[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, K, rel. 2): non mutagenic up to 5000 μg/plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100

- HL/CAT chromosome aberration test (OECD 473, GLP, K, rel. 1): non clastogenic up to cytotoxic concentrations.

- MLA In Vitro Mammalian Cell Gene Mutation Test (OECD 476, GLP, K, rel. 1): non mutagenic up to cytotoxic concentrations

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:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

OECD guideline compliant as at 1987

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

as at 1987

Deviations:

yes

Remarks:

only 4 strains tested (E. Coli not tested), only plate incorporation assay used.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

TA1535: his G46 rfa- delta uvrB-
TA1537: his C3076 rfa- delta uvrB-
TA98: his D3052 rfa- delta uvrB- R+
TA100: his G46 rfa- delta uvrB- R+

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

Plate incorporation assay up to 10,000 µg Fe/plate + and - S9. Pre-incubation assay

Vehicle / solvent:

Vehicle used: distilled water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene for all strains

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation);

DURATION
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours concurrent with exposure

SELECTION AGENT (mutation assays): minimal agar

NUMBER OF REPLICATIONS: doses were tested in triplicate in the plate incorporation assay . 5 dose levels were tested.

DETERMINATION OF CYTOTOXICITY
- Method:
• one Salmonella strain (TA98), based on the assumption that all strains used show a similar toxic response with and wo activation.
• concentrations: 0, 1.6, 8, 40, 200, 1000 and 5000 µg/plate.
• analysis of appearance of a complete bacterial lawn as seen under a dissecting microscope after 24 hours incubation at 37°C.

Evaluation criteria:

A statistically significant increase in the mean number of revertants that exceeds twice the concurrent negative control, plus evidence of a dose response relationship.

Statistics:

• analysis of variance on each set of data to obtain the F-statistic
• in case of statistical significance (p<0.05) and dose related increases correlation coefficient is calculated for the response range and the significance of the result is determined from standard tables.

Species / strain:

E. coli WP2 uvr A pKM 101

Remarks:

E. coli WP2 uvrA, not used in this study

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Remarks:

AMINOL A15 LA 438-7 (FB-4136)

Positive controls validity:

not applicable

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: at 5000 µg/plate with and without activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: at 5000 µg/plate with and without activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: at 5000 µg/plate with and without activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: at 5000 µg/plate with and without activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

GENOTOXIC EFFECTS:

- The test item caused restricted growth of the background lawns for all four strains, both with and without S-9, at 5000 µg/plate.

- No dose related increases in revertant rates were seen with any of the strains except for TA1537 and TA98 without activation at the highest dose. The effects occur only at the highest dose and are deemed rather survivors of the cytotoxic effect than mutants. In addition the effects are neither dose related nor reproducible in experiment 2 and therefore not regarded as a positive mutagenic response.

- High concentrations of the test item caused decreases in numbers of revertants (except for the two concentrations nentioned above).

- All solvent (negative) controls gave counts of revertants within normal ranges

- All positive controls gave numbers of induced revertants within expected ranges demonstrating the sensitivity of the assay and the metabolising activity of the S-9 mix.

PRECIPITATION CONCENTRATION: None reported

Table 1: Pretest

Strain	% S-9	Concentration of Test Substance (µg/plate)*
		0	1.6	8	40	200	1000	5000
TA98	0	+	+	+	+	+	+	+
TA98	10	+	+	+	+	+	+	+

* These are actual amounts of test substance added and correspond to solutions of test compound with concentrations from 16 µg/ml to 50 mg/ml.

+ = restricted growth

+ = normal growth

Table 2: MEAN NUMBER OF REVERTANTS PER PLATE FOR THE TEST ITEM

Strain	% S-9	Concentration of Test Substance (µg/plate)
		0	8	40	200	1000	5000	PC
TA1535	0	9.0	13.0	9.0	6.7	6.0	1.7	322.3
TA1537	0	3.0	5.0	3.7	4.0	1.7	1.7	1013.0
TA98	0	10.7	11.7	5.3	5.3	4.7	3.7	173.3
TA100	0	93.0	88.3	80.7	77.0	72.0	57.3	287.3
TA1535	10	11.0	12.3	7.7	10.7	7.0	4.7	95.7
TA1537	10	5.7	3.3	6.0	5.0	3.3	3.3	35.7
TA98	10	16.0	13.7	11.3	9,7	11.0	9.0	523.0
TA100	10	92.0	86.0	89.7	80.3	72.7	72.0	639.7

PC = Positive control

Table3: MEAN NUMBER OF REVERTANTS PER PLATE FOR THE TEST ITEM

Strain	% S-9	Concentration of test substance (µg/plate)
		0	8	40	200	1000	5000	PC
TA1535	0	5.3	8.3	4.7	6.3	4.7	1.7(a)	262.3
TA1537	0	6.3	7.0	5.7	5.3	2.7	58.3(a)	925.7
TA98	0	10.0	13.7	10.0	9.0	9.0	25.0(a)	138.0
TA100	0	112.0	106.7	81.3	84.0	67.3	58.0(a)	218.3
TA1535	10	10.0	9.7	8.7	6.7	4.3	3.7(a)	186.7
TA1537	10	6.7	4.3	4.3	4.3	2.3	3.3(a)	34.0
TA98	10	13.0	8.7	14.7	10.3	4.3	5.3(a)	138.3
TA100	10	104.0	94.7	81.7	89.7	72.7	67.7(a)	369.3

PC = Positive control

a = Reduced background lawn

Conclusions:

Under the test conditions, test substance is not mutagenic with and without metabolic activation in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, up to 5000 µg/plate.

Executive summary:

The test item was tested in vitro by the Ames plate incorporation method for its ability to induce mutations in four histidine dependent auxotrophic mutants of Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 according to OECD 471 as at 1987.

Two independent mutation tests were performed, each in both the presence and absence of a metabolic activation system (S-9 mix) derived from the livers of Aroclor 1254 treated rats. The bacteria were exposed to the test material dissolved in Dimethylsulphoxide, which was also the solvent control. Based on the results of a preliminary toxicity rangefinder, 5000 µg/plate of AA 15 was chosen as the highest dose level to be used, with lower dose levels of 1000, 200, 40 and 8 µg/plate.

All solvent (negative) controls gave counts of revertants within normal ranges.

All positive controls gave numbers of induced revertants within expected ranges demonstrating the sensitivity of the assay and the metabolising activity of the S-9 mix.

The test item produced no significant increases in the number of revertants, with any of the tester strains, in either of the two experiments performed in both the presence and absence of metabolic activation, when assayed up to 5000 µg/plate.

Under the test conditions, test substance is not mutagenic in Salmonella typhimurium strains with and without metabolic activation, up to 5000 µg/plate.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23/12/1999-14/08/2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to the OECD Guideline and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC Guidelines: Methods for the Determination of Toxicity. Other Effects-Mutagenicuty (in vitro mammalian cytogenetic test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: CPMP/ICH/174/95 Note for Guidance on Genotoxicity: A standard Battery for Genotoxicity Testing of Pharmaceuticals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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):

CELLS USED
Human peripheral blood was obtained by venipuncture from healthy donors known to be without any medication and collected in heparinised vessels. Small inocula of whole blood (0.5mL) were added to tubes containing 5 mL of complete culture medium. The tubes were sealed and incubated at 37°C with occasional shaking to prevent clumping.

MEDIA USED
- Complete medium:
500 mL chromosome medium 1A with phytohemagglutinin (Gibco)
5 mL penicillin/streptomycin (10000 IU/mL)
- Treatment medium:
500 mL Ham's F-10
13.1 mL Fetal Calf Serum

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

The preparation of the Aroclor 1254 induced rat S9 fraction was carried out according to MARON & AMES and stored in liquid nitrogen util used. The SA was collected from 20 - 30 rats.

Test concentrations with justification for top dose:

The concentrations employed were chosen based on the result of a cytotoxicity study. In this preliminary experiment, without (24-hour exposure) and with metabolic activation (4-hour exposure), a nearly complete cytotoxicity was noted at a concentration of 1000 µg/mL medium whereas a complete cytotoxicity was observed starting from a concentration of 2500 µg/mL medium onwards.

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk


DURATION
- Preincubation period: 4 hours
- Exposure duration:
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):


SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):


NUMBER OF REPLICATIONS: duplicate cultures were used per sampling point employing blood from two different donors.


NUMBER OF CELLS EVALUATED:


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:


OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:


OTHER:

Statistics:

The assessment was carried out by a comparison of the number of chromosome aberrations of the samples with those of the solvent control, using the exact test of R.A. FISHER (p ≤ 0.05) as recommended by the UKEMS guidelines.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the present test conditions, the substance tested up to cytotoxic concentrations in the absence and presence of metabolic activation is not clastogenic and not aneugenic in human lymphocytes.

Executive summary:

Test samples of the substance were assayed in an in vitro cytogenetic study, using human lymphocyte cultures, both in the presence and absence of metabolic activation by a rat liver post-mitochonrial fraction (S9 mix) from Aroclor-1254 induced animals. DMSO served as the vehicle. The concentrations employed were chosen on the results of a cytotoxicity study. In this preliminary experiment, without (24 -hours exposure) and with metabolic activation (4 -hour exposure), a nearly complete cytotoxicity was noted at a concentration of 1000µg/ml medium whereas a complete cytotoxicity was observed starting from a concentration of 2500µg/ml medium onwards.Hence, the top concentration employed in this study was 250µg/mL using a 4 -hours and a 24 -hour exposure without metabolic activation. For the 4 -hour exposure (with metabolic activation), the top concentration employed was also 250µg/mL. Treatment with the concentrations of 62.5, 125 or 250µg/mL medium resulted in pronounced cytotoxicity in the experiment without metabolic activation (250µg/mL: 4 -h exposure; 62.5µg/mL: 24 -h exposure). Treatment with the concnetration of 125 or 250µg/mL resulted in moderate to severe cytotoxicity in the experiment with metabolic activation (4 -h exposure). The test was carried out employing 2 exposure times without S9: 4 and 24 hours, and 1 exposure time with S9:4 hours. The harvesting time was 24 hours after start of exposure. The incubation procedure took place in the dark. The study was conducted in duplicate. Mitomycin C and cyclophosphamide were employed as positive control chemicals in the absence and presence of metabolic activation, respectively. Test without metabolic activation (4 -and 24 -hour exposure): The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with the substance (15.625, 31.25, 62.5, 125 or 250 µg/mL medium) in the absence of metabolic activation ranged from 1.0% to 4.1% in the two independent experiments with blood from different donors. Severe cytotoxicity was noted at the concentration of 250µg/mL (mitotic index: 0.23and 0.13, respectively). The result are considered to be within the nominal range of the negative control where a mean incidence of chromosomal aberrations (excluding gaps) of 0.0% or 1.5% was observed after a 4 -hour or 24 -hour exposure.

Test with metabolic activation (4 -hour exposure): The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with the substance (15.625, 31.25, 62.5, 125 or 250µg/mL medium), in the presence of metabolic activation, ranged from 1.5% to 2.5% in the two independent experiments. Only at the severely cytotoxic concentration of 250µg/mL (mitotic index: 0.07) during the 4-hour exposure, a slight increase was noted in the number of aberrations to 5.2%. It is known that high cytotoxicity causes artefacts in the form of aberrations in "in vitro" chromosomal tests. Hence, the increase at the concentration of 250µg/mL is considered as artefact and not substance-related.

The results are considered to be within the nominal range of the negative control where a mean incidence of chromosomal aberrations (excluding gaps) of 0.5% was observed. The incidence of chromosomal aberrations (excluding gaps) of the controls was for the last 30 experiments 0.0%-5.0%.

Under the present test conditions, the substance tested up to cytotoxic concentrations in the absence and presence of metabolic activation, employing two exposure times (without S9) and one exposure time (with S9), revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage in human lymphocytes.

In the same test, mitomycin C and cyclophosphamide induced significant damage.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06/10/2008-22/12/2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to the OECD Guideline and in compliance with GLP

Qualifier:

according to guideline

Guideline:

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

Guideline:

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

Principles of method if other than guideline:

experiment I: 4 hours treatment with and without metabolic activation
experiment II: 24 hours treatment without metabolic activation and 4 hours treatment with metabolic activation
experiment IIA (repeat experiment): 24 hours treatment without metabolic activation

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

On the day of the experiment (immediately before treatment), the test item was dissolved in acetone (E. MERCK, 64293 Darmstadt; Germany, purity 99.5 %). The final concentration of acetone in the culture medium was 0.5 % (v/v).

Target gene:

Thymidine Kinase (TK) locus of heterozygous L5178Y/TK +/- cells to TK-/- mutants

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

other: Clone 3.7.2C

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Test concentrations with justification for top dose:

Experiment I:
without S9 mix: 0.9; 1.9; 3.8; 7.5; 15.0; 30.0 µg/mL (4 hours treatment)
with S9 mix: 1.9; 3.8; 7.5; 15.0; 30.0; 60.0 µg/mL (4 hours treatment)
Experiment II:
without S9 mix: 1.3; 2.5; 5.0, 10.0; 20.0; 30.0 µ/mL (24 hours treatment)
with S9 mix: 5.0; 10.0; 20.0; 40.0; 60.0; 80.0 µg/mL (4 hours treatment)
Experiment IIA:
without S9 mix: 20.0; 30.0; 40.0; 50.0; 60.0 µg/mL (24 hours treatment)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: solubility properties

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: dissolved in medium


DURATION
- Exposure duration:
experiment I: 4 hours with and without metabolic activation in experiment 1;
experiment II: 24 hours without metaoblic activation and 4 hours treatment with metabolic activation;
experiment IIA: 24 hours treatment without metabolic activation

- Expression time (cells in growth medium): 48 hours

- Selection time (if incubation with a selection agent): 10 to 15 days

SELECTION AGENT (mutation assays): RPMI 1640 medium by addition of 5 µg/mL TFT

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5 x 10 exp. 6 cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10 exp. 6 cells above the
corresponding solvent control or negative control, respectively.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in negative
and/or vehicle con¬trols and the mutation rates of all negative and/or vehicle controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth, and the cloning efficiency 1 is less than 10 % of the vehicle control
unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used
to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and
dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.

Statistics:

Linear regression analysis (least squares) using SYSTAT11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA)

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

Positive controls validity:

valid

Additional information on results:

precipitation occurred in experiment I at 30 µg/mL without metabolic activation

Summary Table

	conc. µg	S9	total	colonies/		total	colonies/
	per mL	mix	growth	10^6 cells	threshold	growth	10^6 cells	threshold
Column	1	2	3	4	5	6	7	8
Experiment I / 4 h treatment			culture I			culture II
Solv. control with acetone		-	100.0	110	236	100.0	187	313
Pos. control with MMS	19.5	-	10.2	1059	236	34.8	378	313
Test item	0.9	-	106.2	167	236	100.4	171	313
Test item	1.9	-	78.4	231	236	80.3	274	313
Test item	3.8	-	127.4	120	236	130.9	190	313
Test item	7.5	-	57.6	292	236	124.1	155	313
Test item	15.0	-	46.6	210	236	81.8	140	313
Test item	30.0 (p)	-	culture was not continued^#			culture was not continued^#
Solv. control with acetone		+	100.0	107	233	100.0	113	239
Pos. control with CPA	3.0	+	44.7	190	233	42.4	242	239
Pos. control with CPA	4.5	+	22.6	335	233	15.0	304	239
Test item	1.9	+	culture was not continued^##			culture was not continued^##
Test item	3.8	+	100.9	85	233	107.7	103	239
Test item	7.5	+	92.8	91	233	95.3	90	239
Test item	15.0	+	83.4	90	233	80.8	145	239
Test item	30.0	+	98.1	68	233	75.3	93	239
Test item	60.0	+	5.0	177	233	63.5	98	239
Experiment II / 24 h treatment			culture I			culture II
Solv. control with acetone		-	100.0	149	275	100.0	130	256
Pos. control with MMS	13.0	-	64.5	352	275	15.6	822	256
Test item	1.3	-	culture was not continued^##			culture was not continued^##
Test item	2.5	-	173.9	107	275	99.6	135	256
Test item	5.0	-	154.6	147	275	60.8	265	256
Test item	10.0	-	97.4	186	275	66.1	224	256
Test item	20.0	-	123.5	157	275	66.8	156	256
Test item	30.0	-	65.2	202	275	69.0	115	256
Experiment II / 4 h treatment			culture I			culture II
Solv. control with acetone		+	100.0	95	221	100.0	130	256
Pos. control with CPA	3.0	+	39.6	256	221	38.0	241	256
Pos. control with CPA	4.5	+	7.9	869	221	16.8	485	256
Test item	5.0	+	81.8	106	221	culture was not continued^##
Test item	10.0	+	71.8	124	221	96.7	193	256
Test item	20.0	+	82.5	131	221	76.8	212	256
Test item	40.0	+	88.3	89	221	81.7	213	256
Test item	60.0	+	52.8	118	221	61.3	230	256
Test item	80.0	+	culture was not continued^#			6.1	214	256
Experiment IIA / 24 h treatment			culture I			culture II
Solv. control with acetone		-	100.0	106	232	100.0	128	254
Pos. control with MMS	13.0	-	5.3	688	232	1.5	804	254
Test item	20.0	-	26.5	162	232	36.2	230	254
Test item	30.0	-	10.2	216	232	13.8	271	254
Test item	40.0	-	3.2	142	232	4.6	205	254
Test item	50.0	-	culture was not continued^#			culture was not continued^#
Test item	60.0	-	culture was not continued^#			culture was not continued^#

Threshold = number of mutant colonies per 10^6 cells of each solvent control plus 126

# culture not continued due to exceedingly strong toxic effects

## culture was not continued since a minimum of four concentrartions is required by the guidelines

p precipitation was observed in culture I

 

Conclusions:

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.
Therefore, the test item is considered to be non-mutagenic in this mouse lymphoma assay at the TK locus.

Executive summary:

An in vitro mammalian cell gene mutation test was performed according to the OECD test guideline 476 and in compliance with GLP to investigate the potential of the test substance to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in three independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

In experiment I and II without metabolic activation the required toxicity range of approximately 10-20 % was not reached. Therefore, a repeat experiment was performed (experiment IIA) using modified concentrations. In experiment IIA the treatment period was 24 hours without metabolic activation.

The highest applied concentration in the pre-experiment (4000 µg/mL) was chosen with regard to the molecular weight of the test item corresponding to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxic effects.

The following dose levels were selected in the main experiments:

Experiment I:      

without S9 mix:   0.9; 1.9; 3.8; 7.5; 15.0; 30.0  µg/mL (4 hours treatment)

with S9 mix:          1.9; 3.8; 7.5; 15.0; 30.0; 60.0 µg/mL (4 hours treatment)

Experiment II:

without S9 mix:  1.3; 2.5; 5.0, 10.0; 20.0; 30.0 µ/mL (24 hours treatment)

with S9 mix:        5.0; 10.0; 20.0; 40.0; 60.0; 80.0 µg/mL (4 hours treatment)

Experiment IIA:

without S9 mix:  20.0; 30.0; 40.0; 50.0; 60.0 µg/mL (24 hours treatment)

No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Therefore, the test item is considered to be non-mutagenic in this mouse lymphoma assay at the TK locus.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Table 7.6/1: Summary of genotoxicity tests

 

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1	Ames Test (OECD 471) K, rel. 2	Gene mutation	TA 1535, TA 1537, TA 98, TA 100	-S9 +S9	Up to limit concentration	-S9 : non mutagenic +S9 : non mutagenic
2	HL/CAT (OECD 473) K, rel. 1	chromosome aberration	Human Lymphocytes	-S9 +S9	Up to cytotoxic concentrations	-S9 : non clastogenic +S9 : non clastogenic
3	MLA (OECD 476) K, rel.1	gene mutation	L5178Y	-S9 +S9	Up to cytotoxic concentrations	-S9 : non mutagenic +S9 : non mutagenic

 

Gene mutation Assays (Tests n° 1 & 3):

A Bacterial Reverse mutation Assay (Ames test) was performed similarly to OECD guideline No. 471 with the substance (See Table 7.6/1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test conditions, with any dose of the substance, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies.The substance is therefore considered as non-mutagenic in S. typhimurium according to the Ames test.

A cell mutation assay at the thymidine kinase locus (TK +/-) in mouse lymphoma L5178Y cells was performed according to the OECD guideline No. 476. The assay was performed in three independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid. Under the tes conditions, the substance is therefore considered as non-mutagenic in mouse lymphoma cells at the TK locus.

Chromosomal aberration (Test n°2)

The clastogenic potential of the substance was determined using an in vitro chromosome aberration test in human lymphocytes (OECD guideline No. 473), which measures the potential of a substance to increase the incidence of structural chromosome aberrations in cultured human lymphocytes.

None of the dose levels up to the cytotoxicity limit with the substance, either in the presence or absence of metabolic activation, induced significant toxicological increases in the frequency of cells with aberrations in either of three experiments. The substance 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 substance 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 for human health according to the Regulation (EC) No. 1272/2008.

Self classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.