(E)-3-methyl-5-cyclopentadecen-1-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test: non mutagenic (OECD 471, GLP, K, rel. 1) up to 5000 µg/plate in S. typhimurium TA 1537, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA.

- ML/TK test: non mutagenic (OECD 476, GLP, K, rel. 1) up to cytotoxicity limit.

- CA/HL test: non clastogenic (OECD 473, GLP, K, rel. 1) up to cytotoxicity limit

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2005-06-1 to 2005-08-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 471 and in compliance with GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 2nd December 2002. Signed on 13 February 2003.

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene for Salmonella and tryptophan gene for E.coli

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from Sprague-Dawley rats treated with phenobarbitone/-naphtoflavone

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
Range-finding test: 50, 150, 500, 1500 and 5000 μg/plate.
Main test: 50, 150, 500, 1500 and 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test substance immiscible in water. Well known solvent/vehicle not reacting with the test substance.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

See table 7.6.1/1

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

See table 7.6.1/1

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

With S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: ca. 48 hours at 37°C

NUMBER OF REPLICATIONS: triplicate plates per dose level

DETERMINATION OF CYTOTOXICITY
- Method: growth assessment of the bacterial background lawn

OTHER EXAMINATIONS:
- Other: Observations of precipitate of the test substance

OTHER: ACCEPTANCE CRITERIA: The reverse mutation assay was considered valid if the following criteria were met:
1. All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (according to historical control 2003 & 2004).
2. The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
3. All tester strain cultures should be in the approximate range of 1 to 9.9 billion bacteria per ml.
4. Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
5. There should be a minimum of four non-toxic test material dose levels.
6. There should be no evidence of excessive contamination.

Rationale for test conditions:

Tested up to limit concentrations

Evaluation criteria:

Dose-related increase in revertant frequency over the dose range tested and/or reproducible at one or more concentrations in at least one bacterial strain with or without metabolic activation.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

none

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not applicable
- Effects of osmolality: not applicable
- Evaporation from medium: Test material vapour pressure (0.04 Pa) is too low to expect a significant effect of evaporation on test results. Test material is not classified as volatile according to the criteria of the Directive 1999/13/EC.
- Water solubility: Test material was solubilized in DMSO to improve solubility
- Precipitation: a precipitate (oily in appearance) was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: the test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-).

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and positive controls. The comparison was made with the historical control ranges for 2003 and 2004 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: the test material caused no visible reduction in the growth of the bacterial background lawn at any dose level, although slight decreases in revertant colony frequency were noted to several of the Salmonella strains at the upper dose level

Remarks on result:

mutagenic potential (based on QSAR/QSPR prediction)

Preliminary toxicity Test:

The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

 

The number of revertant colonies for the preliminary toxicity test were:

Metabolic Activation	Strain	Dose (μg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	133	120	141	138	142	136	133	120	107	120P	107P
+	TA100	87	82	98	94	113	87	94	126	89	80P	76P
-	WP2uvrA-	21	18	18	22	15	22	25	20	17	25P	16P
+	WP2uvrA-	29	15	21	24	17	13	18	22	26	22P	27P

Mutation test:

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 (See Graphs & Tables of results in “Attached background material”) and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5 (See Graphs & Tables of results in "Attached background material").

The test material caused no visible reduction in the growth of the bacterial background lawn although a slight decrease in the frequency of revertant colonies was noted to the majority of the Salmonella strains at the upper dose levels. The test material was, therefore, tested up to the maximum recommended dose level of 5000ug/plate. A precipitate (oily in appearance) was observed at and above 1500ug/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Conclusions:

The test item is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA-.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E.coli strain WP2 uvrA^- were exposed the test material diluted in DMSO both in the presence and absence of a metabolic activation system (10% liver S9 in standard co-factors) using the plate incorporation method. The dose range for the range-finding test was determined in a preliminary toxicity assay and was 15 to 5000 µg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn although slight decrease in the frequency of revertant colonies was noted to the majority of the Salmonella strains at the upper dose levels. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A precipitate (oily in appearance) was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

 

Under the test condition, the test material is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA^-.

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

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 1995-04-24 to 1995-08-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 473 and in compliance with GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 31 January 1994. Signed on 16 March 1994

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Fresh whole blood cultures
- Type and identity of media: Eagle's MEM, supplemented with sodiumbicarbonate, L-glutamine, penicillin, streptomycin, amphotericin B and 15% FCS.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data but not relevant
- Periodically checked for karyotype stability: no data but not relevant
- Periodically "cleansed" against high spontaneous background: not relevant but all donors used are validated for spontaneous aberration rate

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from male Sprague-Dawley rats receiving intraperitoneally 500 mg/kg bw of Aroclor 1254

Test concentrations with justification for top dose:

- First experiment: +S9: 17.81 to 2280 µg/mL (10mM), -S9: 4.45 to 71.25 µg/mL.
- Second experiment (20h recovery): + S9: 17.81 to 213.8 µg/mL, -S9: 4.45 to 53.44 µg/mL.
- Second experiment (44h recovery): + S9: 35.63 to 213.8 µg/mL, -S9: 8.91 to 53.44 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: test substance not sufficiently water soluble

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

500 µg/mL in DMSO

Positive control substance:

ethylmethanesulphonate

Remarks:

Without S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

25 µg/mL in culture medium without serum

Positive control substance:

cyclophosphamide

Remarks:

With S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Preincubation period: Cells cultured for 48 hours before exposure.
- Exposure duration: Experiment 1: 4h (with S9), 20h (without S9)
Experiment 2: 4h (with S9), 20h and 44h (without S9)
- Expression time (cells in growth medium): Experiment 1: 16h (after wash-off S9)
Experiment 2: 16h and 40h (after S9 wash-off S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours in Experiment 1, 20 and 44 hours in Experiment 2.

SPINDLE INHIBITOR (cytogenetic assays): Demecolcine (Colcemid) 0.1 µg/mL
STAIN (for cytogenetic assays): Gurrs Giemsa R66 for five minutes

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per duplicate culture, 200 per dose level

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (2000 lymphocytes)

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes (included in polyploid cell frequency)

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations markedly exceeded that seen in the concurrent control,
either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

Fisher's exact test or Chi-squared test

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not measured.
- Effects of osmolarity: Maximum dose level limited to 10mM, so no osmolarity effects.
- Evaporation from medium: the test material is not volatile
- Water solubility: The test substance was not sufficiently soluble in water for water to be used as the vehicle.
- Precipitation: None observed.

COMPARISON WITH HISTORICAL CONTROL DATA: The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were consistent with acceptance criteria. The study was therefore considered valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The mitotic index for Experiment 1 demonstrates that metaphases were present up to 35.63 µg/mL in the 20-hour treatment without S9 and there was a sharp toxicity dose-response curve with no scorable metaphases at 71.25 µg/mL. A toxic response was also apparent in the 20 hour with S9 treatment group in which metaphase cells were present at up to 142.5 µg/mL.
In Experiment 2, toxicity was similar to that observed in Experiment 1. However, the intermediate dose levels that were used achieved a more desirable toxicity response curve that resulted in approximately 50% mitotic inhibition in both the 20-hour harvest groups and in the 44-hour without S9 group.

Table 7.6.1/1 Results of chromosome analysis (mean) – Experiment 1 without metabolic activation

		Control	8.91 µg/mL	17.81µg/mL	35.63µg/mL	EMS 500
Cytotoxicity		no	no	no	no	yes
Chromatid aberrations (/200)	gaps	3	2	2	2	31
	breaks	2	3	1	0	32
	interchanges	0	2	0	0	3
Isochromatid aberrations (/200)	breaks	0	1	0	1	5
	interchanges	0	0	0	0	0
Frequency of aberrant cell (%)	+ Gaps	5	8	3	3	71
	- Gaps	2	6	2	1	40
Mitotic index(% of control)		100	80	86	97	32
Polyploidy(%)		0	1	2	2	1

Table 7.6.1/2 Results of chromosome analysis (mean) – Experiment 1 with metabolic activation

		Control	36.25 µg/mL	71.25µg/mL	142.5µg/mL	CP25
Cytotoxicity		no	no	no	no	yes
Chromatid aberrations (/200)	gaps	1	3	3	1	8
	breaks	0	0	0	1	8
	interchanges	0	0	0	0	0
Isochromatid aberrations (/200)	breaks	0	0	0	0	2
	interchanges	0	0	0	0	0
Frequency of aberrant cell (%)	+ Gaps	1	3	3	2	18
	- Gaps	0	0	0	1	10
Mitotic index(% of control)		100	116	87	81	40
Polyploidy(%)		0	0	0.5	0	0.5

Table 7.6.1/3 Results of chromosome analysis (mean) – Experiment 2 (20-hour harvest) without metabolic activation

		Control	8.91 µg/mL	17.81µg/mL	35.63µg/mL	53.44 µg/mL	EMS 500
Cytotoxicity		no	no	no	yes	yes	yes
Chromatid aberrations (/200)	gaps	4	1	1	2	4	27
	breaks	1	0	0	2	2	33
	interchanges	0	0	0	0	0	2
Isochromatid aberrations (/200)	breaks	0	0	0	0	0	5
	interchanges	0	0	0	0	0	0
Frequency of aberrant cell (%)	+ Gaps	5	1	1	4	6	67
	- Gaps	4	0	0	2	2	40
Mitotic index(% of control)		100	103	82	58	35	62
Polyploidy(%)		0	1	0.5	1	0.5	1.3

 

Table 7.6.1/4 Results of chromosome analysis (mean) – Experiment 2 (20-hour harvest) with metabolic activation

		Control	71.25µg/mL	142.5µg/mL	213.8 µg/mL	CP25
Cytotoxicity		no	no	yes	yes	yes
Chromatid aberrations (/200)	gaps	1	1	3	2	18
	breaks	4	2	0	2	12
	interchanges	0	0	0	0	4
Isochromatid aberrations (/200)	breaks	0	0	0	0	3
	interchanges	0	0	0	0	0
Frequency of aberrant cell (%)	+ Gaps	5	3	3	4	37
	- Gaps	4	2	0	2	19
Mitotic index(% of control)		100	97	57	63	37
Polyploidy(%)		1	0	0	0.5	0

Table 7.6.1/5 Results of chromosome analysis (mean) – Experiment 2 (44-hour harvest) without metabolic activation

		Control	53.44 µg/mL
Cytotoxicity		no	yes
Chromatid aberrations (/200)	gaps	0	1
	breaks	1	2
	interchanges	0	0
Isochromatid aberrations (/200)	breaks	0	0
	interchanges	0	0
Frequency of aberrant cell (%)	+ Gaps	1	3
	- Gaps	1	2
Mitotic index(% of control)		100	40
Polyploidy(%)		0.5	0

Table 7.6.1/6 Results of chromosome analysis (mean) – Experiment 2 (44-hour harvest) with metabolic activation

		Control	213.8 µg/mL
Cytotoxicity		no	no
Chromatid aberrations (/200)	gaps	0	1
	breaks	0	0
	interchanges	0	0
Isochromatid aberrations (/200)	breaks	0	1
	interchanges	0	0
Frequency of aberrant cell (%)	+ Gaps	0	2
	- Gaps	0	1
Mitotic index(% of control)		100	113
Polyploidy(%)		1	1

 

Conclusions:

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

Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline No 473 and in compliance with GLP, human primary lymphocyte cultures were exposed to the test material diluted in ethanol. Four treatment conditions were used, ie. 4 hours exposure with the addition of an induced rat liver homogenate metabolising system with cell harvest after 16 and 40-hour expression periods and 20 and 44 hour continuous exposures in the absence of activation. The dose range for evaluation was selected from a series of 8 dose levels on the basis of toxicity.

 

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

 

The test material was moderately toxic and did not induce any toxicologically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included some dose levels that induced approximately 50 % mitotic inhibition.

 

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

 

This study is considered as acceptable and satisfies the requirement for in vitro mammalian chromosome aberration assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2001-07-23 to 2001-09-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 476 and in compliance with GLP

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 28 February 2000. Signed on 26 April 2000

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Source: L5178Y TK+/- 3.7.2c mouse lymphoma cell line obtained from Dr J Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK.
- Type and identity of media: RPMI 1640 with 20% donor horse serum (R20) and without serum (R20).
- Properly maintained: yes, stored in liquid nitrogen at -196°C.
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes, by culturing in THMG medium for 24 hours. For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

PB/betaNF S9 (S9 mix) from male Sprague-Dawley rats treated with phenobarbital/β-naphtoflavone

Test concentrations with justification for top dose:

Preliminary toxicity test: 36.94 to 2364 µg/mL, due to toxicity repeated with 0.625 to 120 µg/mL.
Exp 1: without S9-mix, six doses levels 2.5 to 25 µg/mL
Exp 1: with S9-mix, six doses levels 2.5 to 40 µg/mL
Exp 2: without S9-mix, six doses levels 2.5 to 25 µg/mL
Exp 2: with S9-mix, six doses levels 5 to 40 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test substance not sufficiently soluble in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

at 800 µg/mL in Exp 1 and 150 µg/mL in Exp 2

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

at 2.5 µg/mL

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: no
- Exposure duration: 3 hours in Experiment 1, 3 hours with metabolic activation cultures and 24 hours without metabolic activation in Experiment 2
- Expression time (cells in growth medium): two days
- Selection time (if incubation with a selection agent): 10 to 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): NA

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2, both with and without metabolic activation

NUMBER OF CELLS EVALUATED: the cells were counted and processed to give 1* 10EXP6 cells/mL in 10 mL aliquots in R10 medium

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - After the preliminary toxicity test, the cultures were incubated and sub-cultured after 24 hours by counting and diluting to 2 x 10EXP5 cells/mL. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate suspension growth values (SG). The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a % Relative Suspension Growth Value (%RSG).

OTHER
- Other: Determination Plating Efficiency: Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the plating efficiency (P.E.) was calculated using the zero term of the Poisson distribution [P(0)] method.
[P(0) = number of negative wells / total wells plated]
[(P.E.% = -lnP(0) x 100)/( number of well/cells)]
- Other: Determination of Calculation of Mutation Frequency - M.F. per survivor = [(-In P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium. The experimental data was analysed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS.

Evaluation criteria:

For a test material to give a 'significant' result then 2 or more of the following criteria should be met:
i) A greater than three-fold increase in the mutant frequency per survivor over the negative control value.
ii) A dose-related increase in the mutant frequency per survivor.
iii) An increase in the absolute number of mutants.
A test material may be reported as equivocal if only one of the above criteria is met. Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director's discretion.

Statistics:

The experimental data was analysed using a dedicated computer program which follows the statistical guidelines recommended by UKEMS.

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:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no change in pH at maximum dose level
- Effects of osmolality: not increased by more than 50 mOsm.
- Precipitation: not seen

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was performed on cell cultures at 5 x 10EXP5 cells/mL, using a 3-hour exposure time both with and without metabolic activation (S9), and at 1.5 x 10EXP5 cells/mL using a continuous 24-hour exposure without S9. The dose range used in the preliminary toxicity test was initially 36.94 to 2364 µg/mL. However, the test material proved to be very toxic and the preliminary toxicity test was repeated using the dose range 0.625, 1.25, 2.5, 5, 10, 20 and 40 µg/mL in the 3-hour without-S9 exposure group, 2.5, 5, 10, 20, 40, 80 and 120 µg/mL in the 3-hour with-S9 exposure group and 0.625, 1.25, 2.5, 5, 10, 20 and 30 µg/mL in the 24-hour exposure without S9. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted using a coulter counter and then serially diluted to 10 cells/mL.

COMPARISON WITH HISTORICAL CONTROL DATA: Vehicle control mutation frequencies were within historical ranges (25 to 150.10EXP-6 viable cells). Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment 1 and 2, there was evidence of toxicity with the test material in both the presence and absence of metabolic activation, as indicated by the %RSG and Day 2 (%V) viabilities; this was confirmed by the dose-related decrease in relative total growth (RTG) values.

Table 7.6.1/1: Preliminary toxicity test – Relative Suspension Growth (RSG)

Dose (µg/mL)			% RSG (-S9) 3-hour exposure	% RSG (+S9) 3-hour exposure	%RSG (-S9) 24-hour exposure
0			100	100	100
0.625	2.5	0.625	108	104	105
1.25	5	1.25	124	117	105
2.5	10	2.5	104	107	90
5	20	5	120	90	97
10	40	10	45	9	83
20	80	20	29	0	34
40	120	30	0	NP	1

NP = not plated due to excessive toxicity

 

Table 7.6.1/2: Mutagenicity test – Experiment 1

Treatment (µg/mL)	-S9 (3-hour)				Treatment (µg/mL)	+S9 (3-hour)
	%RSG	RTG	MF	Proportion small colony mutants		%RSG	RTG	MF	Proportion small colony mutants
0	100	1.00	77.66	0.66	0	100	1.00	51.19	0.54
2.5	104	1.12	67.81	0.47	2.5	88	1.10	49.66	0.48
5	99	0.95	76.08	0.57	5	95	1.05	61.46	0.42
10	95	0.95	73.71	0.43	10	87	1.34	48.60	0.41
15	76	1.10	62.16	0.43	20 $$	77	(0.67)	(56.08)	0.23
20	38	0.49	80.05	0.52	30	10	0.86	57.25	0.50
25 X	1	0.04	142.06	0.61	40 $$	1	(0.10)	(49.31)	0.81
Linear Trend                                 NS					Linear Trend                                    NS
EMS					CP
800	66	0.33	1581.91	0.29	2.5	69	0.28	1081.09	0.77

X = treatment excluded from test statistics due to toxicity

$$ = Treatment excluded from analysis due to excessive heterogeneity

 

Table 7.6.1/3: Mutagenicity test – Experiment 2

Treatment (µg/mL)	-S9 (24-hour)				Treatment (µg/mL)	+S9 (3-hour)
	%RSG	RTG	MF	Proportion small colony mutants		%RSG	RTG	MF	Proportion small colony mutants
0	100	1.00	64.11	0.36	0	100	1.00	59.13	0.52
2.5	94	1.02	57.53	0.29	5	97	0.94	68.43	0.58
5	81	1.10	57.35	0.27	10	99	0.93	51.02	0.43
10	74	1.12	55.58	0.39	20	95	0.87	74.86	0.41
15	73	0.90	43.94	0.28	30	66	0.66	54.27	0.34
20	55	0.85	54.59	0.33	35 $$	12	(0.12)	(64.33)	0.32
25	22	0.47	47.71	0.14	40 $,X	2
Linear Trend                                 NS					Linear Trend                                    NS
EMS					CP
150	56	0.60	1115.81	0.24	2.5	66	0.22	1068.55	0.73

X = treatment excluded from test statistics due to toxicity

$$ = Treatment excluded from analysis due to excessive heterogeneity

 

Conclusions:

The test material did not induce any statistically significant or dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Executive summary:

In a mammalian cell gene mutation assay performed according to the OECD test guideline No. 476 and in compliance with GLP, L5178Y mouse lymphoma cells were exposed to the test material diluted in DMSO at 6 dose levels, in duplicate in the presence and absence of metabolic activation (S9-mix). The entire experiment was repeated to confirm the result of the first experiment. Three-hour exposures were used both with and without activation (S9) in Experiment 1. In Experiment 2 the exposure time without activation was increased to 24 hours. The dose range of test material, plated out for expression of mutant colonies, was selected on the results of a preliminary toxicity test and was 2.5 to 25 µg/mL in the absence of S9 and 2.5 to 40 µg/mL in the presence of S9 for the first experiment. For the second experiment the dose range was 5 to 40 µg/mL with activation and 2.5 to 25 µg/mL without activation. The maximum dose level used was limited by test material-induced toxicity.

 

The vehicle (solvent) controls gave acceptable levels of mutant frequencies for the L5178Y cell line at the TK +/- locus.

 The positive control treatments, both in the absence and presence of metabolic activation, induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any statistically significant or dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Table 7.6/1: Summary of genotoxicity tests

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1   Safepharm, 2005	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537 TA 98 TA 100 WP2 uvrA	-S9 +S9	Up to 5000 µg/plate	-S9 : non mutagenic +S9 : non mutagenic
2   Toxicol, 1991	Ames Test (OECD 471) S, rel. 2	Gene mutation	TA 1535, TA 1537 TA 98 TA 100	-S9 +S9	Up to 2000 µg/plate	-S9 : non mutagenic +S9 : non mutagenic
3   Safepharm, 2001	ML/TK test (OECD 476) K, rel. 1	Gene mutation	mouse lymphoma L5178Y cells	-S9 +S9	Up to 40 µg/mL	-S9 : non mutagenic +S9 : non mutagenic
4   Safepharm, 1995	CA/HL test (OECD 473) K, rel. 1	Chromosomal aberration	Human Lymphocytes	-S9 +S9	Up to 213.8 µg/mL	-S9 : non clastogenic +S9 : non clastogenic

 

Gene mutation Assays (Tests n° 1-3):

Two Bacterial Reverse mutation Assays (Ames test) were performed according to OECD 471 test guidelines with the test material (See Table 1). Test n°1 was selected as the key study. Test 2 was used as supporting data because only four strains of bacteria were used. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains in both tests, with any dose of the test material, either with or without metabolic activation. Both tests indicate that the test material does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The test material is therefore considered as non-mutagenic according to the Ames test.

Inability to produce gene mutation was confirmed in mammalian cells using an in vitro forward mutation assay in mouse lymphoma TK L5178Y cells (ML/TK test) (Test n°3). None of the dose levels up to the cytotoxicity limit with the test material, either with or without metabolic activation, induced significant mutant frequency increases in the initial or repeat tests. The test material does not induce forward mutations at the TK locus in L5178Y mouse lymphoma cells under activation and non activation conditions whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. The test material is therefore considered as negative for inducing forward mutations at the TK locus in L5178Y mouse lymphoma cells under activation and non-activation conditions used in this assay. This result confirms the results of both Ames tests and extends the non-mutagenic effect of the test material from bacteria to mammalian cells.

 

Chromosomal aberration (Test n°4)

The clastogenic potential of the test material was determined using an in vitro chromosome aberration test in human lymphocytes, which measures the potential of a substance to increase the incidence the of structural chromosome aberrations in cultured human lymphocytes.

None of the dose levels up to the cytotoxicity limit with the test material, either with or without metabolic activation, induced significant increases in the frequency of cells with aberrations in either of two experiments. The test material does not induce structural aberrations in the chromosomes of human lymphocytes under activation and non-activation conditions, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells. The test material is therefore considered as negative for inducing chromosomal mutations in human lymphocyte cells under activation and non-activation conditions used in this assay.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008 (CLP).

Self-classification:

Based on the available information, no additional classification is proposed regarding genetic toxicity according to the CLP and to the GHS.