2-hydroxypropyl [(1R,2S,5R)-2-isopropyl-5-methyl-cyclohexyl] carbonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay/Ames test: negative (equivalent or similar to OECD 471; GLP)

In vitro mammalian cell micronucleus test: negative (OECD 487; GLP)

In vitro mammalian cell gene mutation assay: negative (OECD 476; GLP)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-08-10 to 2016-11-07

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:

2014-09-26

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-09-14

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human (peripheral)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: healthy human donors (non-smoking; not receiving medication)

- Suitability of cells: lymphocytes of the donors have been shown to respond well to stimultation of proliferation with phytohemeagglutinine (PHA) and to positive control substance. All donors had previously established low incidence of micronuclei in their peripheral blood lymphocytes.

- Sex, age and number of blood donors: two female donors (30 and 28 years old, respectively; Experiment 1A and Experiment 2, respectively); one male donor (26 years old; Experiment 1B)

- Whether whole blood or separated lymphocytes were used: blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hours after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (mixture 1:1) supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin, the mitogen PHA, 10 % fetal bovine serum, 10 mM HEPES and the anticoagulant heparin.
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Cytokinesis block (if used):

4 µg/mL cytochalasin B (exposure duration: 20 hours)

Metabolic activation:

with and without

Metabolic activation system:

S9-mix: S9 supernatant, MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM), and NADP (4 mM) in sodium-ortho-phosphate buffer (100 mM, pH 7.4)

Test concentrations with justification for top dose:

Preliminary cytotoxicity test/Experiment 1A: 13.4, 23.4, 40.9, 71.6, 125, 219, 384, 671, 1175, and 2056 µg/mL (with and without metabolic activation; 4 hours)
Experiment 1B: 82.9, 108, 140, 182, 237, 308, and 400 µg/mL (with metabolic activation; 4 hours)
Experiment 2: 2.6, 4.5, 8.0, 13.9, 24.4, 42.6, 74.6, 131, 229, and 400 µg/mL (without metabolic activation; 20 hours)
Please refer to the field "Rationale for test conditions" below.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (culture medium with 0.5 % vehicle)
- 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 0.5 % DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: demecolcin

Details on test system and experimental conditions:

DETERMINATION OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI)
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison with the controls by counting 500 cells per culture.

EXPERIMENTAL DESIGN
1) Pre-experiment/Experiment 1A/1B (pulse treatment):
- the preliminary cytotoxicity test was designated Experiment 1A, since the cultures fulfilled the requirements for cytogenetic evaluation.
- 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control.
- all cell cultures were set up in duplicate.
- exposure time was 4 hours (with and without S9 mix) and the preparation interval was 40 hours after start of the exposure.
- NOTE: the experimental part with S9 mix was repeated due to an increase in micronucleate cells and was designated as Experiment 1B.

- about 48 hours after seeding 2 blood cultures (10 mL each) were set up for each test item concentration.
- culture medium was replaced with serum-free medium containing the test item.
- for the treatment with metabolic activation 50 μL S9 mix/mL culture medium was added.
- after 4 hours the cells were spun down by centrifugation and the supernatant was discarded.
- cells were resuspended in and washed with "saline G" (pH 7.2, containing NaCl, KCl, glucose • H2O, Na2HPO4 • 2 H2O and KH2PO4).
- washing procedure was repeated once.
- cells were resuspended in complete culture medium with 10 % FBS (v/v) and cultured for a 16-hour recovery period.
- Cytochalasin B (4 μg/mL) was added and the cells were cultured another approx. 20 hours until preparation.

- Experiment 2 (continuous treatment):
- about 48 hours after seeding 2 blood cultures (10 mL each) were set up for each test item concentration.
- culture medium was replaced with complete medium (with 10 % FBS) containing the test item.
- after 20 hours the cells were spun down by centrifugation and the supernatant was discarded.
- cells were re-suspended in and washed with "saline G".
- washing procedure was repeated once.
- cells were re-suspended in complete culture medium containing 10 % FBS (v/v).
- Cytochalasin B (4 μg/mL) was added and the cells were cultured another approx. 20 hours until preparation.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- cultures were harvested by centrifugation 40 hours after beginning of treatment.
- cells were spun down by centrifugation and the supernatant was discarded.
- cells were re-suspended in approx. 5 mL saline G and spun down by centrifugation.
- cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes.
- 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts:1 part) was added to the hypotonic solution and cells were resuspended.
- After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold.
- slides were prepared by dropping the cell suspension in fresh fixative onto a microscope slide.
- cells were stained with Giemsa.
- evaluation of the slides was performed using microscopes with 40 x objectives.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976)*. The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus.

NUMBER OF CELLS EVALUATED:
- at least 1000 binucleate cells/culture were scored for cytogenetic damage (% micronucleated cells)
- 500 cells/culture were scored for CBPI (% cytostasis)

*Reference:
- Countryman P.I. and Heddle J.A. (1976) The production on micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Research, 41, 321-332.

Rationale for test conditions:

In the preliminary cytotoxicity test, the highest treatment concentration in this study, 2056 μg/mL was chosen with regard to the purity (97.3%) of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.
In the pre-test for toxicity, phase separation of the test item was observed at the end of treatment at 219 μg/mL and above in the absence of S9 mix and at 384 μg/mL and above in the presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment 1A. The experimental part with S9 mix was repeated due to an increase in micronucleate cells (Experiment 1B).
Clear toxic effects were observed in Experiment 1A after 4 hours treatment with 219 μg/mL and above in the absence of S9 mix and with 384 μg/mL in the presence of S9 mix. Additionally, clear toxic effects were observed in Experiment 1B in the presence of S9 mix with 308 μg/mL and above. Therefore, 400 μg/mL (without S9 mix) were chosen as top concentration in Experiment 2.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be negative if, in all of the experimental conditions examined:
− none of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− there is no concentration-related increase
− the results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% confidence interval)

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be positive if, in any of the experimental conditions examined:
− at least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− the increase is concentration-related in at least one experimental condition
− the results are outside the range of the laboratory historical solvent control data (95 % confidence interval)

Statistics:

Chi square test (α < 0.05).

Key result

Species / strain:

lymphocytes: human (peripheral)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

NOTE: the following concentrations were evaluated for micronucleated cells:
Experiment 1A :23.4, 40.9, and 71.6 µg/mL (without metabolic activation; 4 hour exposure)
Experiment 1A :71.6, 125, and 219 µg/mL (with metabolic activation; 4 hour exposure)
Experiment 1B: 140, 182, and 237 µg/mL (with metabolic activation; 4 hour exposure)
Experiment 2: 24.4, 42.6, and 74.6 µg/mL (without metabolic activation; 20 hour exposure)

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no relevant influence on pH was observed
- Effects of osmolarity: no relevant influence on osmolarity was observed
- Phase separation:
Experiment 1A: phase separation of the test item in the culture medium was observed at 219 μg/mL and above in the absence of S9 mix and at 384 μg/mL and above in the presence of S9 mix at the end of treatment.
Experiment 1B: no phase separation was observed.
Experiment 2: phase separation occurred in the absence of S9 mix at 400 μg/mL and above at the end of treatment.

INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI
In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration. Concentrations showing clear cytotoxic effects, however, were not evaluable for cytogenetic damage.

CYTOGENETIC:
Experiment 1A and 2: in the absence of S9 mix no relevant increases in the number of micronucleate cells were observed after treatment with the test item.

In Experiment 1A in the presence of S9 mix, the highest evaluated concentration (219 μg/mL) showed a statistical significant increase in the number of micronucleate cells of 1.30 % (value exceeded historical control range: 0.08 – 1.20 %). In Experiment 1B the finding could not be confirmed. Therefore, the finding of Experiment 1A was not reproducible and can be considered as biologically irrelevant.

Please also refer for further information on results to the field "Attached background material" below.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Please refer to the field "Any other information on results incl. tables" below

Historical laboratory control data

Percentage of micronucleated cells in human lymphocyte cultures (2014-2015)

Solvent Control without S9
Micronucleated cells in %
	Pulse treatment (4/40)	Continous treatment (20/40)
No. of experiments	50*	54**
Mean	0.61	0.55
95% Ctrl limit	0.07 - 1.15	0.05 - 1.05
1x SD	0.27	0.25
2x SD	0.54	0.50
Min	0.15	0.05
Max	1.25	1.43

 

*Aqueous solvents – 23 Experiments; Organic solvents – 27 Experiments

**Aqueous solvents – 24 Experiments; Organic solvents – 30 Experiments

Solvent Control with S9
Micronucleated cells in %
	Pulse treatment (4/40)
No. of experiments	67*
Mean	0.64
95% Ctrl limit	0.08 - 1.20
1x SD	0.28
2x SD	0.56
Min	0.15
Max	1.35

 *Aqueous solvents – 24 Experiments; Organic solvents – 43 Experiments

 

Aqueous solvents: DMEM/Han’s F12, Deionised water (10% v/v)

Organic solvents: DMSO (0.5 or 1.0%), Acetone, Ethanol and THF (0.5%)

Positive Control without S9
Micronucleated cells in %
	Pulse treatment (4/40)	Continous treatment (20/40)
	MMC	Demecolcin
No. of experiments	50	54
Mean	11.66	3.55
95% Ctrl limit	1.48 - 21.85	1.69 - 5.41
1x SD	5.09	0.93
2x SD	10.18	1.86
Min	4.15	2.10
Max	24.00	6.40

Positive Control with S9
Micronucleated cells in %
	Pulse treatment (4/40)
	CPA
No. of experiments	81
Mean	4.80
95% Ctrl limit	0.88 - 8.73
1x SD	1.96
2x SD	3.92
Min	2.25
Max	11.30

Conclusions:

The substance tested non-clastogenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.

Executive summary:

An in vitro mammalian cell micronucleus test was performed with the test item dissolved in DMSO using peripheral human lymphocytes. The test procedure was according to the method described in OECD guideline 487 (2014).

The assay was performed in three independent experiments with the following test concentrations and exposure durations:

Preliminary cytotoxicity test/Experiment 1A: 13.4, 23.4, 40.9, 71.6, 125, 219, 384, 671, 1175, and 2056 µg/mL (with and without metabolic activation; 4 hours)

Experiment 1B: 82.9, 108, 140, 182, 237, 308, and 400 µg/mL (with metabolic activation; 4 hours)

Experiment 2: 2.6, 4.5, 8.0, 13.9, 24.4, 42.6, 74.6, 131, 229, and 400 µg/mL (without metabolic activation; 20 hours)

Positive and solvent controls were run concurrently.

The cells were prepared 40 hours after start of treatment with the test item. During the preparation period cytochalasin B had been added to the cell cultures to ensure that there were binucleate cells to be evaluated for micronuclei.

In each experimental group two parallel cultures were analysed. At least 1000 binucleate cells per culture were scored for cytogenetic damage (% micronucleated cells). To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis.

In Experiment 1A, phase separation of the test item in the culture medium was observed at 219 μg/mL and above in the absence of S9 mix and at 384 μg/mL and above in the presence of S9 mix at the end of treatment. In addition, phase separation occurred in Experiment 2 in the absence of S9 mix at 400 μg/mL and above at the end of treatment. In Experiment 1B no phase separation was observed. No relevant influence on osmolarity or pH was observed.

The following concentrations were evaluated for cytogenetic damage:

Preliminary cytotoxicity test/Experiment 1A: 23.4, 40.9, and 71.6 µg/mL (without metabolic activation; 4 hours)

Preliminary cytotoxicity test/Experiment 1A: 71.6, 125, and 219 µg/mL (with metabolic activation; 4 hours)

Experiment 1B: 140, 182, and 237 µg/mL (with metabolic activation; 4 hours)

Experiment 2: 24.4, 42.6, and 74.6 µg/mL (without metabolic activation; 20 hours)

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration. Concentrations showing clear cytotoxic effects, however, were not evaluable for cytogenetic damage.

In Experiment 1A and 2 in the absence of S9 mix no relevant increases in the number of micronucleate cells were observed after treatment with the test item.

In Experiment IA in the presence of S9 mix, the highest evaluated concentration (219 μg/mL) showed a statistical significant increase in the number of micronucleate cells of 1.30 % (value exceeded historical control range (0.08 – 1.20 %)). In Experiment 1B the finding could not be confirmed. Therefore, the finding of Experiment 1A was not reproducible and can be considered as biologically irrelevant.

The positive and negative controls were considered to be valid.

The substance tested non-clastogenic under the conditions of the study.

According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance is considered to have not a mutagenic potential.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1993-10-26 to 1993-11-12

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Deviations from the OECD 471 (1997): toxic effects not appropriately considered in dose setting, occasionally leading to only 4 analysable concentrations. Unusual solvent and vehicle control values for TA 1535

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983-05-26

Deviations:

yes

Remarks:

toxic effects not appropriately considered in dose setting, occasionally leading to only 4 analysable concentrations. Unusual solvent and vehicle control values for TA 1535

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 1987-12-07

Type of assay:

bacterial reverse mutation assay

Target gene:

TA1537: his C 3076
TA98 and TA1538: his D 3052
TA1535 & TA100: his G 46

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (containing 10 % S9): distilled water (0.335 mL); phospahte buffer (0.2 M, pH 7.4; 0.5 mL); NADP (0.1 M; 0.04 mL); glucose-6-phosphate (1 M; 0.005 mL); MgCl2/KCl (0.4 M/1.65M; 0.02 mL; S9 fraction (0.1 mL)

Test concentrations with justification for top dose:

Experiment 1: 15, 50, 150, 500, 1500, and 5000 µg/plate (without metabolic activation)
Experiment 1: 50, 150, 500, 1500, and 5000 µg/plate (with metabolic activation)
Experiment 2: 5, 15, 50, 150, 500, and 1500 µg/plate (without metabolic activation)
Experiment 2: 15, 50, 150, 500, and 1500 µg/plate (with metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell titer (viable cells/mL):
Experiment 1 (with and without metabolic activation):
TA1535: 3.2 x10^9 (316, 321, 320)
TA1537: 1.2 x 10^9 (115, 126, 120)
TA1538: 0.7 x 10^9 (57,72,75; without S9)(64, 82, 72; with S9)
TA98: 3.3 x 10^9 (314, 335, 332)
TA100: 1.4 x 10^9 (118, 148, 167)

Experiment 2 (with and without metabolic activation):
TA1535: 3.5 x10^9 (327, 375, 357)
TA1537: 1.0 x 10^9 (87, 103, 107)
TA1538: 0.7 x 10^9 (64, 64, 76)
TA98: 3.5 x 10^9 (363, 336, 341; without S9)(363, 336, 342; with S9)
TA100: 3.0 x 10^9 (285, 316, 294)

MAIN EXPERIMENT (two independent experiments)
- Exposure duration: 48 hours at 37 °C in the dark
- Number of replications: 3 replicates for each experimental point
- the number of revertant colonies (his+ revertants) were counted and the plates were examined for the existence of a normal background lawn and/or precipitates as well as microscopically for microcolony growth.
- when there is any question about the nature of colonies scored as revertants and when positive mutagenic results are obtained, the genotype of revertant colonies are spot-checked by picking and streaking on histidine free plates.

DETERMINATION OF CYTOTOXICITY
An initial toxicity test was performed with the test item.

Rationale for test conditions:

The test concentrations were chosen according to an initial toxicity test performed with the test item.

Evaluation criteria:

not specified

Statistics:

χ²-test

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Please refer to the field "Additional information" below

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

MAIN STUDY:
The number of spontanoeus revertants observed using each of the five strains was very close to those previously established in the laboratory and was within the range obtained by Ames et al. (1975)* as well as reported by De Serres and Shelby (1979)*.

The test substance did not increase the spontaneous mutation frequency of the tester strains both in the absence and presence of a metabolic activation system. Similarly the estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level, using a χ²-test (Mohn and Ellenberger, 1977)*, did not reveal a significant effect at any of the test points.

INFORMATION ON CYTOTOXICITY:
- with metabolic activation: bacteriotoxic effects were observed with all strains at 5000 µg/plate
- without metabolic activation: bacteriotoxic effects were observed with strains TA1537, TA1538, and TA98 at 500 µg/plate, with strain TA1535 at 1500 and with TA100 at 5000 µg/plate.

Please also refer to the field "Attached background material" below.

*References:
- Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 31: 347 - 364.
- De Serres, F.J., and M.D. Shelby (1979) Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay, Mutation Res., 64: 9 - 165.
- Mohn, G.R. and J. Ellenberger (1977) The use of E. coli K12/343/113 (lambda) as a multi-purpose indicator strain in various mutagenicity testing procedures, in: B.J. Kilbey (Ed.), Handbook of mutagenicity test procedures, Elsevier, Amsterdam, pp. 95-118.

Conclusions:

The substance tested non-mutagenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.

Executive summary:

A reliable bacterial reverse mutation assay equivalent or similar to OECD 471 (1983) was conducted. Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were treated with the substance using the plate incorporation method at five or six dose levels in triplicate with and without metabolic activation. Two experiments were conducted with the following concentrations:

Experiment 1: 15, 50, 150, 500, 1500 and 5000 µg/plate (without metabolic activation)

Experiment 1: 50, 150, 500, 1500 and 5000 µg/plate (with metabolic activation)

Experiment 2: 5, 15, 50, 150, 500 and 1500 µg/plate (without metabolic activation)

Experiment 2: 15, 50, 150, 500 and 1500 µg/plate (with metabolic activation)

In the concentration range investigated, the test item did not show mutagenic activity with and without a metabolic activation system.

In the presence of the metabolizing system bacteriotoxic effects were observed with all strains at 5000 µg/plate. Furthermore, in the absence of

the metabolizing system bacteriotoxic effects were observed with strains TA1537, TA1538, and TA98 at 500 µg/plate, with strain TA1535 at 1500 amd with TA100 at 5000 µg/plate.

Thus, the substance tested non-mutagenic under the conditions of this study.

According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-08-11 to 2016-11-15

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)

Version / remarks:

2016-07-29

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-09-14

Type of assay:

other: in vitro mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany
- Suitability of cells: the high proliferation rate and a good cloning efficiency of untreated cells (as a rule more than 50 %) both necessary for appropriate performance of the study, recommended the use of this cell line.
- Doubling time: 12 - 16 hours in stock cultures
- Methods for maintenance in cell culture: thawed stock cultures were propagated at 37 °C in plastic flasks. About 2-3×10^6 cells were seeded into each flask with MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin and amphotericin B. The cells were sub-cultured once or twice weekly. All incubations were done at 37 °C with 1.5 % carbon dioxide (CO2) in humidified air.
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media: for seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin, 10% FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix: S9 supernatant, MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM), and NADP (4 mM) in sodium-ortho-phosphate buffer (100 mM, pH 7.4)

Test concentrations with justification for top dose:

Pre-experiment: 16.1, 32.1, 64.3, 128.5, 257.0, 514.0, 1028.0, and 2056.0 µg/mL (4 hour treatment; with and without metabolic activation)
Experiment 1: 4.0, 8.0, 16.0, 32.0, 48.0, and 64.0 µg/mL (4 hour treatment; without metabolic activation)
Experiment 1: 16.0, 32.0, 64.0, 128.0, 192.0, and 256.0 µg/mL (4 hour treatment; with metabolic activation)
Experiment 2: 80.0, 120.0, 160.0, 200.0, 240.0, and 260.0 µg/mL (4 hour treatment; with metabolic activation)
Please also refer for information on the justification for the top dose to the field "Rationale for test conditions" below.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (final concentration in the culture medium: 0.5 % (v/v))
- Justification for choice of solvent/vehicle: the solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

PRE-TEST ON TOXICITY
- a pre-test was performed in order to determine the concentration range for the mutagenicity experiment.
- general culture conditions and experimental conditions were the same as described for the mutagenicity experiment below.
- pre-experiment was performed in the presence and absence (4 hour treatment) of metabolic activation.
- test medium was checked for precipitation or phase separation at the beginning and at the end of treatment (4 hours) prior to removal to the test item.
- colony forming ability of approx. 500 single cells (duplicate cultures/concentration level) after treatment with the test item was observed and compared to the controls.
- toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

- osmolarity and the pH-value were determined in culture medium of the solvent control and of the maximum concentration in the pre-experiment without metabolic activation.

MAIN EXPERIMENT - Experiment 1 and 2
Seeding:
- two to four days after sub-cultivation stock cultures were trypsinized at 37 °C.
- then, complete culture medium with 10% FBS was added and a single cell suspension was prepared.
- trypsin concentration for all sub-culturing steps was 0.2% in saline.
- prior to the trypsin treatment the cells were rinsed with PBS.
- approx. 0.7 to 1.2×10^7 were seeded..
- cells were grown for 24 hours prior to treatment.

Treatment:
- after 24 hours the medium was replaced with serum-free medium containing the test item, solvent or positive controls, either without or with S9 mix.
- 4 hours after treatment, the medium was replaced with complete medium following washing steps with "saline G".
- after the end of treatment the cells were trypsinised as described above and sub-cultivated.
- at least 2.0×10^6 cells/experimental point (concentration series plus controls) were subcultured in medium.
- two flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (cloning efficiency I) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.
- colonies used to determine the cloning efficiency I were fixed and stained 6 to 8 days after treatment as described below.
- three or four days after first sub-cultivation approx 2.0×10^6 cells/experimental point were sub-cultivated in medium.
- following the expression time of 7 days five cell culture flasks were seeded with about 4 to 5×10^5 cells each in medium containing 6-TG.
- two flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (cloning efficiency II).
- cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days.
- colonies were stained with 10% methylene blue in 0.01% KOH solution.
- stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

NUMBER OF REPLICATIONS: duplicates

Rationale for test conditions:

In the pre-experiment a relevant cytotoxic effect (relative cloning efficiency of 50% or below) was observed at 64.3 μg/mL and above in the absence of metabolic activation and at 257.0 μg/mL and above in the presence of metabolic activation. Furthermore, phase separation occurred at 257.0 μg/mL and above after 4 hours treatment with and without metabolic activation.
The dose range of the first main experiment was set according to data generated in the pre-experiment. The dose range of the second main experiment was based on data generated in the first main experiment.

Evaluation criteria:

A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups (significance if p-value < 0.05). Both, biological and statistical significance was considered together.
A t-test was performed to evaluate a significant increase of the mutation frequency at test points exceeding the 95% confidence interval (significance if p-value < 0.05).

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Please refer to the field "Additional information on results" below.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

NOTE: the main experiments were evaluated at the following concentrations with replicate:
Experiment 1: 4.0, 8.0, 16.0, 32.0, and 48.0 µg/mL (4 hour treatment; without metabolic activation)
Experiment 1: 16.0, 32.0, 64.0, and 128.0 µg/mL (4 hour treatment; with metabolic activation)
Experiment 2: 80.0, 120.0, and 160.0 µg/mL (4 hour treatment; with metabolic activation)

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH (pre-experiment): no relevant shift of pH of the medium even at the maximum concentration of the test item.
- Effects of osmolarity (pre-experiment): no relevant shift of osmolarity of the medium even at the maximum concentration of the test item.

- Phase separation:
pre-experiment: phase separation occurred at 257.0 μg/mL and above after 4 hours treatment with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment a relevant cytotoxic effect (relative cloning efficiency of 50% or below) was observed at 64.3 μg/mL and above in the absence of metabolic activation and at 257.0 μg/mL and above in the presence of metabolic activation.

INFORMATION ON CYTOTOXICITY (MAIN EXPERIMENT):
- Experiment 1: relevant cytotoxic effects (cloning efficiency I below 50%) occurred at 48.0 μg/mL without metabolic activation. In the presence of metabolic activation no relevant cytotoxicity was noted at the highest analysable concentration of 128.0 μg/mL. Exceedingly severe cytotoxicity occurred at the next higher concentration of 192 μg/mL. To cover the toxic range with metabolic activation a second experiment was performed.
Experiment 2: cytotoxicity was noted at 120 μg/mL with metabolic activation. The recommended toxic range of approx. 10-20% relative adjusted cloning efficiency I was covered.

GENOTOXICTY (MAIN EXPERIMENT):
No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration.

The 95% confidence interval was exceeded at several concentrations in culture I of the first experiment with and without metabolic activation. However, the mutation frequency of culture II remained within the confidence interval at all evaluated concentrations. A t-test run at any one of the experimental points exceeding the 95% confidence interval was not-significant at all occasions with a single exception. A significant t-test was noted at 64.0 μg/mL with metabolic activation. This result however, was judged as biologically irrelevant as it was not reproduced at any other, even higher, concentration and there was no dose-dependent increase as indicated by the non-significant trend test.

No significant dose dependent trend of the mutation frequency was determined in experiment 1 and 2 (linear regression analysis (least squares)).

In the main experiment with and without S9 mix the range of the solvent controls was from 15.9 up to 30.7 mutants/10^6 cells; the range of the groups treated with the test item was from 13.9 up to 47.1 mutants/10^6 cells. The highest solvent control value of 30.7 mutants/10^6 cells exceeded the 95% confidence interval but the mutation frequency of the parallel culture and the mean value of both cultures (25.0 and 30.7, equal to a mean of 27.9) was fully acceptable.

Ethylmethane sulfonate (300 μg/mL) and 7,12-dimethylbenz(a)anthracene (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

HISTORICAL CONTROL DATA
Please refer to the field "Any other information on results incl. tables" below

Historical Data 

2012 – 2015
Number of mutant colonies per 10^6cells
without metabolic activation (4 hours treatment time)
	Positive control ethylmethane sulfonate 150 and 225 µg/mL	Solvent control (medium, acetone, water, DMSO, ethanol, THF)
Range:	53.9 - 889.0	1.6 - 42.8
Mean value:	153.0	15.0
Standard deviation:	88.5	7.4
95% confidence interval	--	0.2 - 29.7
Number of studies:	147	147
with metabolic activation (4 hours treatment)
	Positive control DMBA 1.1 and 2.2 µg/mL	Solvent control (medium, acetone, water, DMSO, ethanol, THF)
Range:	59.6 - 2042.6	2.4 - 44.2
Mean value:	424.6	14.6
Standard deviation:	291.4	7.0
95% confidence interval	--	0.6 - 28.7
Number of studies:	142	142

The 95% confidence interval is derived from the mean value plus/minus 2 times the standard deviation.

Conclusions:

The substance tested non-mutagenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.

Executive summary:

An in vitro mammalian cell gene mutation assay was performed with the test item dissolved in DMSO using Chinese hamster lung fibroblasts (V79). The test procedure was according to the method described in OECD guideline 476 (2016). The assay was performed in two independent experiments, using two parallel cultures/concentration. The first main experiment was performed with and without metabolic activation and a treatment period of 4 hours (test item concentrations: 4.0, 8.0, 16.0, 32.0, 48.0, and 64.0 µg/mL (without S9) or 16.0, 32.0, 64.0, 128.0, 192.0, and 256.0 µg/mL (with S9)). The second experiment was performed with a treatment period of 4 hours in the presence of metabolic activation (test item concentrations: 80.0, 120.0, 160.0, 200.0, 240.0, and 260.0 µg/mL). Solvent control and positive controls were also run concurrently.

Relevant cytotoxic effects occurred in the first experiment at 48.0 μg/mL without metabolic activation. In the presence of metabolic activation no relevant cytotoxicity was noted at the highest analysable concentration of 128.0 μg/mL. Exceedingly severe cytotoxicity occurred at the next higher concentration of 192 μg/mL. In the second experiment cytotoxicity was noted at 120 μg/mL with metabolic activation. No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration.

The substance tested non-mutagenic under the conditions of the study.

According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

The substance was not observed to be mutagenic in a reliable bacterial reverse mutation assay (equivalent or similar to OECD 471), an in vitro mammalian cell micronucleus test (OECD 487) or an in vitro mammalian cell gene mutation assay (OECD 476).

.

Justification for classification or non-classification

Genetic toxicity in vitro

The test substance should be considered void of genotoxicity based on a bacterial reverse mutation assay (equivalent or similar OECD 471), an in vitro mammalian cell micronucleus test (OECD 487) and an in vitro mammalian cell gene mutation assay (OECD 476). Thus, the test substance has no mutagenic potenital and does not require classification according to Regulation (EC) No 1272/2008 and subsequent adaptations.