6,8-Dioxabicyclo[3.2.1]octan-4-one, (1S,5R)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial mutagenicity (Ames test): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 (OECD Test Guideline 471) (Harlan Cytotest Cell Research GmbH, 2014d).

Cytogenicity in mammalian cells: negative with and without metabolic activation in cultured peripheral human lymphocytes (OECD Test Guideline 487) (Harlan Cytotest Cell Research GmbH, 2014e).

Gene mutation (mouse lymphoma L5178Y) cells): negative with and without metabolic activation (OECD Test Guideline 476) (Covance Laboratories Ltd, 2018c).

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:

21 May - 12 Jun 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997

Deviations:

yes

Remarks:

Only 2-Aminoanthracene was used as positive control in the presence of S9 mix.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Only 2-Aminoanthracene was used as positive control in the presence of S9 mix.

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

nitroreductase deficient

Remarks:

for uvrB- strains TA 1537, TA 98, TA 1535, TA 100

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from phenobarbital/β-naphtoflavone induced rat liver

Test concentrations with justification for top dose:

1st experiment: 3, 10, 33, 100, 333, 1000, 2500, and 5000 μg/plate with and without metabolic activation.
2nd experiment: 33, 100, 333, 1000, 2500, and 5000 μg/plate with and without metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionized water- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (2-AA)

Remarks:

+S9: 2-aminoanthracene (2-AA; 2.5 or 10 µg/plate, in DMSO, all strains); -S9: 4-NOPD (10 or 50 µg/plate, in DMSO, TA1537/TA98), sodium azide (NaN3, 10 µg/plate, in water, TA1535/TA100); methylmethanesulfonate (MMS, 2 µg/plate, in water, TA102)

Details on test system and experimental conditions:

Experiment I: in agar (plate incorporation)
Experiment II: pre-incubation
DURATION Experiment I - Exposure duration: 48 h
DURATION Experiment II - Preincubation period: 60 min
NUMBER OF REPLICATIONS: each concentration was tested in triplicates in two independent experiments both with and without S9 mix

DETERMINATION OF CYTOTOXICITY:
Method: inspection of the bacterial background growth and reduction in the number of revertant colonies

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 TA 102) 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.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
Water solubility: The test item is dissolvable in water.
Precipitation: No precipitation of the test item occurred up to the highest investigated dose.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment I. Since no toxic effects were observed 5000 μg/plate were chosen as maximal concentration.

Pre-Experiment for Toxicity

To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I (plate incorporation test). Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. The pre-experiment is reported as main experiment I, since the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

Table 1: Test Results of Experiment 1

EXPERIMENT 1
S9-Mix	Without
Test item (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	TA 102
Verhicle control	17.7	10.7	24.7	175.0	443.3
Untreated control	2.0	1.2	8.7	166.7	468.7
Cyrene™ (3)	18.3	12.7	25.0	168.3	453.0
Cyrene™ (10)	18.0	11.7	27.3	183.7	447.0
Cyrene™ (33)	17.7	9.3	28.0	183.0	435.7
Cyrene™ (100)	19.7	9.0	23.0	174.7	440.7
Cyrene™ (333)	15.7	9.0	27.7	175.0	455.0
Cyrene™ (1000)	17.7	9.7	22.7	172.0	424.7
Cyrene™ (2500)	15.0	11.0	24.0	188.0	467.3
Cyrene™ (5000)	14.3	13.7	30.3	178.0	465.0
NaN3 (10)	2940.3			1906.7
4-NOPD (50)		58.3
4-NOPD (10)			273.7
MMS (2.0 µL/plate)					5586.7
S9-Mix	With
Test item (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	TA 102
Vehicle control	16.7	24.0	38.3	177.0	595.3
Untreated control	15.3	20.0	36.7	170.0	618.3
Cyrene™ (3)	14.7	21.3	42.7	181.0	573.3
Cyrene™ (10)	15.3	24.0	41.3	184.7	587.7
Cyrene™ (33)	13.3	20.3	38.7	187.7	651.0
Cyrene™ (100)	12.0	20.0	34.3	185.3	690.7
Cyrene™ (333)	14.7	19.7	41.3	179.7	603.0
Cyrene™ (1000)	13.7	20.3	46.3	177.0	595.7
Cyrene™ (2500)	15.3	20.7	35.7	174.3	589.3
Cyrene™ (5000)	17.0	15.0	35.7	167.3	645.7
2-AA (2.5)	548.3	376.0	3912.0	3957.7
2-AA (10.0)					1384.7
2 -AA: 2 -aminoanthracene; 4 -NOPD: 4 -nitro-o-phenylene-diamine; NaN3: sodium azide; MMS: methylmethanesulfonate

Table 2: Test Results of Experiment 2

EXPERIMENT 2
S9-Mix	Without
Test item (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	TA 102
Vehicle control	12.0	11.3	26.7	152.0	362.3
Untreated control	9.7	7.7	31.0	161.3	407.7
Cyrene™ (33)	12.7	11.0	25.3	159.7	366.3
Cyrene™ (100)	12.3	12.7	29.3	164.0	394.0
Cyrene™ (333)	13.3	9.0	29.7	165.3	351.7
Cyrene™ (1000)	17.3	8.7	29.7	165.3	391.7
Cyrene™ (2500)	13.0	12.7	33.3	163.0	395.7
Cyrene™ (5000)	14.0	11.3	29.7	152.3	425.3
NaN3 (10)	2758.7			1706.3
4-NOPD (50)		53.3
4-NOPD (10)			242.0
MMS (2.0 µL/plate)					1776.7
S9-Mix	With
Test item (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	TA 102
Vehicle control	11.0	16.0	43.0	200.0	526.7
Untreated control	15.3	11.7	42.7	160.7	534.3
Cyrene™ (33)	14.7	14.0	44.3	158.7	550.3
Cyrene™ (100)	11.7	19.7	42.7	156.3	447.3
Cyrene™ (333)	10.7	20.0	41.3	178.7	577.0
Cyrene™ (1000)	10.0	16.0	35.0	180.3	530.7
Cyrene™ (2500)	13.0	15.0	41.3	176.3	517.3
Cyrene™ (5000)	14.7	12.7	43.7	188.0	593.3
2-AA (2.5)	505.3	131.0	3724.3	2360.7
2-AA (10.0)					5146.7
2 -AA: 2 -aminoanthracene; 4 -NOPD: 4 -nitro-o-phenylene-diamine; NaN3: sodium azide; MMS: methylmethanesulfonate

Conclusions:

Cyrene™ has been tested in a valid bacterial reverse mutation assay, conducted according to OECD Test Guideline 471 (1997) and in compliance with GLP, using Salmonella typhimurium strains S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102. No increase in the number of revertants was observed in any test strain, with or without metabolic activation when tested up to limit concentration. Appropriate positive, negative and solvent controls were added and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Apr - 26 Jun 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD guideline 487, adopted 26 September 2014

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU method B.49

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: DMEM/Ham's F12 (mixture 1:1), supplemented with:200 mM GlutaMAX™penicillin/streptomycin (100 U/mL/ 100 µg/mL) mitogen PHA (3 µg/mL)10% fetal bovine serum (FBS) During pulse exposure to the test item (4 h treatment) DMEM/Ham's F12 was used without FBS supplementation. For continuous exposure to the test item complete DMEM/Ham's F12 was used.10 mM HEPES anticoagulant heparin (125 U.S.P. - U/mL)

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from Phenobarbital/β-naphthoflavone induced rat liver

Test concentrations with justification for top dose:

Experiment I, 4h exposure: 8.3, 14.6, 25.5, 44.6, 78.0, 136.5, 238.8, 418.0, 731.4 and 1280.0 µg/mL with and without S9 mix
Experiment II, 4 h exposure: 78.0, 136.5, 238.8, 418.0, 731.4 and 1280.0 µg/mL with S9 mix;
Experiment II, 20 h exposure: 8.3, 14.6, 25.5, 44.6, 78.0, 136.5, 238.8, 418.0, 731.4 and 1280.0 µg/mL without S9 mix

Vehicle / solvent:

Deionised water final concentration of solvent in the culture medium: 10%
Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

culture medium with 10.0 % deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: demecolcin

Remarks:

- S9 mix: mitomycin C (MMC, 2.0 µg/mL, in deionised water, pulse treatment), demecolcin (150.0 ng/mL, in deionised water, continuous treatment); + S9 mix: cyclophosphamide (CPA, 15.0 µg/mL, in 0.9% NaCL [w/v])

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium without FBS for pulse exposure with S9 mix, with FBS for continuous exposure without S9 mix
DURATION:
Exposure duration:
Experiment I: exposure period 4 h with and without S9 mix
Experiment II: exposure period 4 h with S9 mix and 20 h without S9 mix
Fixation time (start of exposure up to harvest of cells): 40 h
ACTIN POLYMERISATION INHIBITOR: Cytochalasin B (cytoB; 4 µg/mL)STAIN: Giemsa
NUMBER OF REPLICATIONS: In two independent experiments 2 replications for each concentration (each with or without S9 mix) were performed.
NUMBER OF CELLS EVALUATED: 2000 binucleated cells per concentration in cytoB-treated cultures
DETERMINATION OF CYTOTOXICITY- Method: Cytokinesis-block proliferation index (CBPI)

Evaluation criteria:

A test item can be classified as non-clastogenic and non-aneugenic if:
-    the number of micronucleated cells in all evaluated dose groups is in the range of the historical laboratory control data and
-    no statistically significant or concentration-related increase of the number of micronucleated cells is observed in comparison to the respective solvent control.   A test item can be classified as clastogenic and aneugenic if:
-    the number of micronucleated cells is not in the range of the historical laboratory control data and
-    either a concentration-related increase in three test groups or a statistically significant increase in the number of micronucleated cells is observed  If the above mentioned criteria for the test item are not clearly met, the test item will be classified as equivocalor a confirmatory experiment may be performed. However, results may remain questionable regardless of the number of times the experiment is repeated. An increase in the number of micronucleated mononucleate cells may indicate that the test item has aneugenic potential.

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: With and without S9 mix, no clear cytotoxicity was observed up to the highest test concentration. Only experiment I at 1280 µg/mL - S9 mix showed 46.3% cytostasis. However, clear cytotoxic effects could only be observed in one of the two cultures.

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant influence was observed. The pH for the test substance was 7.6.
- Effects of osmolality: No relevant influence was observed. The osmolarity for the test substance was approximately 300 mOsm.
- Precipitation: No visible precipitation of the test item in culture medium was observed

RANGE-FINDING/SCREENING STUDIES:
Test item concentrations ranging from 8.3 to 1280.0 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, no precipitation of the test item was observed. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.Using a reduced Cytokinesis-block proliferation index (CBPI) as an indicator for toxicity in Experiment I, no cytotoxic effects were observed after 4 h treatment in the absence and presence of S9 mix. Therefore, 1280.0 µg/mL was chosen as top treatment concentration for Experiment II.
COMPARISON WITH HISTORICAL CONTROL DATA:The micronucleus rates of the cells after treatment with the test item (0.20 – 0.75% micronucleated cells) were within the range of the laboratory historical control data
ADDITIONAL INFORMATION ON CYTOTOXICITY:Cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate. In the absence and presence of S9 mix, no clear cytotoxicity was observed up to the highest required concentration. In Experiment I in the absence of S9 mix 46.3% cytostasis was observed at the highest applied concentration. However, clear cytotoxic effects could only be observed in one of the two cultures.

Table 1           Summary of results of the in vitro micronucleus test in human lymphocytes with the test item

Exp.	Preparation interval	Test item concentration	Proliferation index	Cytostasis in %*	Micronucleated cells
		in µg/mL	CBPI		in %**
Exposure period 4 h without S9 mix
I	40 h	deionised water 10%	1.91		0.50
		MMC 2.0	1.18	80.5	6.35#
		418.0	1.88	4.3	0.20
		731.4	1.93	n.c.	0.25
		1280.0	1.49	46.3	0.30
Exposure period 20 h without S9 mix
II	40 h	deionised water	1.84		0.55
		Demecolcin 0.15	1.40	52.2	3.25#
		418.0	1.90	n.c.	0.35
		731.4	1.90	n.c.	0.35
		1280.0	1.82	1.9	0.75
Exposure period 4 h with S9 mix
I	40 h	deionised water 10%	2.02		0.50
		CPA 15.0	1.61	40.0	3.15#
		418.0	1.94	8.1	0.25
		731.4	1.89	13.1	0.35
		1280.0	1.94	7.9	0.45
II	40 h	deionised water 10%	2.07		0.50
		CPA 15.0	1.66	38.5	5.20#
		418.0	2.01	5.5	0.60
		731.4	1.95	11.4	0.65
		1280.0	2.04	3.1	0.45
CPA: Cyclophosphamide; MMC: Mitomycin C *       For the positive control groups and the test item treatment groups the values are related to the solvent controls **     The number of micronucleated cells was determined in a sample of 2000 binucleated cells #       The number of micronucleated cells is statistically significantly higher than corresponding control values n.c.   Not calculated as the CBPI is equal or higher than the solvent control value

Conclusions:

Cyrene™ has been tested for ability to cause chromosome aberrations in cultured peripheral human lymphocytes in an in vitro cytogenicity study, conducted according to OECD Test Guideline 487 and in compliance with GLP. No increase in the number of cells with aberrations was observed either with or without metabolic activation in cultured peripheral human lymphocytes. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of this study.  

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study Initiation Date: 26 October 2017 - Study Completion Date: 8 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: : In Vitro L5178Y Gene Mutation Assay at the hprt locus

Target gene:

HPRT locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. were stored as frozen stocks in liquid nitrogen.
Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free.
For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated at 37±1ºC. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 used was obtained from Molecular Toxicology Incorporated, USA, prepared from male SD rats induced with Aroclor 1254. The S-9 was supplied as lyophilized S-9 mix (MutazymeTM), stored frozen at <-20°C.

Test concentrations with justification for top dose:

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 40.06 to 1282 µg/mL (equivalent to 10 mM at the highest concentration tested).

In the Mutation Experiment ten concentrations, ranging from 100 to 1282 µg/mL in the absence of S-9 and from 25 to 1282 µg/mL in the presence of S-9, were tested.

Preliminary solubility data indicated that Cyrene™ was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations up to at least 153.8 mg/mL.
The solubility limit in culture medium was in excess of 1538 µg/mL, as indicated by a lack of any visible precipitation, at this concentration either immediately upon addition or following a period of 21 hours’ incubation at 37ºC.
A maximum concentration of 1282 µg/mL was therefore selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to 10 mM, an acceptable maximum recommended concentration according to current regulatory test guidelines (OECD, 2016).
Concentrations selected for the Mutation Experiment were based on the results of this cytotoxicity Range-Finder Experiment.

Vehicle / solvent:

Anhydrous analytical grade dimethyl sulphoxide (DMSO)

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

Test System:
The test system was labelled with the study number, test article concentration, positive and vehicle control, presence and absence of S 9.
Cytotoxicity Range-Finder Experiment:
Treatment of cell cultures for the cytotoxicity Range-Finder Experiment was as described below for the Mutation Experiment. However, single cultures only were used and positive controls were not included. The final treatment volume was 20 mL.
Following 3 hour treatment, cells were centrifuged (200 g), washed with tissue culture medium 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 incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air for 8 days. Wells containing viable clones were identified by eye using background illumination and counted.

Mutation Assay:
Treatment of Cell Cultures:
At least 107 cells in a volume of 17.8 mL of RPMI 5 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 0.2 mL vehicle, test article or positive control solution was added. S-9 mix or 150 mM KCl was added as described. Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL.
After 3 hours’ incubation at 37±1°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 the concentrations adjusted to 2 x 105 cells/mL. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival as described.
Osmolality and pH measurements on post-treatment media were taken in the cytotoxicity Range-Finder Experiment.
Plating for Survival:
Following adjustment of the cultures to 2 x 105 cells/mL after treatment, samples from these were diluted to 8 cells/mL.
Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells, averaging 1.6 cells/well). The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% 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 105 cells/mL in readiness for plating for 6TG resistance. Samples from these were diluted to 8 cells/mL.
Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well). The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (9 to10 days). Wells containing viable clones were identified by eye using background illumination and counted.
Plating for 6TG Resistance:
At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 105 cells/mL. 6TG (1.5 mg/mL) was diluted 100-fold into these suspensions to give a final concentration of 15 µg/mL. Using a multichannel pipette, 0.2 mL of each suspension was placed into each well of 4 x 96-well microtitre plates (384 wells at 2 x 104 cells/well). Plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (14 to 15 days) and wells containing clones were identified as above and counted.

Statistics:

All calculations were performed by computer using validated software. From the zero term of the Poisson distribution the probable number of clones/well (P) on microtitre plates in which there are EW empty wells (without clones) out of a total of TW wells is given by:
P = -ln (EW/TW).
Cloning Efficiency (CE) in any given culture is therefore:
CE = P/No of cells plated per well
and as an average of 1.6 cells/well were plated on all survival and viability plates,
CE = P/1.6.
Percentage Relative Survival (%RS) in each test culture was determined by comparing plating efficiencies in test and control cultures thus:
%RS = [CE (test)/CE (control)] x 100.
To take into account any loss of cells during the 3 hour treatment period, percentage relative survival values for each concentration of test article were adjusted.

Mutant Frequency (MF) is usually expressed as "mutants per 106 viable cells". In order to calculate this, the cloning efficiencies of both mutant and viable cells in the same culture were calculated:
MF = [CE (mutant)/CE (viable)] x 106.
From the formulae given and with the knowledge that 2 x 104 cells were plated/well for mutation to 6TG resistance,
CE (mutant) = P (mutant)/2 x 104
CE (viable) = P (viable)/1.6
where, in each case, P = -ln (EW/TW).
Therefore,
MF = [P (mutant)/2 x 104] x [1.6/P (viable)] x 106
= {-ln [EW/TW (mutant)]/-ln [EW/TW (viable)]} x 80.
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No marked changes in pH were observed in the Range-Finder at the highest concentrations tested (1282 µg/mL) as compared to the concurrent vehicle controls (measured data not reported).
- Data on osmolality: No marked changes in osmolality were observed in the Range-Finder at the highest concentrations tested (1282 µg/mL) as compared to the concurrent vehicle controls (measured data not reported).

RANGE-FINDING/SCREENING STUDIES (if applicable): In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 40.06 to 1282 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentrations to give >10% relative survival (RS) were 641 µg/mL in the absence of S-9 and 1282 µg/mL in the presence of S-9, which gave 22% and 21% RS, respectively.

STUDY RESULTS
In the Mutation Experiment ten concentrations, ranging from 100 to 1282 µg/mL in the absence of S-9 and from 25 to 1282 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest two concentrations (1000 and 1282 µg/mL) in the absence of S-9 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 750 µg/mL in the absence of S-9 and 1282 µg/mL in the presence of S-9 which gave11% and 49% RS, respectively.

Mutation
Following 3 hour treatment in the absence and presence of S-9 no statistically significant increases in MF, compared to the vehicle control, were observed following treatment with Cyrene™ at any concentration analysed and there were no significant linear trends.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency (see table 2)

HISTORICAL CONTROL DATA: All concentrations were within the upper limit of the historical range generated by the last twenty experiments performed in this laboratory (1.55 to 6.61 in the absence of S-9 and 2.43 to 6.17 in the presence of S-9).

Table 1: Range-Finder Experiment - 3 Hour Treatment in the Absence and Presence of S-9

Concentration	3 Hour Treatment –S‑9	3 Hour Treatment +S‑9
µg/mL	%RS	%RS
0	100	100
40.06	92	84
80.13	71	95
160.3	58	98
320.5	43	109
641	22	79
1282	7	21

Table 2: Mutation Experiment - 3 Hour treatment in the Absence and Presence of S-9

3 Hour Treatment –S-9			3 Hour Treatment +S-9
Concentration	%RS	MF §	Concentration	%RS	MF §
µg/mL			µg/mL
0	100	1.36	0	100	2.24
100	62	2.48 NS	25	97	2.42 NS
200	42	1.65 NS	50	97	5.73 NS
400	29	1.82 NS	75	110	2.34 NS
500	20	0.85 NS	100	105	2.78 NS
600	19	1.54 NS	200	89	3.12 NS
650	17	1.88 NS	400	84	3.61 NS
700	18	1.50 NS	600	69	3.72 NS
750	11	2.20 NS	800	64	3.39 NS
NQO 0.15	53	20.05	1000	52	1.40 NS
NQO 0.2	48	16.55	1282	49	3.32 NS
			B[a]P 2	51	28.99
			B[a]P 3	47	39.59

Linear trend: Not significant (-/ +S-9)

Conclusions:

Cyrene™ has been tested in a valid in vitro mammalian mutagenicity study, conducted according to OECD Test Guideline 476 and in compliance with GLP, using mouse lymphoma L5178Y cells. Cyrene™ did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested for 3 hours up to cytotoxic concentrations in the absence of a metabolic activation system (S9) and up to a concentration equivalent to 10 mM in the presence of metabolic activation system. Appropriate solvent, negative and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Cyrene™ has been tested in a valid bacterial reverse mutation assay, conducted according to OECD Test Guideline 471 (1997) and in compliance with GLP, using Salmonella typhimurium strains S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102. No increase in the number of revertants was observed in any test strain, with or without metabolic activation when tested up to limit concentration. Appropriate positive, negative and solvent controls were added and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test (Harlan Cytotest Cell Research GmbH, 2014d).

Cyrene™ has been tested for its ability to cause chromosome aberrations in cultured peripheral human lymphocytes in an in vitro cytogenicity study, conducted according to OECD Test Guideline 487 and in compliance with GLP. No increase in the number of cells with aberrations was observed either with or without metabolic activation in cultured peripheral human lymphocytes. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of this study (Harlan Cytotest Cell Research GmbH, 2014e).  

Cyrene™ has been tested in a valid in vitro mammalian mutagenicity study, conducted according to OECD Test Guideline 476 and in compliance with GLP, using mouse lymphoma L5178Y cells. Cyrene™ did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested for 3 hours up to cytotoxic concentrations in the absence of a metabolic activation system (S9) and up to a concentration equivalent to 10 mM in the presence of metabolic activation system. Appropriate solvent, negative and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of this study (Covance Laboratories Ltd, 2018c).  

Justification for classification or non-classification

Based on the available data for Cyrene™, no classification for genetic toxicity is required according to Regulation (EC) No 1272/2008.