Registration Dossier

Administrative data

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 TG 471) (Harlan, 2014e).

Cytogenicity in mammalian cells: negative with and without metabolic activation in cultured peripheral human lymphocytes (OECD TG 487) (Harlan, 2014f).

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

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 May - 12 Jun 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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:
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. certificate)
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:
Negative with and without metabolic activation.
Executive summary:

Cyrene™ was tested in a valid bacterial reverse mutation assay, according to the OECD TG 471 (1997), and under 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.

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:
other: OECD guideline 487, adopted 26 September 2014
Deviations:
no
Qualifier:
according to
Guideline:
other: EU method B.49
Deviations:
no
GLP compliance:
yes (incl. certificate)
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:
Negative with and without metabolic activation.
Executive summary:

Cyrene™ was tested for ability to cause chromosome aberrations in cultured peripheral human lymphocytes according to OECD TG 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.  

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)
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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

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 (see following table).

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

No marked changes in osmolality or 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).

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 were750 µg/mL in the absence of S-9 and 1282 µg/mL in the presence of S-9 which gave11% and 49% RS, respectively (see following table).

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)

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. 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).

Conclusions:
It is concluded that Cyrene™ did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested for 3 hours up to toxic concentrations in the absence of a rat liver metabolic activation system (S-9) and up to a concentration equivalent to 10 mM in the presence of S-9.
Executive summary:

Cyrene™ was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3 hour treatment incubation period was used for each experiment.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from40.06 to 1282µg/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.

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 concentrations analysed to determine viability and 6TG resistance were750 µg/mL in the absence of S-9 and 1282 µg/mL in the presence of S-9 which gave11% and 49% RS, respectively.

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by 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.

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. 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).

It is concluded that Cyrene™ did not induce mutation at the hprtlocus of L5178Y mouse lymphoma cellswhen tested for 3 hours up to toxic concentrationsin the absence ofa rat liver metabolic activation system (S-9)and up to a concentration equivalent to 10 mM in the presence of S-9.

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, according to the OECD TG 471 (1997), and under 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, 2014e).

Cyrene™ has been tested for ability to cause chromosome aberrations in cultured peripheral human lymphocytes according to OECD TG 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, 2014f).  

Cyrene™ has been tested for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells in a study conducted according to OECD TG 476 and in compliance with GLP. The test substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested for 3 hours up to toxic concentrations in the absence of a rat liver metabolic activation system (S-9) and up to a concentration equivalent to 10 mM in the presence of S-9 (Covance, 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.