	0	0.40	0.40
	B	215	259	26	1.62		4	0	0	0.40
280	A	203	280	17	1.63	96	5	0	0	0.50	0.75
	B	174	305	21	1.69		10	0	0	1.00
350	A	205	275	20	1.63	99	3	0	0	0.30	0.50
	B	164	306	30	1.73		7	0	0	0.70
420	A	368	129	3	1.27	46	1	0	0	0.10	0.15
	B	322	176	2	1.36		2	0	0	0.20
CP 5	A	369	131	0	1.26	41	35	2	1	3.80	5.05***
	B	345	153	2	1.31		59	3	1	6.30
Experiment 2 – 24 h Exposure Without Metabolic Activation (S9)
0	A	71	366	63	1.98	100	3	0	0	0.30	0.25
	B	83	341	75	1.98		2	0	0	0.20
52.5	A	114	333	53	1.88	95	2	0	0	0.20	0.10
	B	79	354	67	1.98		0	0	0	0.00
105	A	186	282	32	1.69	78	6	0	0	0.60	0.60
	B	125	329	46	1.84		6	0	0	0.60
210	A	264	232	4	1.48	56	2	0	0	0.20	0.25
	B	209	270	21	1.62		3	0	0	0.30
245	A	339	158	3	1.33	35	2	1	0	0.30	0.20
	B	332	162	6	1.35		1	0	0	0.10
DC 0.075	A	204	233	63	1.72	72	20	7	3	3.00	3.65***
	B	199	256	45	1.69		31	8	4	4.30

 

MMC = Mitomycin C; CP = Cyclophosphamide; DC = Demecolcine; *** = P<0.001

Conclusions:

Under the test conditions, test material was 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 test material in the presence and absence of a metabolic activation system (2% S9 mix).

In Experiment 1, test material was tested at the concentrations of 0, 52.5, 105, 210, 280, 350 and 420 μg/mL (without S9-mix) and 0, 52.5, 105, 210, 280, 350 and 420 μg/mL (with S9-mix) for a 4 h exposure time. In Experiment 2, test material was tested at the concentrations of 0, 26.25, 52.5, 105, 210, 245, 280 and 350 μg/mL (without S9-mix) for 24 h continuous exposure. At the end of the exposure period, the cell cultures were washed and then incubated for a further 28 h in the presence of Cytochalasin B. After harvesting, the cells were then treated with a hypotonic solution, fixed, stained and examined for micronuclei. Preliminary toxicity test was performed before the main test.

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. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

 

The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, in either of the two experiments using a dose range which included a dose level which achieved approximately 50% reduction in cytokinesis block proliferation index (CBPI) in the 4 h exposure group in the presence of S9 and in the 24 h exposure group. The 4-hour exposure group in the absence of S9 was tested to toxic dose levles but due to the steepness of the toxicity curve, optimum toxicity as not achieved.

 

Under the test conditions, test material was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

November 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

Salmonella/microsome mutagenicity test according to a method of Ames et al. (1975) and comparable to the OECD guideline 471 with the following deviation: five strains of Salmonella typhimurium was assessed (TA98, TA100, TA35, TA37 and TA38).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only five strains of Salmonella typhimurium was assessed (TA98, TA35, TA37, TA38 and TA100).

Principles of method if other than guideline:

Salmonella/microsome mutagenicity test according to a method of Ames et al. (1975) and comparable to the OECD guideline 471 with the following deviation: five strains of Salmonella typhimurium was assessed (TA98, TA100, TA35, TA37 and TA38).

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

S.typhimurium: Histidine gene

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 1538 TA 98 and TA 100 bacteria

Details on mammalian cell type (if applicable):

Provided by Dr. B.N. Ames, Berkeley, California, USA.
Fresh overnight cultures (16 hours) containing about 10e9 bacteria per mL are mixed with DMSO and divided into 0.5 mL aliquots in sterile polypropylene vials. They are quickly frozen on dry ice and subsequently stored at -80°C. Part of the overnight culture is retained and checked for numbers of spontaneous revertants, histidine requirement and sensitivity to ampicilline, crystal violet and UV radiation. In addition, the induced reversion properties of the strains are checked using reference mutagens.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: in-house preparation dated to 28 July 1999
- method of preparation of S9 mix: Male Wistar rats (from Central Institute for the Breeding of Laboratory Animals) were injected intraperitoneally with a single dose of Aroclor 1254 (nominal dose of 500 mg/kg bw) in soya bean oil (20% w/v) and killed, five days later, by decapitation and exsanguination. The extracted liver homogenate was centrifuged for 10 min at 9,000 g. The supernatant, which is called S9, was collected and divided into small aliquots in sterile polypropylene vials that are quickly frozen in dry ice and stored in liquid nitrogen.
- concentration or volume of S9 mix and S9 in the final culture medium: On the day of use, aliquots of S9 were thawed and mixed with a NADPH generating system. The final concentration of the various ingredients in the S9-mix were: MgCl2 8 mM; KCl 33 mM; G-6-P 4 mM; NADP 4 mM in sodium phosphate buffer 0.05 M (pH 7.4). The S9-mix contained 10% S9. The S9-mix was kept on ice until use.

Test concentrations with justification for top dose:

The dose range used in the mutagenesis assay is based on a preliminary test performed to assess the toxicity of the compound for the bacteria. If possible the lowest toxic dose is taken as the highest dose for the mutagenesis assay. In the case of questionable results the plate incorporation assay is partly repeated and/or a number of revertant colonies is checked for histidine requirement and for other strain characteristics if appropriate.


Mutagenicity test: with and without S9-mix: 0, 0.0037, 0.011, 0.033, 0.1 and 0.3 µg/plate ; direct plate incorporation method

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Methanol
- Formulation preparation: Appropriate solutions of the test material were prepared in methanol immediately before use.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

with S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: Hycanthone methanesulphonate: 12.5 µg/0.1 mL water/plate for TA 1537, TA 1538 and TA 98

Remarks:

Without S9-mix

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: All four strains were provided by Dr. B.N. Ames (University of California Berkeley, USA) and stocked in the freezer.
To obtain cultures for mutagenesis testing, nutrient broth is inoculated with a thawed aliquot of the appropriate bacterial culture (0.1 mL per 10 mL nutrient bouillon) and grown up overnight with shaking at 37°C for 16 hours. The viable count of each culture is determined by plating appropriate dilutions on nutrient broth agar plates. The bacterial cultures are stored in a refrigerator at 5°C for up to four days.

The procedure used in this assay has been described in detail by Ames et al. (1975).

METHOD OF APPLICATION: In agar (plate incorporation method)
To 2 ml molten top agar containing 0.6% agar, 0.5% NaCl and 0.5 mM L-histidine. HCl/0.5 mM biotin, maintained at 46°C, were added subsequently: 0.1 ml of a fully grown culture of the appropriate strain, 0.1 ml of the appropriate test substance solution or of the negative or positive control substance, and 0.5 ml S9-mix for assay with metabolic activation. The ingredients were thoroughly mixed and the mix was immediately poured onto minimal glucose agar plates.

DURATION
- Incubation period: 72 h at 37 °C

NUMBER OF REPLICATIONS:
- Mutation study: Triplicate

NUMBER OF CELLS EVALUATED: Not specified.
Subsequently the incubation, the his+ revertants were counted. The background lawn of bacterial growth was examined microscopically to determine any growth-diminidhing or growth-enhancing effects by the test substance, if a two-fold or greater increase in the mean number of his+ revertants was observed.

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the toxicity was performed on the basis of a reduction in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth.

ADDITIONNAL CHECKS
S9, S9-mix and test product are checked for sterility.
Positive controls include direct as well as indirect mutagens.

Evaluation criteria:

Not mentionned.

Statistics:

Not mentionned.

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At the higher doses, the test liquid was clearly toxic for the bacteria as revealed by a less dense background lawn of bacterial growth and a decrease in the number of revertant colonies.

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Incorporation of the test product with the bacteria at levels up to 0.3 mg per plate did not increase the numbers of his+ revertants with any of the five tester strains, either in the presence or in the absence of S9-mix.

At the lower dose levels tested there were no signs that chemical toxicity interfered with the mutagenicity testing: the background lawn of bacterial growth in control and test plates was comparable. At higher doses, the test liquid was clearly toxic for the bacteria as revealed by a less dense background lawn of bacterial growth and a decrease in the number of revertant colonies.

From the present results it can be concluded that Rhubafurane did not reveal any mutagenic activity in the plate incorporation assay with S.typhimurium TA 1535, TA 1537, TA 1538, TA 98 or TA 100 in the presence or absence of the liver microsome activation system under the test conditions employed in this evaluation.

Table 1. Evaluation of Rhubafurane in the Salmonella/microsome mutagenicity test

mg test liquid per 0.1 mL methanol per plate	S9-mix added	Numbers of his+ revertants (mean of 3 plates +/- SD) with test
		TA 1535	TA 1537	TA 1538	TA 98	TA 100
0	no	36±5	13±5	18±2	29±2	164±26
0.0037	no	32±7	15±8	19±5	28±7	149±11
0.011	no	44±22	12±4	23±1	32±3	127±14
0.033	no	36±1	11±4	14±2	26±7	117±8
0.1	no	14±4	13±1	14±4	37±9	131±12
0.3	no	9±4†	12±4†	17±4†	22±2†	105±15†
0	yes	36±3	24±7	37±8	52±5	186±18
0.0037	yes	30±15	19±3	31±10	53±10	138±28
0.011	yes	35±4	18±2	42±13	58±11	151±9
0.033	yes	28±10	18±2	35±4	50±11	154±19
0.1	yes	20±4	23±2	31±2	43±12	149±15
0.3	yes	9±4†	14±6†	29±7†	44±1†	134±14†
Positive controls
	no	343±13	124±28	222±40	181±28	323±3
	yes	59±2	41±6	292±1	230±88	422±60
Number of bacterial per mL
		1 x 10e9	8 x 10e8	8 x 10e8	2 x 10e9	1 x 10e9

Test performed: 12.11.1980

S9-prepared: 23.09.1980

†: background lawn of bacterial growth less dense than in concomitant control plates

Conclusions:

Under the test conditions, the test item is not considered as mutagenic in S. typhimurium (TA 1535, TA 1537, TA 1538, TA 98 and TA 100) strains.

Executive summary:

In a Salmonella/microsome mutagenicity test according to a method of Ames et al. (1975) and comparable to the OECD guideline 471, five strains of Salmonella typhimurium was assessed (TA98, TA100, TA35, TA37 and TA38) were exposed to the test item. 

The dose range used in the mutagenesis assay is based on a preliminary test performed to assess the toxicity of the compound for the bacteria and appropriate solutions of the test material were prepared in methanol.The following concentrations were used in a plate incorporation method with and without S9-mix: 0, 0.0037, 0.011, 0.033, 0.1 and 0.3 µg/plate.

Metabolic activation system used in this study was 10 % (v/v) S9 mix. S9 fraction was prepared from livers of male Wistar rats induced with Aroclor 1254 (500 mg/kg bw) by intraperitoneal route. Vehicle and positive control groups were also included in mutagenicity tests.

Incorporation of the test product with the bacteria at levels up to 0.3 mg per plate did not increase the numbers of his+ revertants with any of the five tester strains, either in the presence or in the absence of S9-mix.

At the lower dose levels tested, there were no signs that chemical toxicity interfered with the mutagenicity testing: the background lawn of bacterial growth in control and test plates was comparable. At higher doses, the test liquid was clearly toxic for the bacteria as revealed by a less dense background lawn of bacterial growth and a decrease in the number of revertant colonies.

From the present results it can be concluded that Rhubafurane did not reveal any mutagenic activity in the plate incorporation assay with S.typhimurium TA 1535, TA 1537, TA 1538, TA 98 or TA 100 in the presence or absence of the liver microsome activation system under the test conditions employed in this evaluation.

Under the test conditions, Rhubafurane is not considered as mutagenic in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, in both the absence and the presence of the S9-mix.

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 n°	Test / GuidelineReliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1  BioReliance, 2021	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537, TA 98,TA 100, WP2 uvrA	-S9+S9	15.0, 50.0, 150, 500, 1500 and 5000 µg per plate in DMSO	-S9: non mutagenic +S9: non mutagenic
2  TNO, 1980	Ames Test (eq. to OECD 471) S, rel. 2	Gene mutation	TA 1535, TA 1537, TA 1538, TA 98,TA 100	-S9+S9	0, 0.0037, 0.011, 0.033, 0.1 and 0.3 µg/plate (in methanol)	-S9: non mutagenic +S9: non mutagenic
3 Harlan, 2014	Micronucleus (OECD 487) K, rel.1	in vitro mammalian cell micronucleus test	Human lymhocytes	-S9+S9	See below	-S9: non clastogenic +S9: non clastogenic
4 Covance, 2021	L5178Y cells/HPRT (OECD 476) K, rel.1	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&2):

In the first reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA1535, TA1537, TA98, TA100 and E.coli strain WP2 uvrA were exposed to test material both in the presence and absence of metabolic activation system (10% liver S9-mix) using the plate incorporation method. The first phase, the initial toxicity-mutation assay was used to establish the dose range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone, positive controls and eight dose levels of the test substance (1.50 to 5000 µg/plate), in duplicate, in the presence and absence of Aroclo-rinduced rat liver S9. Dose levels for the confirmatory mutagenicity assay were based upon post-treatment toxicity.

The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test substance.  TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone, positive controls and six dose levels of the test substance (15.0 to 5000 µg/plate), in triplicate, in the presence and absence of Aroclor-induced rat liver S9.  Vehicle (dimethyl sulphoxide) and positive control groups were also included in mutagenicity assay.

In the initial toxicity-mutation assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. No precipitate was observed. Toxicity was observed at 5000 µg per plate with most conditions. Several increases in revertant frequencies were observed with WP2 uvrA in the absence of S9 activation.  However, the values were within the 95% control limits.  Since, the vehicle control mean revertant value was towards the lower end of the range, the treated plates only appear to have greater fold increase.  Moreover there was no dose dependent trend.  As such these increases were not considered to be biologically significant. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Based upon these results, the maximum dose tested in the confirmatory mutagenicity assay was 5000 µg per plate.

In the confirmatory mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. No precipitate was observed. Toxicity was observed beginning at 1500 or at 5000 µg per plate with most conditions. Several increases in revertant frequencies were observed with TA1537 in the absence of S9 activation.  However, the values were within the 95% control limits.  Since, the vehicle control mean revertant value was towards the lower end of the range, the treated plates only appear to have greater fold increase.  Moreover there was no dose dependent trend.  As such these increases were not considered to be biologically significant. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

These results indicate that Rhubafuran was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

 

In addition, a second Salmonella/microsome mutagenicity test according to a method of Ames et al. (1975) and comparable to the OECD guideline 471 was perfomed in five strains of Salmonella typhimurium  (TA98, TA100, TA35, TA37 and TA38) exposed to the test item. 

The dose range used in the mutagenesis assay is based on a preliminary test performed to assess the toxicity of the compound for the bacteria and appropriate solutions of the test material were prepared in methanol.The following concentrations were used in a plate incorporation method with and without S9-mix: 0, 0.0037, 0.011, 0.033, 0.1 and 0.3 µg/plate.

Metabolic activation system used in this study was 10 % (v/v) S9 mix. S9 fraction was prepared from livers of male Wistar rats induced with Aroclor 1254 (500 mg/kg bw) by intraperitoneal route. Vehicle and positive control groups were also included in mutagenicity tests.

Incorporation of the test product with the bacteria at levels up to 0.3 mg per plate did not increase the numbers of his+ revertants with any of the five tester strains, either in the presence or in the absence of S9-mix.

At the lower dose levels tested, there were no signs that chemical toxicity interfered with the mutagenicity testing: the background lawn of bacterial growth in control and test plates was comparable. At higher doses, the test liquid was clearly toxic for the bacteria as revealed by a less dense background lawn of bacterial growth and a decrease in the number of revertant colonies.

From the present results it can be concluded that Rhubafurane did not reveal any mutagenic activity in the plate incorporation assay with S.typhimurium TA 1535, TA 1537, TA 1538, TA 98 or TA 100 in the presence or absence of the liver microsome activation system under the test conditions employed in this evaluation.

Under the test conditions, Rhubafurane is not considered as mutagenic in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, in both the absence and the presence of the S9-mix.

 

Micronucleus test (Test n°3)

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to test material in the presence and absence of a metabolic activation system (2% S9 mix).

In Experiment 1, test material was tested at the concentrations of 0, 52.5, 105, 210, 280, 350 and 420 μg/mL (without S9-mix) and 0, 52.5, 105, 210, 280, 350 and 420 μg/mL (with S9-mix) for a 4 h exposure time. In Experiment 2, test material was tested at the concentrations of 0, 26.25, 52.5, 105, 210, 245, 280 and 350 μg/mL (without S9-mix) for 24 h continuous exposure. At the end of the exposure period, the cell cultures were washed and then incubated for a further 28 h in the presence of Cytochalasin B. After harvesting, the cells were then treated with a hypotonic solution, fixed, stained and examined for micronuclei. Preliminary toxicity test was performed before the main test.

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. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, in either of the two experiments using a dose range which included a dose level which achieved approximately 50% reduction in cytokinesis block proliferation index (CBPI) in the 4 h exposure group in the presence of S9 and in the 24 h exposure group. The 4-hour exposure group in the absence of S9 was tested to toxic dose levles but due to the steepness of the toxicity curve, optimum toxicity as not achieved.

Under the test conditions, test material was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

 

HPRT test (Test n°4)

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 11.72 to 1500 µg/mL (limited by precipitation of the formulated test article in culture medium). The highest concentration to give >10% relative survival (RS) was 187.5 µg/mL, which gave 83% and 75% RS in the absence and presence of S-9, respectively.

In the Mutation Experiment thirteen concentrations, ranging from 100 to 400 µg/mL, were tested in the absence of S-9 and twelve concentrations, ranging from 50 to 450 µg/mL, were tested in the presence of S‑9. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 320 µg/mL in the absence of S-9 and 350 µg/mL in the presence of S-9, which gave 14% and 18% 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 were considered acceptable for addition to the laboratory historical vehicle control database and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) without S-9 and benzo(a)pyrene (B[a]P) with S-9. Therefore, the study was accepted as valid.

When tested up to toxic concentrations in the absence and presence of S-9, no statistically significant increases in MF were observed following treatment with Rhubafuran at any concentration tested and there were no statistically significant linear trends. This was indicative of a negative result under both treatment conditions

 

It is concluded that Rhubafuran 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.

Justification for classification or non-classification

Harmonised classification

The test material has no harmonised classification for human health according to the Regulation (EC) No. 1272/2008 (CLP).

Self classification

No additional classification is proposed regarding in vitro genetic toxicity according to the criteria of Annex I to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.