Octadecanoic acid, 1-(2-hydroxy-2-methylpropoxy)-2,2,6,6-tetramethyl-4-piperidinyl ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two Ames tests, an HPRT assay and a chromosome aberration test were performed according GLP and OECD guideline 471, 473 and 476 to evaluate the mutagenic abd clastogenic potential of the test substance. The test item did not induce mutations or chromosome aberrations in vitro. Therefore, the substance is not considered to be mutagenic under the conditions of these tests.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HGPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- high proliferation rate (doubling time of about 12 - 16 hours)
- high plating efficiency (about 90%)
- karyotype with a modal number of 20 chromosomes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from phenobarbital and β-naphthoflavone induced rat liver

Test concentrations with justification for top dose:

1st Experiment
without S9 mix (4-hour exposure period)
0; 3.13; 6.25; 12.5; 25.0; 50.0; 100.0; 200.0 μg/mL
with S9 mix (4-hour exposure period)
0; 3.13; 6.25; 12.5; 25.0; 50.0; 100.0; 200.0 μg/mL

2nd Experiment
without S9 mix
0; 12.5; 25.0; 50.0; 100.0; 200.0 μg/mL
with S9 mix
0; 18.8; 37.5; 75.0; 150.0; 200.0 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofurane (THF)
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water and other commonly used vehicles (e.g. DMSO, ethanol, acetone), tetrahydrofurane (THF) was selected as the vehicle, which had been demonstrated to be suitable in the CHO/HPRT assay. The final concentration of the vehicle THF in the culture medium was 0.5% (v/v).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

with and without S9 mix

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: methylcholanthrene (with metabolic activation, 20 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- pretreatment: During the week prior to treatment, spontaneous HPRT-deficient mutants were eliminated by pretreatment with "HAT" medium.
- Exposure duration: 20-24h
- Expression time (cells in growth medium): 4 and 24h --> washing and first cytotox determination, 1. passage day 5, days 7-9 2. passage into selection medium (TG medium) and second cytotox determination
- Fixation time (start of exposure up to fixation or harvest of cells): from day 16 drying, fixation, staining and counting of the selected colonies

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2 independent experiments

NUMBER OF CELLS EVALUATED: number of colony per flask was counted and recorded

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
pH, osmolarity, solubility, cel morphology

Evaluation criteria:

A finding is assessed as positive if the following criteria are met:
• Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range (see Appendix 5).
• Evidence of reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship. Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 106 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significant increased above the concurrent negative control and is within our historical negative control data range.

Statistics:

Due to the clearly negative findings, a statistical evaluation was not carried out.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In the pretest for toxicity based on the solubility properties of the test substance in the most suitable vehicle 2500 μg/mL (approx. 5 mM) was used as top concentration both with and without S9 mix at 4-hour exposure time and without S9 mix at 24-hour exposure time. Higher concentrations could not be formulated in a suitable solvent. The pretest was performed following the method described for the main experiment. The cloning efficiency 1 (survival) was determined as toxicity indicator for dose selection and various parameters were checked for all or at least for some selected doses. In the pretest the parameters pH value and osmolarity were not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. In culture medium test substance precipitation occurred at 78.1 μg/mL and above at the end of treatment in the absence and presence of S9 mix. After 4 and 24 hours treatment in the absence and presence of S9 mix no cytotoxicity indicated by reduced relative cloning efficiency of about or below 20% was observed.

In this study, no relevant increase in the number of mutant colonies was observed either without S9 mix or after the addition of a metabolizing system. In both experiments after 4 and 24 hours treatment with the test substance the values for the corrected mutation frequencies (MFcorr.: 0.00 – 9.68 per 10⁶ cells) were close to the respective vehicle control values (MFcorr.: 0.50 – 6.92 per 10⁶ cells) and clearly within the range of the historical negative control data (without S9 mix: MFcorr.: 0.00 – 15.95 per 10⁶ cells; with S9 mix: MFcorr.: 0.00 – 15.68 per 10⁶ cells). The positive control substances EMS (without S9 mix; 300 μg/mL) and MCA (with S9 mix; 20 μg/mL) induced clearly increased mutant frequencies as expected. The values of the corrected mutant frequencies (without S9 mix: MFcorr.: 108.87 – 381.88 per 10⁶ cells; with S9 mix: MFcorr.: 48.01 – 72.64 per 10⁶ cells) were clearly within the historical positive control data range (without S9 mix: MFcorr.: 48.83 – 1338.10 per 10⁶ cells; with S9 mix: MFcorr.: 26.29 – 413.54 per 10⁶ cells).

Table 5: Summary of results

Exp.	Exposure period	Test groups	S9 mix	Prec.*	Genotoxicity** MFcorr. [per 10^6cells]	Cytotoxicity***
						CE1 [%]	CE2 [%]
1	4 h	Vehicle control 3.13 µg/mL 6.25 µg/mL 12.50 µg/mL 25.00 µg/mL 50.00 µg/mL 100.00 µg/mL 200.00 µg/mL Positive control	- - - - - - - - -	- - - - - + + + -	0.50 n.c. n.c. 2.12 0.00 1.19 4.44 2.56 108.87	100.0 100.9 94.7 89.9 94.9 91.1 93.8 96.2 96.2	100.0 n.c. n.c. 117.4 86.4 99.2 95.4 100.3 94.1
2	24 h	Vehicle control 12.50 µg/mL 25.00 µg/mL 50.00 µg/mL 100.0 µg/mL 200.00 µg/mL Positive control	- - - - - - -	- - - + + + -	1.58 5.84 1.58 2.02 1.54 2.90 381.88	100.0 107.9 101.0 91.4 88.6 92.3 71.7	100.0 85.9 80.2 79.6 82.3 95.7 70.5
1	4 h	Vehicle control 3.13 µg/mL 6.25 µg/mL 12.50 µg/mL 25.00 µg/mL 50.00 µg/mL 100.00 µg/mL 200.00 µg/mL Positive control	+ + + + + + + + +	- - - - - + + + -	3.46 n.c. n.c. 5.10 1.84 0.47 1.97 3.17 72.64	100.0 96.5 89.8 97.7 86.9 96.8 95.4 93.3 94.4	100.0 n.c. n.c. 92.6 93.7 96.9 96.7 90.6 91.2
2	4 h	Vehicle control 18.80 µg/mL 37.50 µg/mL 75.00 µg/mL 150.00 µg/mL 200.00 µg/mL Positive control	+ + + + + + +	- - - + + + -	6.92 1.44 0.00 0.35 9.68 4.45 48.01	100.0 107.9 99.5 110.7 108.4 117.2 124.3	100.0 98.2 102.0 103.9 95.9 79.5 88.7

* Precipitation in culture medium at the end of exposure period

** Mutant frequency MFcorr.: mutant colonies per 10⁶ cells corrected with the CE2 value

*** Cloning efficiency related to the respective vehicle control

n.c. Culture was not continued since a minimum of only four analysable concentrations is required

Table 6: Cytotoxicity data - 1st Experiment without S9 mix; 4 -hour exposure period

Test groups		Cell density (x 10^5/mL) At 1^stsub-culture	CE1(survival) (4 h after treatment; about 200 cells/flask seeded)				CE2(viability) (at the end of the expression period; about 200 cells/flask seeded)
			Cells Flask 1	Cells Flask 2	Cloning efficiency [%]		Cells Flask 1	Cells Flask 2	Cloning efficiency [%]
					Abs.	Rel.			Abs.	Rel.
Vehicle control*	A	3.76	109	125	66.3	100.0	142	137	63.1	100.0
	B	6.19	155	141			110	115
3.13 µg/mL	A	5.75	116	142	66.9	100.9	n.c.	n.c.	n.c.	n.c.
	B	6.13	132	145			n.c.	n.c.
6.25 µg/mL	A	5.52	115	110	62.8	94.7	n.c.	n.c.	n.c.	n.c.
	B	6.26	132	145			n.c.	n.c.
12.50 µg/mL	A	5.56	126	111	59.6	89.9	137	128	74.1	117.4
	B	6.46	136	103			138	189
25.00 µg/mL	A	5.35	114	112	62.9	94.9	135	95	54.5	86.4
	B	7.95	128	149			110	96
50.00 µg/mL	A	5.23	117	123	60.4	91.1	111	108	62.6	99.2
	B	6.94	124	119			149	132
100.00 µg/mL	A	6.43	109	131	62.2	93.8	119	109	60.2	95.4
	B	7.72	138	119			125	128
200.00 µg/mL	A	5.18	109	125	63.8	96.2	153	142	63.3	100.3
	B	6.86	134	142			114	97
300.00 µg/mL EMS	A	5.52	120	122	63.8	96.2	116	103	59.4	94.1
	B	6.51	133	135			150	106

*THF 0.5 % (v/v)

Table 7: Cytotoxicity data - 1st Experiment with S9 mix; 4 -hour exposure period

Test groups		Cell density (x 10^5/mL) At 1^stsub-culture	CE1(survival) (4 h after treatment; about 200 cells/flask seeded)				CE2(viability) (at the end of the expression period; about 200 cells/flask seeded)
			Cells Flask 1	Cells Flask 2	Cloning efficiency [%]		Cells Flask 1	Cells Flask 2	Cloning efficiency [%]
					Abs.	Rel.			Abs.	Rel.
Vehicle control*	A	4.60	155	114	65.8	100.0	119	140	64.6	100.0
	B	5.34	135	122			127	130
3.13 µg/mL	A	5.34	150	90	63.5	96.5	n.c.	n.c.	n.c.	n.c.
	B	5.99	133	135			n.c.	n.c.
6.25 µg/mL	A	6.42	112	127	59.1	89.8	n.c.	n.c.	n.c.	n.c.
	B	6.31	123	110			n.c.	n.c.
12.50 µg/mL	A	6.50	133	153	64.3	97.7	137	105	59.8	92.6
	B	6.33	102	126			117	119
25.00 µg/mL	A	6.13	111	107	57.2	86.9	99	143	60.5	93.7
	B	6.00	122	117			103	139
50.00 µg/mL	A	6.69	136	119	63.7	96.8	128	109	62.6	96.9
	B	5.90	121	133			159	104
100.00 µg/mL	A	6.99	127	115	62.8	95.4	104	114	62.5	96.7
	B	6.89	109	151			136	146
200.00 µg/mL	A	7.06	128	116	61.4	93.3	75	111	58.5	90.6
	B	6.44	128	119			146	136
20.00 µg/mL MCA	A	6.80	129	110	62.1	94.4	98	139	58.9	91.2
	B	5.16	125	132			116	118

*THF 0.5 % (v/v)

Table 8: Cytotoxicity data - 2nd Experiment without S9 mix; 24 -hour exposure period

Test groups		Cell density (x 10^5/mL) At 1^stsub-culture	CE1(survival) (24 h after treatment; about 200 cells/flask seeded)				CE2(viability) (at the end of the expression period; about 200 cells/flask seeded)
			Cells Flask 1	Cells Flask 2	Cloning efficiency [%]		Cells Flask 1	Cells Flask 2	Cloning efficiency [%]
					Abs.	Rel.			Abs.	Rel.
Vehicle control*	A	4.67	147	127	76.4	100.0	187	182	90.3	100.0
	B	7.38	156	181			177	176
12.5 µg/mL	A	6.46	170	177	82.4	107.9	194	181	77.6	85.9
	B	7.55	157	155			125	120
25.0 µg/mL	A	7.20	157	138	77.2	101.0	151	151	72.4	80.2
	B	7.61	182	140			127	150
50.0 µg/mL	A	6.57	134	145	69.8	91.4	161	139	71.9	79.6
	B	7.60	130	149			145	130
100.0 µg/mL	A	6.94	123	137	67.7	88.6	132	156	74.3	82.3
	B	7.94	148	133			163	143
200.0 µg/mL	A	7.51	135	151	70.5	92.3	193	194	86.4	95.7
	B	8.25	135	143			174	130
300.00 µg/mL EMS	A	5.65	129	106	54.8	71.7	125	120	63.7	70.5
	B	7.07	107	96			115	149

*THF 0.5 % (v/v)

Table 9: Cytotoxicity data - 2nd Experiment with S9 mix; 4 -hour exposure period

Test groups		Cell density (x 10^5/mL) At 1^stsub-culture	CE1(survival) (4 h after treatment; about 200 cells/flask seeded)				CE2(viability) (at the end of the expression period; about 200 cells/flask seeded)
			Cells Flask 1	Cells Flask 2	Cloning efficiency [%]		Cells Flask 1	Cells Flask 2	Cloning efficiency [%]
					Abs.	Rel.			Abs.	Rel.
Vehicle control*	A	6.25	110	134	61.7	100.0	158	149	78.6	100.0
	B	5.90	126	123			163	158
18.8 µg/mL	A	6.59	135	132	66.6	107.9	170	150	77.2	98.2
	B	6.39	128	137			160	137
37.5 µg/mL	A	6.83	138	108	61.4	99.5	165	183	80.2	102.0
	B	6.31	122	123			141	152
75.0 µg/mL	A	6.02	113	124	68.3	110.7	162	166	81.7	103.9
	B	6.10	139	170			165	160
150.0 µg/mL	A	6.15	145	112	66.9	108.4	167	150	75.4	95.9
	B	5.19	126	152			147	140
200.0 µg/mL	A	6.30	144	149	72.3	117.2	127	123	62.5	79.5
	B	6.30	145	140			130	120
20.00 µg/mL MCA	A	6.86	128	155	76.7	124.3	155	137	69.7	88.7
	B	7.02	180	150			130	135

*THF 0.5 % (v/v)

Conclusions:

Under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Executive summary:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogeneous metabolic activation). Based on the solubility properties of the test substance and according to an initial range-finding cytotoxicity test for the determination of the experimental doses the following doses were tested and the doses in bold type were evaluated in this study:

1st Experiment

without S9 mix (4 -hour exposure period)

0; 3.13; 6.25; 12.5; 25.0; 50.0; 100.0; 200.0 µg/mL

with S9 mix (4 -hour exposure period)

0; 3.13; 6.25; 12.5; 25.0; 50.0; 100.0; 200.0 µg/mL

2nd Experiment

without S9 mix

0; 12.5; 25.0; 50.0; 100.0; 200.0 µg/mL

with S9 mix

0; 18.8; 37.5; 75.0; 150.0; 200.0 µg/mL

After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line.

Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations. In this study in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest applied concentration. On the basis from the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies both without S9 mix and / or after adding a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his, trp

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: rfa wall mutation, deletion of the uvrB gene, contain the R-factor plasmid, pKM101

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: uvrA DNA repair deficiency

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from aroclor induced rat liver

Test concentrations with justification for top dose:

The doses tested were 33.3,100, 333, 1000,3330, and 5000 µg per plate in both the presence and absence of S9 mix.

Vehicle / solvent:

- Vehicle: ethanol

Untreated negative controls:

yes

Remarks:

cells and medium

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene (TA 100, TA 1535, TA 1537, WP2uvrA, with S9 mix), ICR-191 (TA 1537, without S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation

DURATION
When S9 mix was not required, 100 /µL of tester strain and 100 /µL of vehicle or test article dose were added to 2.5 mL of molten selective top agar (maintained at 45 ± 2°C). When S9 mix was required, 500 /µL of S9 mix, 100 /uL of tester strain and 100 /µL of vehicle or test article dose were added to 2.0 mL of molten selective top agar. After the required components had been added, the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar contained in a 15 x 100 mm petri dish. After the overlay had solidified, the plates were inverted and incubated for 52 ± 4 hours at 37 ± 2°C. Positive control articles were plated using a 50 /µL plating aliquot.

NUMBER OF REPLICATIONS: in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Dose Rangefinding Assay
The growth inhibitory effect (cytotoxicity) of the test article to the test system was determined in order to allow the selection of appropriate doses to be tested in the mutagenicity assay.

Design
The dose rangefinding assay was performed using tester strains TAIOO and WP2MvrA both in the presence and absence of S9 mix. Ten doses of test article were tested at one plate per dose. The test article was checked for cytotoxicity up to a maximum concentration of 5 mg per plate.

Evaluation criteria:

Assay Evaluation Criteria
Once the criteria for a valid assay had been met, responses observed in the assay were evaluated as follows:
Tester Strains TA98, TA100, and WP2uvrA. For a test article to be considered positive, it had to produce at least a 2-foId increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.
Tester Strains TA1535 and TA1537. For a test article to be considered positive, it had to produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.

Statistics:

For all replicate platings, the mean revertants per plate and the standard deviation were calculated. The results of these calculations are presented in tabular form in the Data Tables section of this report. The historical control data are presented after the data tables.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: see above in study design

COMPARISON WITH HISTORICAL CONTROL DATA: yes, data were presented after data tables

ADDITIONAL INFORMATION ON CYTOTOXICITY: no

Table 1: Mutagenicity assay results - Summary of mean revertants per plate with standard deviation (SD), Experiment 1

	Dose/plate	TA 98		TA 100		TA 1535		TA 1537		WP2 uvrA		Background lawn
		Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Microsomes: rat liver vehicle control		34	3	109	3	16	4	10	2	19	1	Normal
Test article	33.3 µg 100 µg 333 µg 1000 µg 3330 µg 5000 µg	30 34 32 33 31 31	7 3 5 5 1 6	108 104 107 92 91 103	15 6 15 6 2 19	15 12 15 15 17 11	2 2 2 3 4 3	6 7 8 6 7 5	2 1 3 1 1 2	16 15 19 17 15 15	3 1 8 1 5 5	Normal Normal Normal Normal / slight precipitate Normal / moderate precipitate Normal / moderate precipitate
Positive control		461	27	1034	218	156	16	187	28	242	26	Normal
Microsomes: none vehicle control		26	3	108	13	11	2	6	2	17	4	Normal
Test article	33.3 µg 100 µg 333 µg 1000 µg 3330 µg 5000 µg	21 18 22 19 10 15	4 4 3 4 2 7	97 105 96 88 91 72	7 10 3 5 6 2	11 11 12 11 13 12	2 2 1 1 6 1	6 8 5 7 3 4	2 2 1 3 2 2	18 20 18 18 20 15	4 6 1 8 3 7	Normal Normal Normal / slight precipitate Normal / moderate precipitate Normal / moderate precipitate Normal / moderate precipitate
Positive control		173	13	657	60	509	6	585	12	186	19	Normal

Table 2: Mutagenicity assay results - Summary of mean revertants per plate with standard deviation (SD), Experiment 2

	Dose/plate	TA 98		TA 100		TA 1535		TA 1537		WP2 uvrA		Background lawn
		Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Microsomes: rat liver vehicle control		35	8	141	4	15	4	7	3	19	4	Normal
Test article	33.3 µg 100 µg 333 µg 1000 µg 3330 µg 5000 µg	37 33 51 37 34 34	4 6 3 3 8 5	125 132 124 127 114 116	4 8 11 13 8 23	19 15 20 20 17 11	2 1 2 6 1 4	9 7 9 9 10 9	2 3 1 4 3 2	18 19 22 22 18 13	2 6 3 3 6 1	Normal Normal Normal Normal / slight precipitate Normal / moderate precipitate Normal / moderate precipitate
Positive control		414	40	982	116	126	7	204	26	267	16	Normal
Microsomes: none vehicle control		18	6	102	7	12	4	8	2	19	2	Normal
Test article	33.3 µg 100 µg 333 µg 1000 µg 3330 µg 5000 µg	NC NC NC NC NC NC	- - - - - -	98 99 93 98 75 87	13 17 7 5 7 5	9 11 13 17 13 8	4 3 2 6 3 2	4 7 4 9 5 4	2 1 3 3 3 2	19 21 20 19 16 18	6 4 3 2 6 8	Normal Normal Normal / slight precipitate Normal / moderate precipitate Normal / moderate precipitate Normal / moderate precipitate
Positive control		16	4	538	76	476	64	498	83	244	43	Normal

Conclusions:

The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test substance did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor-induced rat liver (S9)

Executive summary:

The results of the dose rangefinding study were used to select the doses tested in the mutagenicity assay. The doses tested were 33.3, 100, 333,1000, 3330, and 5000 /µg per plate in both the presence and absence of S9 mix. In the initial mutagenicity assay, all data were acceptable and no positive increases in the mean number of revertants per plate were observed with any of the tester strains either in the presence or absence of S9 mix. In the confirmatory assay, all data generated with tester strain TA98 in the presence of S9 mix, and with tester strains TA100, TA1535, TA1537, and WP2uvrA in both the presence and absence of S9 mix, were acceptable, and no positive increases in the mean number of revertants per plate were observed with any of these tester strain/activation condition combinations. In this experiment, the mean positive control value for tester strain TA98 in the absence of S9 mix was not acceptable (did not exhibit at least a 3-fold increase over the mean vehicle control value). For this reason, the test article-treated plates were not scored and tester strain TA98 was retested in the absence of S9 mix in Experiment 21285-Dl. In Experiment 21221-Dl, the mean positive control value for tester strain TA98 in the absence of S9 mix was lower than routinely observed in this laboratory, although it did exhibit at least a 3-fold increase over the mean vehicle control for this tester strain. For this reason, tester strain TA98 was retested in the absence of S9 mix in Experiment 21221-D2. In Experiment 21221-D2, all data generated with tester strain TA98 in the absence of S9 mix were acceptable, and no positive increases in the mean number of revertants per plate were observed. All criteria for a valid study were met.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The CHO cell line was derived from an ovarian biopsy of a female Chinese hamster. The Chinese hamster ovary cells (CHO-WBL) used in this assay were from a permanent cell line and were originally obtained from the laboratory of Dr. S. Wolff, University of California, San Francisco. The cells were subsequently subcloned in this laboratory, and stock cultures stored in liquid nitrogen. The CHO-WBL subclone is a permanent cell line with an average cycle time of 12 to 14 hours and a modal chromosome number of 21.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from aroclor induced rat liver

Test concentrations with justification for top dose:

Initial assay
4.19, 5.98, 8.54, 12.2, 17.4, 24.8, 35.4, 50.5, 72.1, 103, 147, 210, and 300 µg/mL for 3.0 hours with and without S9 and harvested at 20.0 hours after the initiation of treatment. 72.1, 103, 147, and 210 µg/mL without metabohc activation and 50.1, 72.1, 103, and 147 µg/mL with metabolic activation were analyzed for chromosomal aberrations.

Confirmatory assay
6.25 - 200 µg/ml, 17.7h with/out activation, 75 - 200 µg/ml were evaluated

Vehicle / solvent:

The test article was dissolved in ethanol and treated at 1.0% (10.0 nL/mL). The vehicle control cultures were treated with 10.0 µL/mL of Ethanol.
Solubility was evaluated in water and dimethylsulfoxide, and the test article was not soluble or suspended at an acceptable level in either of these two vehicles.

Untreated negative controls:

yes

Remarks:

cells and medium

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- after 1 day culture initiation
- test article added: at 0h
- Exposure duration: 3h and 17.7h
- colcemid added: after 18h
- harvest started: after 20h

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 (100 per culture)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- test for mycoplasma contamination
- karyotype stability

Evaluation criteria:

Assay Evaluation Criteria
The following factors are taken into account in evaluation of the test article data:
• The number and percentages of aberrant cells excluding gaps (-g).
• The number and percentages of aberrant cells including gaps (+g).
• Evidence for increasing amounts of damage with increasing dose, i.e., a dose related increase in aberrations.
The experimental unit is the cell, and therefore the percentage of cells with structural aberrations was the basis for evaluation.

A test article was considered positive for inducing chromosomal aberrations if a significant increase (the difference was considered significant when p<0.01) in the number of cells with chromosomal aberrations is observed at one or more concentrations. The linear trend test evaluated the dose responsiveness. If a significant increase is seen at one or more concentrations, a dose-response should be observed.

A test article was considered negative for inducing chromosomal aberrations if no significant increase was observed in the number of cells with chromosomal aberrations at any of the concentrations.

Statistics:

Statistical analysis employed a Cochran-Armitage test for linear frend and Fisher's Exact Test (Thakur et a l , 1985) to compare the percentage of cells with aberrations in treated cells to the results obtained for the vehicle controls. Statistical analysis was also performed for cells exhibiting polyploidy and/or endoreduplication in order to indicate significant (p <0.01) increases in these events as indicators of possible induction of numerical aberrations; however, the test article was evaluated only for structural aberrations and not for numerical aberrations by this protocol.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Chromosomal Aberrations Assay Without Metabolic Activation

Initial Assay

A precipitate was observed after dosing and prior to washing in the cultures treated with 300 µg/mL, and a slight amount of precipitate was observed prior to harvest in these cultures. A precipitate was observed after dosing and prior to washing in the cultures treated with 210 µg/mL. A slight amount of precipitate was observed after dosing and prior to washing in the cultures treated with 147 µg/mL. No visible signs of toxicity were observed in any of the test cultures. Slight reductions of 15%, 9%, 3%, and 3% were observed in the mitotic indices of the cultures treated with 72.1, 103, 147, and 300 µg/mL as compared with the vehicle control cultures (Table 1). Chromosomal aberrations were analyzed from the cultures treated with 72.1, 103, 147, and 210 µg/mL (Table 2). No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed. Based on the results from the initial assay, the confirmatory aberrations assay was conducted testing concentrations of 6.25, 12.5, 25.0, 50.0, 75.0, 100, 150, and 200 µg/mL. Treatment

period was for 17.7 hours and cultures were harvested 20.0 hours from the initiation of treatment.

Table 1: Assessment of toxicity for chromosomal aberrations assay, 3h-treatment, 20h-harvest, -S9 mix

Treatment		Confluence^a % Solvent Control	% Mitotic Index A culture	% Mitotic Index B culture	Average % Mitotic Index	% Mitotic Index Reduction
Negative control		100	11.3	12.3	11.8	-
Vehicle control	Ethanol 10.0 µL/mL	100	12.4	8.9	10.7	0
Test article	72.1 µg/mL 103 µg/mL 147 µg/mL^b,d 210 µg/mL^c,e 300 µg/mL^c,e,f	100 100 100 100 100	8.8 9.3 8.5 9.0 10.0	9.3 10.0 12.2 12.7 10.8	9.1 9.7 10.4 10.9 10.4	15 9 3 0 3

^a This endpoint is based upon visual observations which are made prior to the harvest of the metaphase cells. At the time of the confluence observation the flasks are also evaluated for the appearance of floating mitotic cells and dead cells.

^b Slight precipitate at dose

^c Precipitate at dose

^d Slight precipitate at wash

^e Precipitate at wash

^f Slight precipitate at harvest

Table 2: Chromosomal Aberrations, cells fixed 20 hours after treatment, 3 hour treatment, without S9

Trial I: Cells Fixed 20.0 Hours After Initiation of Treatment, 3.0 Hour Treatment Metabolic Activation: -S9			% mitotoic index reduction	# Endoreduplicated cells	# Polyploid cells	Judgement^a	Numbers and precentages (%) of cells showing structural chromosomal aberrations								Judgement^c
							Gap	Chromatid Type		Chromosome Type		GT	Totals^b
	Dose level	Cells scored					g	ctb	cte	csb	cse		-g	+g
negative control (McCoy's 5a)		A 100		0	4								0	0
		B 100		0	4						1		1	1
		Total 200									1		1	1
		Average %	---	0	4						0.5		0.5	0.5
solvent control (ethanol)	10 µl/ml	A 100		0	1								0	0
		B 100		0	5								0	0
		Total 200											0	0
		Average %	0	0	3								0	0
Positive Control (MMC)	1.5 µg/ml	A 25		0	4		2	4	10			1	11	11
		B 25		0	5			4	7			5	13	13
		Total 50					2	8	17			6	24	24
		Average %	---	0	4.5	-	4	16	34			12	48	48	+
Test article	72.1 µg/ml	A 100		0	2								0	0
		B 100		0	1								0	0
		Total 200											0	0
		Average %	15	0	1.5	-							0	0	-
	103 µg/ml	A 100		0	2								0	0
		B 100		0	1								0	0
		Total 200											0	0
		Average %	9	0	1.5	-							0	0	-
	147 µg/ml	A 100		0	1								0	0
		B 100		0	3								0	0
		Total 200											0	0
		Average %	3	0	2	-							0	0	-
	210 µg/ml	A 100		0	1			1					1	1
		B 100		0	4								0	0
		Total 200						1					1	1
		Average %	0	0	2.5	-		0.5					0.5	0.5	-

ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange, GT: greater than 5 aberrations

^a Significantly greater in % polyploidy and % endoreduplication than the vehicle control, p ≤ 0.01.

^b -g = #or % of cells with chromosome aberrations; +g = # or % of cells with chromosome aberrations + # or % of cells with gaps.

^c Significantly greater in -g than the vehicle control, p ≤ 0.01

MMC = Mitomycin C

Confirmatory Assay

A slight amount of precipitate was observed after dosing and prior to washing in the cultures treated with 200 µg/mL. A slight amount of precipitate was observed prior to washing in the cultures treated with 150 µg/mL. No visible signs of toxicity were observed in any of the test cultures, except for debris in the cultures treated with 100, 150, and 200 µg/mL. Reductions of 1%, 22%, 40%, and 43% were observed in the mitotic indices of the cultures treated with 25.0, 100, 150, and 200 µg/mL as compared with the vehicle control cultures (Table 3). Chromosomal aberrations were analyzed from the cultures treated with 75.0, 100, 150, and 200 µg/mL (Table 4). No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed. The sensitivity of the cell culture for induction of chromosomal aberrations is shown by the increased frequency of aberrations in the cells exposed to mitomycin C, the positive control agent. The test article is considered negative for inducing chromosomal aberrations, polyploidy, and endoreduplication under nonactivation conditions.

Table 3: Assessment of toxicity for chromosomal aberrations assay, 17.6h-treatment, 20h-harvest, -S9 mix

Treatment		Confluence^a % Solvent Control	% Mitotic Index A culture	% Mitotic Index B culture	Average % Mitotic Index	% Mitotic Index Reduction
Negative control		100	6.5	7.7	7.1	-
Vehicle control	Ethanol 10.0 µL/mL	100	6.9	6.5	6.7	0
Test article	25.0 µg/mL 50.0 µg/mL 75.0 µg/mL 100 µg/mL 150 µg/mL^c 200 µg/mL^b,c	100 100 100 100 100 100	6.3 8.1 6.9 5.7 3.2 3.3	6.8 9.5 7.2 4.6 4.8 4.3	6.6 8.8 7.1 5.2 4.0 3.8	1 0 0 22 40 43

^aThis endpoint is based upon visual observations which are made prior to the harvest of the metaphase cells. At the time of the confluence observation the flasks are also evaluated for the appearance of floating mitotic cells and dead cells.

^bSlight precipitate at dose

^cSlight precipitation at wash

Table 4: Chromosomal Aberrations, cells fixed 20 hours after treatment, 17.6 hour treatment, without S9

Trial II: Cells Fixed 20.0 Hours After Initiation of Treatment, 17.6 Hour Treatment Metabolic Activation: -S9			% mitotoic index reduction	# Endoreduplicated cells	# Polyploid cells	Judgement^a	Numbers and precentages (%) of cells showing structural chromosomal aberrations								Judgement^c
							Gap	Chromatid Type		Chromosome Type		GT	Totals^b
	Dose level	Cells scored					g	ctb	cte	csb	cse		-g	+g
negative control (McCoy's 5a)		A 100		0	0								0	0
		B 100		0	1								0	0
		Total 200											0	0
		Average %	---	0	0.5								0	0
solvent control (ethanol)	10 µl/ml	A 100		0	0								0	0
		B 100		0	0								0	0
		Total 200											0	0
		Average %	0	0	0								0	0
Positive Control (MMC)	0.4 µg/ml	A 25		0	1		1	4	7	2		2	10	10
		B 25		0	2		1	4	9			1	12	12
		Total 50					2	8	16	2		3	22	22
		Average %	---	0	1.5	-	4	16	32	4		6	44	44	+
Test article	75 µg/ml	A 100		0	2								0	0
		B 100		0	0								0	0
		Total 200											0	0
		Average %	0	0	1	-							0	0	-
	100 µg/ml	A 100		0	4								0	0
		B 100		0	2								0	0
		Total 200											0	0
		Average %	22	0	3	-							0	0	-
	150 µg/ml	A 100		0	0								0	0
		B 100		0	0			1					1	1
		Total 200						1					1	1
		Average %	40	0	0	-		0.5					0.5	0.5	-
	200 µg/ml	A 100		0	1								0	0
		B 100		0	0								0	0
		Total 200											0	0
		Average %	43	0	0.5	-							0	0	-

ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange, GT: greater than 5 aberrations

^aSignificantly greater in % polyploidy and % endoreduplication than the vehicle control, p ≤ 0.01.

^b-g = #or % of cells with chromosome aberrations; +g = # or % of cells with chromosome aberrations + # or % of cells with gaps.

^cSignificantly greater in -g than the vehicle control, p ≤ 0.01

MMC = Mitomycin C

Chromosomal Aberrations Assay With Metabolic Activation

Initial Assay

A precipitate was observed after dosing and prior to harvest of the cultures treated with 210 and 300 µg/mL, and a slight amount of precipitate was observed prior to washing in these cultures. A slight amount of precipitate was observed after dosing, and prior to washing and harvest of the cultures treated with 147 µg/mL. A slight amount of precipitate was observed prior to harvest of the cultures treated with 103 µg/mL. No visible signs of toxicity were observed in any of the test cultures, except for debris in the cultures treated with 50.5, 72.1, 103, 147, 210, and 300 µg/mL. Slight reductions of 26%, 8%, and 12% were observed in the mitotic indices of the cultures treated with 50.1, 72.1, and 300 µg/mL as compared with the vehicle control cultures (Table 5). Chromosomal aberrations were analyzed from the cultures treated with 50.5, 72.1, 103, and 147 µg/mL (Table 6). No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed. Based on the results from the initial assay, the confirmatory aberrations assay was conducted testing concentrations of 50.0, 75.0, 100, 150, and 200 µg/mL. Treatment period was for 3.0 hours and cultures were harvested 20.0 hours from the initiation of treatment.

Table 5: Assessment of toxicity for chromosomal aberrations assay, 3h-treatment, 20h-harvest, + S9 mix

Treatment		Confluence^a % Solvent Control	% Mitotic Index A culture	% Mitotic Index B culture	Average % Mitotic Index	% Mitotic Index Reduction
Negative control		100	11.3	12.8	12.1	-
Vehicle control	Ethanol 10.0 µL/mL	100	12.5	14.1	13.3	0
Test article	50.5 µg/mL 72.1 µg/mL 103 µg/mL^e 147 µg/mL^b,d,e 210 µg/mL^c,d,f 300 µg/mL^c,d,f	100 100 100 100 100 100	10.0 11.6 15.2 14.1 12.9 11.8	9.7 12.9 11.7 14.5 14.6 11.6	9.9 12.3 13.5 14.3 13.8 11.7	26 8 0 0 0 12

^aThis endpoint is based upon visual observations which are made prior to the harvest of the metaphase cells. At the time of the confluence observation the flasks are also evaluated for the appearance of floating mitotic cells and dead cells.

^bSlight precipitate at dose

^cPrecipitate at dose

^dSlight precipitate at wash

^eSlight precipitate at harvest

^fPrecipitate at harvest

Table 6: Chromosomal Aberrations, cells fixed 20 hours after treatment, 3.0 hour treatment, with S9

Trial I: Cells Fixed 20.0 Hours After Initiation of Treatment, 3.0 Hour Treatment Metabolic Activation: +S9			% mitotoic index reduction	# Endoreduplicated cells	# Polyploid cells	Judgement^a	Numbers and precentages (%) of cells showing structural chromosomal aberrations								Judgement^c
							Gap	Chromatid Type		Chromosome Type		GT	Totals^b
	Dose level	Cells scored					g	ctb	cte	csb	cse		-g	+g
negative control (McCoy's 5a)		A 100		0	0		1						0	1
		B 100		1	2						2		2	2
		Total 200					1				2		2	3
		Average %	---	0.5	1		0.5				1		1	1.5
solvent control (ethanol)	10 µl/ml	A 100		0	0				1				1	1
		B 100		0	1								0	0
		Total 200							1				1	1
		Average %	0	0	0.5				0.5				0.5	0.5
Positive Control (CP)	5.0 µg/ml	A 25		0	4			2	8		2		9	9
		B 25		0	2		2	1	6		1		7	8
		Total 50					2	3	14		3		16	17
		Average %	---	0	3	-	4	6	28		6		32	34	+
Test article	50.5 µg/ml	A 100		0	2								0	0
		B 100		0	1						1		1	1
		Total 200									1		1	1
		Average %	26	0	1.5	-					0.5		0.5	0.5	-
	72.1 µg/ml	A 100		0	2		1				1		1	2
		B 100		0	2								0	0
		Total 200					1				1		1	2
		Average %	8	0	2	-	0.5				0.5		0.5	1	-
	103 µg/ml	A 100		0	0						1		1	1
		B 100		0	3								0	0
		Total 200									1		1	1
		Average %	0	0	1.5	-					0.5		0.5	0.5	-
	147 µg/ml	A 100		0	0		1						0	1
		B 100		1	0								0	0
		Total 200					1						0	1
		Average %	0	0.5	0	-	0.5						0	0.5	-

ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange, GT: greater than 5 aberrations

^aSignificantly greater in % polyploidy and % endoreduplication than the vehicle control, p ≤ 0.01.

^b-g = #or % of cells with chromosome aberrations; +g = # or % of cells with chromosome aberrations + # or % of cells with gaps.

^cSignificantly greater in -g than the vehicle control, p ≤ 0.01

CP = Cyclophosphamide

Confirmatory assay

A precipitate was observed after dosing and prior to washing of the cultures treated with 200 µg/mL, and a slight amount of precipitate was observed prior to harvest of these cultures. A precipitate was observed after dosing and prior to washing of the cultures treated with 150 µg/mL. A slight amount of precipitate was observed after dosing and prior to washing of the cultures treated with 100 µg/mL. No visible signs of toxicity were observed in any of the test cultures, except for debris in the cultures treated with 150 and 200 µg/mL. A slight reduction of 4% was observed in the mitotic indices of the cultures treated with 200 µg/mL as compared with the vehicle control cultures (Table 7). Chromosomal aberrations were analyzed from the cultures treated with 50.0, 75.0, 100, and 150 µg/mL (Table 8). No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed. The successful activation by the metabolic system is illustrated by the increased incidence of cells with chromosomal aberrations in the cultures induced with cyclophosphamide, the positive control agent. The test article was considered negative for inducing chromosomal aberrations, polyploidy, and endoreduplication under activation conditions.

Table 7: Assessment of toxicity for chromosomal aberrations assay, 3h-treatment, 20h- harvest, + S9 mix

Treatment		Confluence^a % Solvent Control	% Mitotic Index A culture	% Mitotic Index B Culture	Average % Mitotic Index	% Mitotic Index Reduction
Negative control		100	9.0	10.2	9.6	-
Vehicle control	Ethanol 10.0 µL/mL	100	8.3	9.9	9.1	0
Test article	50.0 µg/mL 75.0 µg/mL 100 µg/mL^b,d 150 µg/mL^c,e 200 µg/mL^c,e,f	100 100 100 100 100	9.8 10.5 12.1 10.8 9.2	8.5 10.4 9.0 8.2 8.1	9.2 10.5 10.6 9.5 8.7	0 0 0 0 4

^aThis endpoint is based upon visual observations which are made prior to the harvest of the metaphase cells. At the time of the confluence observation the flasks are also evaluated for the appearance of floating mitotic cells and dead cells.

^bSlight precipitate at dose

^cPrecipitate at dose

^dSlight precipitate at wash

^ePrecipitate at wash

^fSlight Precipitate at harvest

Table 8: Chromosomal Aberrations, cells fixed 20 hours after treatment, 3.0 hour treatment, with S9

Trial II: Cells Fixed 20.0 Hours After Initiation of Treatment, 3.0 Hour Treatment Metabolic Activation: +S9			% mitotoic index reduction	# Endoreduplicated cells	# Polyploid cells	Judgement^a	Numbers and precentages (%) of cells showing structural chromosomal aberrations								Judgement^c
							Gap	Chromatid Type		Chromosome Type		GT	Totals^b
	Dose level	Cells scored					g	ctb	cte	csb	cse		-g	+g
negative control (McCoy's 5a)		A 100		0	0								0	0
		B 100		0	0								0	0
		Total 200											0	0
		Average %	---	0	0								0	0
solvent control (ethanol)	10 µl/ml	A 100		0	2								0	0
		B 100		0	1								0	0
		Total 200											0	0
		Average %	0	0	1.5								0	0
Positive Control (CP)	5.0 µg/ml	A 25		1	2			1	12	1	1		13	13
		B 25		1	2			1	10	3			10	10
		Total 50						2	22	4	1		23	23
		Average %	---	1	2	-		4	44	8	2		46	46	+
Test article	50.0 µg/ml	A 100		0	2								0	0
		B 100		0	3								0	0
		Total 200											0	0
		Average %	0	0	2.5	-							0	0	-
	75.0 µg/ml	A 100		0	0								0	0
		B 100		0	2					1	1		2	2
		Total 200								1	1		2	2
		Average %	0	0	1	-				0.5	0.5		1	1	-
	100 µg/ml	A 100		0	0								0	0
		B 100		0	0								0	0
		Total 200											0	0
		Average %	0	0	0	-							0	0	-
	150 µg/ml	A 100		0	0						1		1	1
		B 100		0	0								0	0
		Total 200									1		1	1
		Average %	0	0	0	-					0.5		0.5	0.5	-

ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange, GT: greater than 5 aberrations

^aSignificantly greater in % polyploidy and % endoreduplication than the vehicle control, p ≤ 0.01.

^b-g = #or % of cells with chromosome aberrations; +g = # or % of cells with chromosome aberrations + # or % of cells with gaps.

^cSignificantly greater in -g than the vehicle control, p ≤ 0.01

CP = Cyclophosphamide

Conclusions:

The test substance was considered to be negative for inducing chromosomal aberrations in CHO cells with and without metabolic activation.

Executive summary:

The objective of this in vitro assay was to evaluate the ability of the test substance to induce chromosomal aberrations in cultured Chinese hamster ovary (CHO) cells with and without exogenous metabolic activation.

The test article was dissolved in ethanol and treated at 1.0 % (10.0 µL/mL). A stock solution of the test substance was prepared at a concentration of 30.0 mg/mL for the assay. The vehicle control cultures were treated with 10.0 µL/mL of Ethanol. The high dose tested, 300 µg/mL, was above the solubility limit in the test system.

In the initial trial of the chromosome aberrations assay, replicate cultures of CHO cells were incubated with 4.19, 5.98, 8.54, 12.2, 17.4, 24.8, 35.4, 50.5, 72.1, 103, 147, 210, and 300 µg/mL for 3.0 hours with and without S9 and harvested at 20.0 hours after the initiation of treatment. Cultures treated with 72.1, 103, 147, and 210 µg/mL without metabolic activation and 50.1, 72.1, 103, and 147 µg/mL with metabolic activation were analyzed for chromosomal aberrations. No increase in cells with chromosomal aberrations, percent polyploidy or endoreduplication was observed at the concentrations analyzed.

In the confirmatory trial, replicate cultures of CHO cells were incubated with 5.25, 12.5, 25.0, 50.0, 75.0, 100, 150, and 200 µg/mL without S9 and 50.0, 75.0, 100, and 150 µg/mL with S9. Treatment periods of 17.7 and 3.0 hours were used without and with S9, respectively, and cultures harvested at 20.0 hours after the initiation of treatment. Cultures treated with 75.0, 100, 150, and 200 µg/mL without S9 and 50.0, 75.0, 100, and 150 µg/mL with S9 were analyzed for chromosomal aberrations. No significant increase in cells with chromosomal aberrations, percent polyploidy or endoreduplication was observed at the concentrations analyzed.

The test substance was considered negative for inducing chromocomal aberrations in CHO cells with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames Test

An Ames test was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test using the S. typhimurium strains TA1535, TA 1537, TA 98, and TA 100, TA 1538 and the E. coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without S9 mix from aroclor induced rat liver and non-induced Syrian hamster liver. The test item was tested at concentrations of 5000, 2500, 1250, 625 and 312.5 µg/plate. No substantial increase in revertant colony numbers of any of the tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation.  Another GLP-conform Ames test according OECD guideline 471 was performed. The tester strains used in this mutagenicity assay were S. typhimurium tester strains TA98, TA100, TA1535, TA1537, and E. coli tester strain WP2uvrA. The assay was conducted with seven doses of test article, dissolved in acetone, in the presence of S9 mix and nine doses of test article in the absence of S9 mix along with concurrent vehicle and positive controls. The doses tested were 5000, 3330, 1000, 333, 100, 33.3, and 10.0 /µg per plate in the presence of S9 mix and 5000, 3330, 1000, 333, 100, 33.3, 10.0, 3.33, and 1.00 /µg per plate in the absence of S9 mix. The test substance did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of metabolic activation.

Chromosome Aberration

In order to evaluate the ability of the test substance to induce chromosomal aberrations in cultured Chinese hamster ovary (CHO) cells with and without exogenous metabolic activation a chromosomal aberration test was performed. The test article was dissolved in ethanol and treated at 1.0% (10.0 µl/ml). The high dose tested, 300 µg/ml, was above the solubility limit in the test system. In the initial trial of the chromosome aberrations assay, replicate cultures of CHO cells were incubated with 4.19 to 300 µg/ml for 3.0 hours with and without S9 and harvested at 20.0 hours after the initiation of treatment. In the confirmatory trial, replicate cultures of CHO cells were incubated with 6.25 to 200 µg/ml without S9 and 50.0 to 200 µg/ml with S9. Treatment periods of 17.7 and 3.0 hours were used without and with S9, respectively, and cultures harvested at 20.0 hours after the initiation of treatment. No increase in cells with chromosomal aberrations, percent polyploidy or endoreduplication was observed at the concentrations analyzed.

HPRT

The test material was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced. Based on the solubility properties of the test substance and according to an initial rangefinding cytotoxicity test doses from 3 -200 µg/ml were tested in presence and absence of a metabolic activation system. On the basis from the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies both without S9 mix

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.