Octahydro-2H-1-benzopyran-2-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECDTG 471): negative
In vitro micronucleus (OECDTG 487): negative
Genemutation in mammalian cells (OECDTG 490): negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 October, 2012 - 01 February, 2012

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:

(1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

- Dose range finding test:
TA 100 and WP2uvrA (without and with S9): 50, 150, 500, 1500 and 5000 µg/plate

- Experiment 1:
Due to cytotoxicity in TA 100, the following dose levels were used:
TA 98, TA 100, TA 1535 and TA 1537 (without and with S9): 15, 50, 150, 500, 1500 and 5000 µg/plate
WP2uvrA (without and with S9): 50, 150, 500, 1500 and 5000 µg/plate

- Experiment 2:
The test substance dose range was the same as experiment 1:
TA 98, TA 100, TA 1535 and TA 1537 (without and with S9): 15, 50, 150, 500, 1500 and 5000 µg/plate
WP2uvrA (without and with S9): 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: The test substance was immiscible in sterile distllied water but was fully miscible in dimethyl sulphoxide at 50 mg/mL in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.

Negative solvent / vehicle controls:

yes

Remarks:

(50 or 100 µL/plate DMSO)

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: With S9 2-aminoanthracene: 1 µg/plate for TA 100, 2 µg/plate for TA 1535 and TA 1537, 10 µg/plate for WP2uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to detrmine the overall result of the study:
1. A dose-related increase in mutant frequency over the range tested (De Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test item will be considered non-mutagenic (negative) in the test system if the abobe criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test substance activity. Results of this type will be reported as equivocal.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to and including 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES:
- In TA 100 toxicity was observed at 5000 µg/plate, both in the absence and presence of S9 in the dose range finder. In strain WP2uvrA no toxicity was observed.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY (MAIN TEST):
- In all Salmonella strains toxicity was observed at 1500 and 5000 µg/plate in the absence of S9-mix and at 5000 µg/plate in the presence of S9-mix. No toxicity was noted to WP2uvrA dosed in the presence of S9-mix although weakened bacterial background lawns were noted to the same strain in the absence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent the test substance being tested up to and including the maximum recommended dose level of 5000 µg/plate.

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay performed according to OECD 471 guideline and GLP principles.

Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 guideline and according to GLP principles. The test was performed in two independent pre-incubation experiments, both in the absence and presence of S9-mix up to and including 5000 µg/plate. Adequate negative and positive controls were included. Toxicity was observed in all Salmonella strains at concentrations of 1500 and 5000 µg/plate in the absence of S9-mix and at concentration of 5000 µg/plate in the presence of S9-mix. In E.coli no toxicity was observed at the concentration of 5000 µg/plate in the presence of S9-mix and slight toxicity was observed at the concentration of 5000 µg/plate in the absence of S9-mix. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in E. coli strain WP2uvrA, both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 September, 2013 - 28 October, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: cultures prepared from blood of a female donor

Details on mammalian cell type (if applicable):

Peripheral blood lymphocytes were cultured in complete medium (RPMI-1640 containing 15% fetal bovine serum, 2mM L-glutamine, 100 units penicillin, 100 μg/mL streptomycin, and 2% phytohemagglutinin) by adding 0.5 mL heparinized blood to a centrifuge tube containing 5 mL of complete medium. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 44-48 hours.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of Aroclor 1254-induced rats

Test concentrations with justification for top dose:

Dose range finding test:
With and without S9-mix, 4 hr exposure; 20 hr recovery: 0.154, 0.462, 1.54, 4.62, 15.4, 46.2, 154, 462 and 1540 μg/mL
Without S9-mix, 24 hr exposure; 0 hr recovery: 0.154, 0.462, 1.54, 4.62, 15.4, 46.2, 154, 462 and 1540 μg/mL
First cytogenetic test:
With and without S9-mix, 4 hr exposure; 20 hr recovery: 250, 500, 750, 1000 and 1540 μg/mL
Without S9-mix, 24 hr exposure; 0 hr recovery: 100, 250, 500, 750, 1000, 1250 and 1540 μg/mL
The following dose levels were selected for scoring of micronuclei:
With and without S9-mix, 4hr exposure; 20 hr recovery: 750, 1000 and 1540 μg/mL
Without S9-mix, 20 hr exposure; 0 hr recovey: 500, 750 and 1250 μg/mL

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: DMSO was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Without S9: Vinblastine 5, 7.5, and 10 ng/mL for a 24 hours exposure period (as the positive control in the non-activated test system for aneugenicity).

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 44-48 hr
- Exposure duration:
Short-term treatment
Without and with S9-mix: 4 hr treatment, 20 hr recovery/harvest time
Continuous treatment
Without S9-mix: 24 hr treatment/harvest time

ARREST OF CELL DIVISION: 6 μg/mL Cytochalasine B
STAIN: Acridine orange

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 1000/culture (binucleated cells)

DETERMINATION OF CYTOTOXICITY
- The cytostasis/cytotoxicity was determined using the cytokinesis-block proliferation index (CPBI index)

Evaluation criteria:

The test substance was considered positive if it induced a statistically significant and dose-dependent increase the frequency of MN-BN cells (p ≤ 0.05). If only one criterion was met (statistically significant OR dose-dependent increase), the result was considered equivocal. If neither criterion was met, the results were considered to be negative.

Statistics:

Statistical analysis of the percentage of micronucleated cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent micronucleated cells of each treatment group with that of the vehicle control. Due to negative results, the Cochran-Armitage test was not required to measure dose-responsiveness.

Key result

Species / strain:

lymphocytes: human peripheral blood primary culture

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH/osmolality:
The osmolality in treatment medium of the highest concentration tested, 1540 μg/mL, was 394 mmol/kg. The osmolality of the vehicle (DMSO) in the treatment medium was 408 mmol/kg. The osmolality of the test substance dose level in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 20%. The pH of the highest concentration of test substance in treatment medium was 7.5.
- Precipitation: Precipitation in the exposure medium was not observed.

RANGE-FINDING/SCREENING STUDIES: Substantial cytotoxicity [50 to 60% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was not observed at any dose levels in the non-activated and S9-activated 4-hour exposure groups. Substantial cytotoxicity was observed at 1540 μg/mL in the non-activated 24-hour exposure group.

CYTOKINESIS BLOCK
- Distribution of mono-, bi- and multi-nucleated cells:
See "Any other information on results incl. tables"

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
See "Any other information on results incl. tables"

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The percentage of micronucleated cells in the MMC (positive control) group (3.8%) was statistically significant (p ≤ 0.01, Fisher's Exact test). The percentage of micronucleated cells in the CP (positive control) group (1.3%) was statistically significant (p ≤ 0.01, Fisher's Exact test). The percentage of micronucleated cells in the VB (positive control) group (1.1%) was statistically significant (p ≤ 0.01, Fisher's Exact test).
- Negative (solvent/vehicle) historical control data: The results for the positive and vehicle controls indicated that all criteria for a valid assay were met.

ADDITIONAL INFORMATION ON CYTOTOXICITY (MAIN TEST):
At the highest test concentration, 1540 μg/mL without S9, cytotoxicity was 11% relative to the vehicle control. At the highest test concentration, 1540 μg/mL with S9, cytotoxicity was 23% relative to the vehicle control. In the 24-hour exposure group, cytotoxicity was observed at the concentrations of 1250 and 1540 µg/mL. In this group, the highest concentration of 1250 µg/mL was chosen for scoring of micronuclei as the reduction in cytokinesis-blocked proliferation index (CBPI) was 51%.

CONCURRENT CYTOTOXICITY TEST USING Octahydrocoumarin

IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION 4-HOUR TREATMENT, 24-HOUR HARVEST

Treatment Condition	Replicate Culture	Total# ofCells	Countpertotalcells Cellswith#ofnuclei			CBPI1	Cytotoxicity2
µg/mL		Counted	1	2	>2
DMSO	A	500	292	185	23	1.450	-
	B	500	303	175	22
Octahydrocoumarin
250	A	500	306	175	19	1.450	0%
	B	500	290	183	27
500	A	500	279	198	23	1.484	-8%
	B	500	282	196	22
750	A	500	301	178	21	1.468	-4%
	B	500	287	178	35
1000	A	500	305	167	28	1.439	2%
	B	500	308	168	24
1540	A	500	322	155	23	1.400	11%
	B	500	318	165	17
MMC,0.4	A	500	331	164	5	1.313	30%
	B	500	368	125	7
MMC,0.5	A	500	366	126	8	1.281	38%
	B	500	367	127	6

1: CBPI = Cytokinesis-Block Proliferation Index

2: Relative to vehicle control

CONCURRENT CYTOTOXICITY TEST USING Octahydrocoumarin

IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION 4-HOUR TREATMENT, 24-HOUR HARVEST

Treatment Condition	Replicate Culture	Total# ofCells	Countpertotalcells Cellswith#ofnuclei			CBPI1	Cytotoxicity2
µg/mL		Counted	1	2	>2
DMSO	A	500	297	188	15	1.436	-
	B	500	298	186	16
Octahydrocoumarin
250	A	500	333	149	18	1.400	8%
	B	500	302	181	17
500	A	500	315	174	11	1.415	5%
	B	500	302	177	21
750	A	500	321	157	22	1.409	6%
	B	500	310	172	18
1000	A	500	314	169	17	1.388	11%
	B	500	328	159	13
1540	A	500	351	136	13	1.335	23%
	B	500	346	135	19
CP,2.5	A	500	390	107	3	1.256	41%
	B	500	358	141	1
CP,5	A	500	410	90	0	1.203	53%
	B	500	392	103	5
CP,7.5	A	500	424	76	0	1.155	64%
	B	500	424	73	3

1: CBPI = Cytokinesis-Block Proliferation Index

2: Relative to vehicle control

CONCURRENT CYTOTOXICITY TEST USING Octahydrocoumarin

IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION 24-HOUR TREATMENT, 24-HOUR HARVEST

Treatment Condition	Replicate Culture	Total# ofCells	Countpertotalcells Cellswith#ofnuclei			CBPI1	Cytotoxicity2
		Counted	1	2	>2
DMSO	A	500	249	190	61	1.597	-
	B	500	266	183	51
Octahydrocoumarin
100 µg/mL	A	500	240	197	63	1.631	-6%
	B	500	252	188	60
250 µg/mL	A	500	250	211	39	1.583	2%
	B	500	246	214	40
500 µg/mL	A	500	254	204	42	1.578	3%
	B	500	248	214	38
750 µg/mL	A	500	286	193	21	1.453	24%
	B	500	286	210	4
1000 µg/mL	A	500	302	196	2	1.397	34%
	B	500	304	195	1
1250 µg/mL	A	500	375	123	2	1.295	51%
	B	500	334	164	2
1540 µg/mL	A	500	456	43	1	1.096	84%
	B	500	449	51	0
VB,5 ng/mL	A	500	255	181	64	1.619	-4%
	B	500	248	194	58
VB,7.5 ng/mL	A	500	253	186	61	1.615	-3%
	B	500	250	193	57
VB,10 ng/mL	A	500	419	64	17	1.168	72%
	B	500	438	54	8

1: CBPI = Cytokinesis-Block Proliferation Index

2: Relative to vehicle control

MICRONUCLEUS ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH Octahydrocoumarin

IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION DEFINITIVE ASSAY: 4-HOUR TREATMENT, 24-HOUR HARVEST

Treatment	Replicate	Total#	Percentageof Micronucleated	AveragePercent Micronucleated
µg/mL	Culture	ofCells Counted	BinucleatedCellsperculture	Binucleated CellsperDose
DMSO	A	1000	0.2%	0.2%
	B	1000	0.2%
Octahydrocoumarin 750	A	1000	0.3%	0.3%
	B	1000	0.3%
1000	A	1000	0.3%	0.4%
	B	1000	0.5%
1540	A	1000	0.0%	0.1%
	B	1000	0.2%
MMC,0.4	A	1000	3.6%	3.8%**
	B	1000	4.0%

* p ≤ 0.05; ** p ≤ 0.01, Fisher's exact test, relative to the solvent control.

MICRONUCLEUS ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH Octahydrocoumarin

IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION DEFINITIVE ASSAY: 4-HOUR TREATMENT, 24-HOUR HARVEST

Treatment	Replicate	Total#	Percentageof Micronucleated	AveragePercent Micronucleated
µg/mL	Culture	ofCells Counted	BinucleatedCellsperculture	Binucleated CellsperDose
DMSO	A	1000	0.5%	0.4%
	B	1000	0.3%
Octahydrocoumarin 750	A	1000	0.2%	0.2%
	B	1000	0.1%
1000	A	1000	0.3%	0.4%
	B	1000	0.4%
1540	A	1000	0.5%	0.5%
	B	1000	0.4%
CP,5	A	1000	1.3%	1.3%**
	B	1000	1.2%

* p ≤ 0.05; ** p ≤ 0.01, Fisher's exact test, relative to the solvent control.

MICRONUCLEUS ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH Octahydrocoumarin

IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION DEFINITIVE ASSAY: 24-HOUR TREATMENT, 24-HOUR HARVEST

Treatment	Replicate	Total#	Percentageof Micronucleated	AveragePercent Micronucleated
	Culture	ofCells Counted	BinucleatedCellsperculture	Binucleated CellsperDose
DMSO	A	1000	0.3%	0.3%
	B	1000	0.2%
Octahydrocoumarin 500 µg/mL	A	1000	0.2%	0.3%
	B	1000	0.3%
750 µg/mL	A	1000	0.1%	0.1%
	B	1000	0.1%
1250 µg/mL	A	1000	0.4%	0.3%
	B	1000	0.1%
VB,7.5 ng/mL	A	1000	1.2%	1.1%**
	B	1000	1.0%

* p ≤ 0.05; ** p ≤ 0.01, Fisher's exact test, relative to the solvent control.

IN VITRO MICRONUCLEUS TEST USING HUMAN PERIPHERAL BLOOD LYMPHOCYTES (HPBL)

HISTORICAL CONTROL VALUES 2010-2012

NON-ACTIVATED ASSAY

Historical Values	MicronucleatedBinucleatedCells
	Negative Control1	PositiveControls
		MMC2	VB3
Mean	0.263%	3.625%	1.729%
StandardDeviation	±0.166%	±1.273%	±0.853%
Range	0.1-1.0%	2.1-5.9%	1.0-4.9%

S9-ACTIVATED ASSAY

Historical Values	MicronucleatedBinucleatedCells
	Negative Control1	PositiveControl
		CP4
Mean	0.233%	1.510%
StandardDeviation	±0.128%	±0.506%
Range	0.0-0.5%	0.8-2.9%

1  Includes distilled water and dimethylsulfoxide (DMSO)

2  MMC=MitomycinC, 0.125 to 0.8 µg/mL

3  VB=VinblastineSulfate, 5 to 150 ng/mL

4   CP=Cyclophosphamide,1 to 15 µg/mL

Conclusions:

An in vitro micronucleus assay with the substance was performed according to OECD 487 guideline and GLP principles, in cultured peripheral human lymphocytes in one experiments. It is concluded that the substance is not clastogenic or aneugenic in human lymphocytes.

Executive summary:

In an in vitro micronucleus assay, cultured peripheral human lymphocytes were exposed to different concentrations of the substance (dissolved in DMSO), in the presence and absence of S9-mix according to OECD 487 guideline and GLP principles. In the cytogenetic assay, the substance was tested up to and including cytotoxic concentrations of 1540 μg/mL for a 4 h exposure time with a 20 h recovery time in the absence and presence of S9 -mix. The substance was tested up to and including the cytotoxic concentration of 1250 μg/mL for a 20 h exposure time in the absence of S9 -mix. Reliable positive and negative controls were included.

The substance did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix. It is concluded that the substance is not clastogenic or aneugenic in human lymphocytes.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 May, 2016 - 13 June, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro gene mutation study in mammalian cells

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium). Exposure medium: medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium). Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 μg/ml trifluorothymidine (TFT).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3 hours treatment: 5.4, 17, 164, 512 and 1540 μg/mL
Without S9-mix, 24 hours treatment: 5.4, 17, 164, 512 and 1540 μg/mL
Experiment 1:
Without and with S9-mix, 3 hours treatment: 0.55, 1.7, 5.4, 17, 52, 164, 512 and 1540 μg/mL
The following dose levels were selected to measure mutation frequencies at the TK-locus :
Without and with S9-mix, 3 hours treatment: 0.55, 1.7, 5.4, 17, 52, 164, 512 and 1540 μg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 33, 63, 125, 250, 500, 650, 800, 1000, 1250, 1400 and 1540 μg/mL
The following dose levels were selected to measure mutation frequencies at the TK-locus:
Without S9-mix, 24 hours treatment: 33, 63, 125, 250, 500, 650, 1000 and 1540 μg/mL

Vehicle / solvent:

Solvent used: DMSO.
- Justification for choice of solvent: The test item was dissolved in dimethyl sulfoxide. DMSO has been accepted and approved by authorities and international guidelines. No correction was made for the purity/composition of the test item.

Untreated negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 μg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth (SG) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and 32-180 (24 hours treatment).
d) The positive control should demonstrate an absolute increase in the total mutation frequency above the spontaneous background MF (an induced MF (IMF) of at least 300 x 10^-6). At least 40% of the IMF should be reflected in the small colony MF. Furthermore, the positive control should have an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent/control (a small colony IMF of at least 150 x 10^-6).

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Statistics:

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In dose levels after 24 hours of treatment without S9-mix.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and of osmolality: The pH and osmolarity at a concentration of 1540 μg/ml were 7.08 and 0.438 Osm/kg respectively (compared to 7.07 and 0.453 Osm/kg in the solvent control).
- Precipitation: Precipitation in the exposure medium was not observed up to and including the dose level of 1540 μg/mL (= 10 mM).

RANGE-FINDING/SCREENING STUDIES:
No toxicity in the suspension growth was observed up to and including the highest test item concentration of 1540 μg/ml (= 0.01 M) compared to the suspension growth of the solvent control both in the absence of S9-mix and presence of S9-mix after 3 hours of treatment. After 24 hours of treatment without S9-mix, the relative suspension growth was 15% at the test item concentration of 1540 μg/ml compared to the relative suspension growth of the solvent control.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Mutation frequency per 10^6 survivors
- S9-mix + S9-mix
3 hour treatment 24 hour treatment
Mean 894 724 1694
SD 247 200 793
n 81 74 108
Upper control limit
(95% control limits) 1431 1216 4045
Lower control limit
(95% control limits) 356 231 657
SD = Standard deviation
n = Number of observations
Distribution historical positive control data from experiments performed between January 2013 and May 2016.

- Negative (solvent/vehicle) historical control data:
Mutation frequency per 106 survivors
- S9-mix + S9-mix
3 hour treatment 24 hour treatment
Mean 83 75 84
SD 22 24 27
n 161 146 210
Upper control limit
(95% control limits) 128 126 141
Lower control limit
(95% control limits) 37 25 28
SD = Standard deviation
n = Number of observations
Distribution historical negative control data from experiments performed between January 2013 and May 2016.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
First experiment:
No severe toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix.
Second experiment:
The dose levels of 800 and 1000 μg/ml showed similar cell growth delay. Therefore, the dose level of 800 μg/ml was not regarded relevant for mutation frequency measurement.
The dose levels of 1250 to 1540 μg/ml showed similar cell growth delay. Therefore, the dose levels of 1250 and 1400 μg/ml were not regarded relevant for mutation frequency measurement.
The relative total growth of the highest test item was 13% compared to the total growth of the solvent controls.

Conclusions:

A mouse lymphoma assay with the substance was conducted according to OECD 490 guideline and GLP principles. It is concluded that the substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions of the study.

Executive summary:

The mouse lymphoma assay with the substance was conducted according to OECD 490 guideline and GLP principles.

In the first experiment, the test item was tested up to concentrations of 1540 μg/mL (~ 0.01 M, recommended in the guidelines) in the absence and presence of S9-mix. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. In the second experiment, the test item was again tested up to concentrations of 1540 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The Relative Total Growth was 13%. Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses.

In the absence of S9-mix, the substance did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the substance did not induce a significant increase in the mutation frequency. It is concluded that the substance is not mutagenic in the mouse lymphoma L5178Y test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 guideline and according to GLP principles. The test was performed in two independent pre-incubation experiments, both in the absence and presence of S9-mix up to and including 5000 µg/plate. Adequate negative and positive controls were included. Toxicity was observed in all Salmonella strains at concentrations of 1500 and 5000 µg/plate in the absence of S9-mix and at concentration of 5000 µg/plate in the presence of S9-mix. In E.coli no toxicity was observed at the concentration of 5000 µg/plate in the presence of S9-mix and slight toxicity was observed at the concentration of 5000 µg/plate in the absence of S9-mix. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in E. coli strain WP2uvrA, both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

In vitro micronucleus:

In an in vitro micronucleus assay, cultured peripheral human lymphocytes were exposed to different concentrations of the substance (dissolved in DMSO), in the presence and absence of S9-mix according to OECD 487 guideline and GLP principles. In the cytogenetic assay, the substance was tested up to and including cytotoxic concentrations of 1540 μg/mL for a 4 h exposure time with a 20 h recovery time in the absence and presence of S9 -mix. The substance was tested up to and including the cytotoxic concentration of 1250 μg/mL for a 20 h exposure time in the absence of S9 -mix. Reliable positive and negative controls were included.

The substance did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix. It is concluded that the substance is not clastogenic or aneugenic in human lymphocytes.

Mouse lymphoma:

The mouse lymphoma assay with the substance was conducted according to OECD 490 guideline and GLP principles.

In the first experiment, the test item was tested up to and including concentrations of 1540 μg/mL (~ 0.01 M, recommended in the guidelines) in the absence and presence of S9-mix. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. In the second experiment, the test item was again tested up to concentrations of 1540 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The Relative Total Growth was 13%. Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses.

In the absence of S9-mix, the substance did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the substance did not induce a significant increase in the mutation frequency. It is concluded that the substance is not mutagenic in the mouse lymphoma L5178Y test system.

Justification for classification or non-classification

Based on the results of the Ames, in vitro micronucleus and mouse lymphoma studies, the substance does not have to be classified for genotoxicity in accordance with Regulation (EC) No 1272/2008 and its updates.