2-Aminoethanol reaction products with cyclohexane and peroxidized N-butyl-2,2,6,6-tetramethyl-4-piperidinamine-2,4,6-trichloro-1,3,5-triazine reaction products

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genotoxic potential of the test material was evaluated in an Ames test, a mouse lymphoma assay and a chromosome aberration assay in mammalian cells (similar to OECD guideline 471, 476 and 473, GLP). The substance did not increase the number of revertants in bacteria, was negative in the mouse lymphoma forward mutation assay and did not induce chromosomal damages or polyploidy in mammalian cells. In the course of a screening Ames assay, a single increase in revertants in one tester strain was observed. The substance identity and purity of the material used in this study was not verified, a confirmatory experiment was not conducted. The study is therefore disregarded. The test item is not considered to be genotoxic.

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:

1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

A continuous cell log was kept to record growth, doubling times, and subculture operations. Laboratory cultures were periodically tested for mycoplasma contamination and karyotype. To reduce the frequency of spontaneous TK-‘. mutants prior to use in the mutation assay, cell cultures were exposed to conditions that selected against the TK +/- phenotype (exposure to methotrexate).
The medium used for this study was RPMI 1640 supplemented with Pluronic F68, L-glutamine, sodium pyruvate, antibiotics, and heat-inactivated horse serum (10% by volume). Treatment medium was Fischers medium with the same media supplements used in the culture medium except that the horse serum concentration was reduced to 5% by volume. Cloning medium consisted of the preceding growth medium with up to 20% horse serum, without Pluronic” F68 and with the addition of BBL purified agar at a final concentration of 0.24% to achieve a semisolid state. Selection medium was cloning medium that contained 3 ug/ml of TFT.

Metabolic activation:

with and without

Metabolic activation system:

rat liver enzymes (S9 fraction, Aroclor induced) and an energy producing system (CORE) comprised of nicotinamide adenine dinucleotide phosphate (NADP., sodium salt) and isocitrate.

Test concentrations with justification for top dose:

5 - 80 µg/ml

Vehicle / solvent:

- insoluble in dimethylsulfoxide (DMSO) at 50 mg/ml and 100 mg/ml
- formed a clear, yellow solution in ethanol at 100 mg/ml. Ethanol was therefore chos’en as the vehicle. The test article was prepared in ethanol at 100x times the highest desired concentration. Lower 100x stocks were prepared by serial dilution with ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h
- Expression time (cells in growth medium): 2 days

NUMBER OF REPLICATIONS: two independent experiments, every sample in triplicate

NUMBER OF CELLS EVALUATED: all colonies are counted

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
- pH
- osmolarity
- solubility
- cell morphology

Evaluation criteria:

- A dose-related or toxicity-related increase in mutant frequency should be observed. It is desirable to obtain this relation for at least three doses, but this depends upon the concentrat,ion steps chosen for the assay and the toxicity at which mutagenic activity appears.

- If the mutant frequency obtained for a single dose at or near the highest, testable toxicity is about two or more times the minimum criterion, the test article will be considered mutagenic in a single trial. Smaller increases at a single dose near the highest testable toxicity will require confirmation by a repeat assay.

- For some test articles, the correla,tion between toxicity and applied concentration is poor. The proportion of the applied test article that effectively interacts with the cells to cause genetic alterations is not always repeatable or under control. Conversely, measurable changes in frequency of induced mutants may occur with concentration changes that cause only small changes in observed toxicity. Therefore, either parameter, applied concentration or toxicity (perceni. relative growth), can be used to establish whether the increase in mutant frequency is related to an increase in effective treatment.

- Treatments that induce less than 10% relative growth are included in the assay, but are not used as primary evidence for mutagenicity as it relates to risk assessment.

A test article is evaluated as non-mutagenic in a single assay only if the minimum increase in mutant frequency is not observed for a range of applied concentrations
that extends to toxicity causing 10% to 20% relative growth or, in the case of relatively nontoxic materials, a range of applied concentrations extending to the maximum of 5000 pglml or to at least twice the solubility limit in medium.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity was observed from 15 ug/ml onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Dose Range Finding Assay

The test article was tested in a preliminary dose range finding assay both with and without S9 metabolic activation. Ten dose levels were used in each case that ranged from 1.95 μg/ml to 1000 μg/ml; vehicle and untreated controls were included under each activation condition (Table 1). The test article was weakly to non-cytotoxic with and without metabolic activation from 1.95 μg/ml to 31.3 μg/ml and treatments from 62.5 μg/ml to 1000 μg/ml were very highly cytotoxic or lethal under both activation conditions. The mutation assays were initiated with treatments based on the results of the cytotoxicity assay.

TABLE 1: Cytotoxicity Assay

	Without S9 Activation		With S9 Activation
Applied concentration (µg/ml)	cell density/ml (x10^5)	% vehicle control	cell density/ml (x10^5)	% vehicle control
negative (media) control	10.5	147.9	13.3	133.0
vehicle control (1% ethanol)	7.1	100.0	10.0	100.0
1.95	NTC	--	NTC	--
3.91	NTC	--	NTC	--
7.81	NTC	--	NTC	--
15.6	12.8	180.3	12.9	129.0
31.3	4.5	63.4	8.3	83.0
62.5	0.0	0.0	0.3	3.0
125	0.0	0.0	0.0	0.0
250	0.0	0.0	0.0	0.0
500	0.0	0.0	0.0	0.0
1000	0.0	0.0	0.0	0.0

NTC = not counted

Mutation Assay

Three non-activation mutation assays were performed with the test article but Trial 2 was unacceptable because of high vehicle control mutant frequencies. In Trial 1 (Table 2), nine dose levels were initiated at 5.00, 10.0, 15.0, 20.0, 30.0, 40.0, 50.0, 60.0 and 80.0 μg/ml. Treatments from 20.0 μg/ml to 80.0 μg/ml were terminated because of excessive cytotoxicity. The remaining three treatments induced little or no cytotoxicity (90.4% to 62.5% relative growths), and a small increase in concentration from 15.0 μg/ml to 20.0 μg/ml was excessively cytotoxic.

In order for a culture to be evaluated as mutagenic in Trial 1 of the non-activation assay, a mutant frequency greater than 92.6 x 10^-6 was required. This threshold value was equal to twice the average mutant frequency of the concurrent vehicle controls. None of the analyzed test item treatments induced a mutant frequency that exceeded the minimum criterion and the test article was considered non-mutagenic in this trial. A confirmatory assay was initiated.

TABLE 2: Mutation assay without activation (Trial 1)

	Daily cell counts (cells/ml, 10^5 units)		Suspension growth	Total mutant colonies	Total viable colonies	Cloning efficiency	Relative growth (%)	Mutant Frequencies (10^-6 units)
	1	2
Vehicle control	17.8	14.6	28.9	97	355	59.2	100.0	54.6
Vehicle control	16.2	14.6	26.3	87	383	63.8	100.0	45.4
Vehicle control	17.4	16.0	30.9	75	386	64.3	100.0	38.9
Vehicle average			28.7			62.4
MMS 5 nl/ml	12.8	16.8	23.9	291	417	69.5	92.8	139.6#
MMS 10 nl/ml	9.2	12.9	13.2	393	187	31.2	23.0	420.3#
MMS 15 nl/ml	9.0	12.3	12.3	317	113	18.8	12.9	561.1#
			relative to vehicle control (%)			relative to vehicle control (%)
Test article 5 µg/ml	15.6	15.3	92.4	82	366	97.8	90.4	44.8
Test article 10 µg/ml	13.0	15.3	77.0	97	404	107.9	83.1	48.0
Test article 15 µg/ml	12.2	12.2	56.7	115	413	110.3	62.5	55.7

Suspension Growth = (Day 1 count / 3) x (Day 2 count) / (3 or Day 1 count if not split back)

Cloning Efficiency = total viable colony count / number of cells seeded x 100

Relative Growth = (Relative suspension growth x relative cloning efficiency) / 100

Mutant Frequency = (total mutant colonies/total viable colonies) x 2x10^-4 (decimal is moved to express the frequency in units of 10^-6)

Vehicle control = 1% ethanol; MMS = Methyl methansulfonate positive control

# mutagenicity exceeds minimum criterion of 92.6 x 10^-6

In Trial 3 of the non-activation assay (Table 3), ten treatments at 5.00, 7.50, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0 and 50.0 μg/ml were initiated and treatments above 25.0 μg/ml were terminated because of excessive cytotoxicity. However, there was a calculation error and the 25.0 μg/ml treatment was actually 20.0 μg/ml and is indicated as such in Table 3. The six analyzed treatments induced a wide range of toxic action (97.5% to 14.8% relative growths). The minimum criterion for a positive response in Trial 3 of the nonactivation assay was 247.1 x 10^-6. None of the assayed treatments induced this level of mutant action and no dose related trend was observed. The test article was therefore considered non-mutagenic without activation in this assay. The background mutant frequency (average vehicle control mutant frequency) was 123.6 x 10^-6, which is slightly above the usual range of 30 x 10-6 to 120 x 10^-6. The range was meant as a guide and all other acceptance criteria were met for this assay. Higher backgrounds are occasionally observed when especially good recovery of mutants occur, therefore, in this cast. the small increase above the usual range was not considered indicative of an unacceptable assay.

TABLE 3: Mutation assay without activation (Trial 3, confirmatory assay)

	Daily cell counts (cells/ml, 10^5 units)		Suspension growth	Total mutant colonies	Total viable colonies	Cloning efficiency	Relative growth (%)	Mutant Frequencies (10^-6 units)
	1	2
Vehicle control	9.5	12.3	13.0	205	444	74.0	100.0	92.3
Vehicle control	12.0	10.7	14.3	302	465	77.5	100.0	129.9
Vehicle control	7.8	14.0	12.1	288	388	64.7	100.0	148.5
Vehicle average			13.1			72.1
MMS 5 nl/ml	7.2	10.8	8.6	973	305	50.8	46.3	638.0#
MMS 10 nl/ml	8.8	6.3	6.2	697	199	33.2	21.8	700.5#
MMS 15 nl/ml	6.0	4.4	2.9	255	48	8.0	2.5	1062.5#
			relative to vehicle control (%)			relative to vehicle control (%)
Test article 5 µg/ml	6.7	13.8	78.4	235	446	103.1	80.3	105.4
Test article 7.5 µg/ml	6.7	11.9	67.6	194	309	71.4	48.3	125.6
Test article 10 µg/ml	6.9	8.3	48.6	240	453	104.7	50.9	106.0
Test article 15 µg/ml	11.1	10.8	101.7	204**	394**	95.9	97.5	103.6
Test article 20 µg/ml	1.6*	5.8	14.8	256	432	99.9	14.8	118.5
Test article 20 µg/ml	3.0*	8.2	20.9	261	571	132.0	27.6	91.4

Suspension Growth = (Day 1 count / 3) x (Day 2 count) / (3 or Day 1 count if not split back)

Cloning Efficiency = total viable colony count / number of cells seeded x 100

Relative Growth = (Relative suspension growth x relative cloning efficiency) / 100

Mutant Frequency = (total mutant colonies/total viable colonies) x 2x10^-4(decimal is moved to express the frequency in units of 10^-6)

Vehicle control = 1% ethanol; MMS = Methyl methansulfonate positive control

# mutagenicity exceeds minimum criterion of 247.1 x 10^-6

* not split back

** dilution error: actually plated 570 cells instead of 600 for total viable colonies and 2.85 x 10⁶ instead of 3 x 10⁶ for total mutant colonies.

Three trials of the activation assay were also initiated but Trial 2 was unacceptable because of excessively high vehicle control mutant frequencies. The results from Trials 1 and 3 are shown in Tables 4 and 5. In Trial 1 (Table 4), nine cultures were dosed with the test article at 5.00, 10.0, 15.0, 20.0, 30.0, 40.0, 50.0, 60.0 and 80.0 μg/ml but doses from 50.0 μg/ml to 80.0 μg/ml were terminated because of excessive cytotoxicity. The remaining six doses were non-cytotoxic to highly cytotoxic (89.4% to 20.1% relative growth). In order for a culture to be evaluated as mutagenic in this assay, a mutant frequency of greater than 102.4 x 10^-6 was required. None of the analyzed doses induced a mutant frequency that exceeded the minimum criterion. The test article was considered non-mutagenic in this trial. Another trial was initiated to confirm these results.

TABLE 4: Mutation assay with activation (Trial 1)

	Daily cell counts (cells/ml, 10^5 units)		Suspension growth	Total mutant colonies	Total viable colonies	Cloning efficiency	Relative growth (%)	Mutant Frequencies (10^-6 units)
	1	2
Vehicle control	13.2	22.1	32.4	119	526	87.7	100.0	45.2
Vehicle control	14.0	18.6	28.9	124	442	73.7	100.0	56.1
Vehicle control	10.7	18.9	22.5	137	524	87.3	100.0	52.3
Vehicle average			27.9			82.9
MCA 2 µg/ml	10.0	15.4	17.1	502	402	67.0	49.5	249.8#
MCA 4 µg/ml	5.8	15.7	10.1	557	393	65.5	28.6	283.5#
			relative to vehicle control (%)			relative to vehicle control (%)
Test article 5 µg/ml	10.2	18.3	74.3	147	492	98.9	73.5	59.8
Test article 10 µg/ml	10.0	24.6	98.0	82	435	87.5	85.8	37.7
Test article 15 µg/ml	11.1	17.3	76.5	98	581	116.8	89.4	33.7
Test article 20 µg/ml	5.5	18.4	40.3	155**	667**	134.1	54.0	46.5
Test article 30 µg/ml	7.8	19.7	61.2	117	424	85.2	52.1	55.2
Test article 40 µg/ml	3.5*	21.8	26.0	81	385	77.4	20.1	42.1

Suspension Growth = (Day 1 count / 3) x (Day 2 count) / (3 or Day 1 count if not split back)

Cloning Efficiency = total viable colony count / number of cells seeded x 100

Relative Growth = (Relative suspension growth x relative cloning efficiency) / 100

Mutant Frequency = (total mutant colonies/total viable colonies) x 2x10^-4(decimal is moved to express the frequency in units of 10^-6)

Vehicle control = 1% ethanol; MCA = Methylchloranthrene positive control

# mutagenicity exceeds minimum criterion of 102.4 x 10^-6

* not split back

** dilution error: actually plated 1010 cells instead of 600 for total viable colonies and 5.05 x 10⁶instead of 3 x 10⁶for total mutant colonies.

In the third activation assay (Table 5), ten doses, at 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 50.0, 60.0 and 70.0 μg/ml were initiated and six doses from 15.0 μg/ml to 40.0 μg/ml were chosen for mutant analysis. However, there was a calculation error and the 25.0 μg/ml treatment was actually 20.0 μg/ml and is indicated as such in Table 5. Treatments above 40.0 μg/ml were excessively cytotoxic, and the 10.0 μg/ml treatment was terminated because a sufficient number of noncytotoxic doses were available for analysis. The selected doses induced a wide range of cytotoxicity (102.9% to 22.2% relative growth). In order for a dose to be considered positive in this assay, a mutant frequency exceeding 260.2 x 10^-6 was required. None of the analyzed test article treatments induced a mutant frequency that exceeded the minimum criterion and no dose-response was observed. These results confirmed the lack of activity observed in Trial 1. The results of the two activation mutation trials were therefore evaluated as negative for inducing forward mutation at the TK locus in mouse lymphoma cells. As was observed with Trial 3 of the nonactivation assay, the background mutant frequency (130.1 x 10^-6) was slightly above the usual range. Since the increase was small and other criteria for an acceptable assay were met, the trial was considered acceptable.

TABLE 5: Mutation assay with activation (Trial 3, confirmatory assay)

	Daily cell counts (cells/ml, 10^5 units)		Suspension growth	Total mutant colonies	Total viable colonies	Cloning efficiency	Relative growth (%)	Mutant Frequencies (10^-6 units)
	1	2
Vehicle control	8.9	16.4	16.2	329	418	69.7	100.0	157.4
Vehicle control	9.9	16.5	18.2	261	407	67.8	100.0	128.3
Vehicle control	10.5	14.3	16.7	250	478	79.7	100.0	104.6
Vehicle average			17.0			72.4
MCA 2 µg/ml	2.0*	8.6	2.9	828	226	37.7	8.9	732.7#
MCA 4 µg/ml	4.9	13.6	7.4	924	260	43.3	26.0	710.8#
			relative to vehicle control (%)			relative to vehicle control (%)
Test article 15 µg/ml	10.6	15.6	108.1	C	325	74.8	80.9	-----
Test article 20 µg/ml	9.2	18.3	110.0	176	307	70.7	77.8	114.7
Test article 20 µg/ml	11.3	15.4	113.7	264 ++	393	90.5	102.9	134.4
Test article 30 µg/ml	7.9	16.3	84.2	280	400	92.1	77.5	140.0
Test article 35 µg/ml	5.8	14.5	55.0	323	427	98.3	54.1	151.3
Test article 40 µg/ml	2.1*	13.1	25.7	294	375	86.3	22.2	156.8

Suspension Growth = (Day 1 count / 3) x (Day 2 count) / (3 or Day 1 count if not split back)

Cloning Efficiency = total viable colony count / number of cells seeded x 100

Relative Growth = (Relative suspension growth x relative cloning efficiency) / 100

Mutant Frequency = (total mutant colonies/total viable colonies) x 2x10^-4(decimal is moved to express the frequency in units of 10^-6)

Vehicle control = 1% ethanol; MCA = Methylchloranthrene positive control

# mutagenicity exceeds minimum criterion of 260.2 x 10^-6

* not split back

C two or more plates contaminated

++ one plate contaminated: value determined by weight proportion

The average cloning efficiencies for the vehicle controls varied from 62.4% and 72.1% without activation to 82.9% and 72.4% with S9 metabolic activation which demonstrated acceptable cloning conditions for the assays. The positive control cultures, MMS (nonactivation) and MCA (activation) induced large increases in mutant frequency that were greatly in excess of the minimum criteria.

In conclusion, the test material was negative in the mouse lymphoma forward mutation assay both with and without S9 metabolic activation under the conditions of testing.

Conclusions:

The test material was evaluated as negative in the mouse lymphoma forward mutation assay both with and without S9 metabolic activation over a wide range of cytotoxicity.

Executive summary:

The objective of this in vitro assay was to evaluate the ability of the test article to induce forward mutations at the thymidine kinase (TK) locus in the mouse lymphoma L5178Y cell line. The test article was soluble in ethanol at 100 mg/ml and soluble in medium up to 40 µg/ml. Higher concentrations of the test item in medium contained a white precipitate. In the preliminary dose range finding assay, cells were exposed to the test article for about four hours in the presence and absence of rat liver S9 metabolic activation. Treatment from 1.95 µg/ml to 1000 µg/ml were initiated. The test article was weakly to non-cytotoxic with and without metabolic activation from 1.95 µg/ml to 31.3 µg/ml and treatments from 62.5 µg/ml to 1000 µg/ml were very highly cytotoxic or lethal. The mutation assays were initiated with treatments based on the results of the cytotoxicity assay. Two non-activation mutation assays were used in the evaluation. In the first non-activation trial, the test article was lethal or excessively toxic above 15.0 µg/ml. The remaining three treatments from 5.00 µg/ml to 15.0 µg/ml induced weak cytotoxicity. None of the three analyzed treatments induced a mutant frequency that exceeded the minimum criterion for a positive response and a small increase in concentration from 15.0 µg/ml to 20.0 µg/ml was excessively cytotoxic. An independent repeat assay was performed to confirm these results. In the independent repeat nonactivation assay, six treatments from 5.00 µg/ml to 20.0 µg/ml were analyzed for mutant induction and a wide range of cytotoxicity was induced. Even in the presence of high cytotoxicity, there was no evidence for a mutagenic response. Two activation mutation trials were also used in the evaluation. In the first activation trial, six treatments from 5.00 µg/ml to 40.0 µg/ml were evaluated for mutant induction. In the confirmatory trial, six treatments from 15.0 µg/ml to 40.0 µg/ml were analyzed. A wide range of cytotoxicity was induced in both trials and no evidence for a mutagenic response was observed. The test material was evaluated as negative in the mouse lymphoma forward mutation assay both with and without S9 metabolic activation over a wide range of cytotoxicity.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-02-02 - 1999-03-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted on 21st July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Exogenous metabolic activation system of mammalian microsomal enzymes derived from Aroclor-induced male rat liver (S9).

Test concentrations with justification for top dose:

10.0, 33.3, 100, 333, 1000, and 5000 µg per plate

Vehicle / solvent:

VEHICLE
- Vehicle used: Ethanol
- Justification for choice of solvent/vehicle: relatively non-toxic, standard vehicle for studies of this type
- Concentrations: 50 mg/mL, which was the most concentrated stock dilution prepared, the test item formed a slightly cloudy, colorless suspension. The test item formed a solution at 10.0 mg/mL and remained a solution in all succeeding dilutions prepared for the mutagenicity assay.

ASSAY
The assay was conducted with six doses of test item in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per dose.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Sodium azide, 4-NQO, 2-nitrofluorene, CR-191

Remarks:

Not requiring metabolic activation (S9 mix)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, benzo[a]-pyrene

Remarks:

Requiring metabolic activation (S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation method)

DURATION
- Preincubation period: none
- Exposure duration: 52 ± 4 hours
- Expression time (cells in growth medium): see exposure duration
- Selection time (if incubation with a selection agent): see exposure duration
- Fixation time (start of exposure up to fixation or harvest of cells): Plates which were not evaluated immediately following the incubation period
were held at 5 ± 3°C until evaluation.

SELECTION AGENT (mutation assays)
- S. typhimurium strains: histidine/biotin solution for selection of histidine revertants
- E. coli strain: tryptophan solution for selection of tryptophan revertants
- Further details on overlay agar for selection of revertants:
Overlay (top) agar was prepared with 0.7% agar (w/v) and 0.5% NaCl (w/v) and was supplemented with 10 mL of 1) 0.5 mM histidine/biotin solution per 100 mL agar for selection of histidine revertants, or 2) 0.5 mM tryptophan solution per 100 mL of agar for selection of tryptophan revertants. When S9 mix was required, 2.0 mL of the supplemented top agar was used in the overlay. However, when S9 mix was not required, water was added to the supplemented top agar (0.5 mL of water per 2 mL of supplemented top agar) and the resulting 2.5 mL of diluted supplemented top agar was used for the overlay.

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED:
- Number of colonies: colony counter and manually (counting revertant colonies)

DETERMINATION OF CYTOTOXICITY
- Method: Bacterial Background Lawn Evaluation

Evaluation criteria:

EVALUATION OF TEST RESULTS

Criteria For A Valid Assay

Tester Strain Integrity
- Salmonella typhimurium: rfa Wall Mutation, pKM101 Plasmid, Characteristic Number of Spontaneous Revertants (TA98 8 – 60; TA100 60 – 240, TA1535 4 – 45, TA1537 2 – 25)

- Escherichia coli: Characteristic Number of Spontaneous Revertants (WP2 uvrA 5 – 40)
Tester Strain Culture Density

Positive Control Values: mean value of the positive control for tester strain in the presence of the S9 mix exhibited at least a 3-fold increase over the mean value of the vehicle control

Cytotoxicity: A minimum of three non-toxic doses were required to evaluate assay data

Criteria For A Positive Response:
- TA98 TA100 and WP2 uvrA - 2-fold increase in the mean revertants per plate over the mean revertants per plate of the appropriate vehicle control
- TA 1535 and TA 1537 - 3-fold increase in the mean revertants per plate over the mean revertants per plate ofthe appropriate vehicle control

Statistics:

No appropriate statistical method is available. Due to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time.

Species / strain:

other: all strains tested

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Slight test item precipitate was observed at the 333 µg per plate dose level and moderate test item precipitate was observed at the 1000 and 5000 µg per plate dose levels. This precipitate did not effect the scoring of these plates. A non-dose responsive 2.1-fold increase was observed with tester strain WP2uvrA in the absence of S9 mix in the initial trial. Due to the lack of a dose response, this increase did not meet the criteria for a positive evaluation. No increase was observed with this strain and activation condition in the confirmatory trial.

Test item: S. typhimurium / E. coli reverse mutation assay
Plate incorporation without S-9 mix: experiment I/II
Dose (µg/plate)	TA100	TA 1535	WP2uvrA	TA 98	TA1537
Solvent control	75 / 77	12 / 11	10 / 17	17 / 13	3 / 6
10.0	75 / 63	10 / 9	19 / 13	24 / 18	7 / 6
33.3	74 / 56	8 / 13	21 / 16	18 / 14	4 / 5
100.0	72 / 62	9 / 13	16 / 14	17 / 14	4 / 5
333.3	82 / 65	10 / 11	17 / 15	18 / 18	4 / 5
1000.0	84 / 69	13 / 6	13 / 10	14 / 14	5 / 3
5000.0	84 / 64	9 / 7	11 / 10	17 / 14	7 / 4
Positive controls
- Sodum azide (2.0 µg/plate)	564 / 530	559 / 499
- 4-NQO (1.0 µg/plate)			631 / 318
- 2-nitrofluorene (1.0 µg/plate)				126 / 138
- ICR-191 (2.0 µg/plate)					859 / 916
Plate incorporation with S-9 mix: experiment I/II
Dose (µg/plate)	TA100	TA 1535	WP2uvrA	TA 98	TA1537
Solvent control	70 / 74	13 / 14	13 / 14	28 / 24	5 / 7
10.0	85 / 85	15 / 14	17 / 12	27 / 26	7 / 8
33.3	84 / 75	10 / 12	18 / 17	36 / 33	5 / 7
100.0	88 / 78	17 / 11	14 / 13	37 / 28	9 / 9
333.3	88 / 88	15 / 12	14 / 19	35 / 28	6 / 6
1000.0	86 / 85	13 / 12	13 / 14	33 / 37	8 / 7
5000.0	81 / 68	13 / 12	9 / 11	26 / 21	3 / 6
Positive control
- 2 -Aminoanthracene (2.5 µg/plate)  - 2 -Aminoanthracene (25 µg/plate)	716 / 787	130 / 121	422 / 336		136 / 133
- benzo[a]-pyrene (2.5 µg/plate)				307 / 249
Revertants/plate (I/II): mean from three plates

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-02-02 - 1999-03-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted on 21st July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No target gene

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: CHO cells were grown in McCoy's 5a culture medium which was supplemented with 10 % fetal bovine serum (FBS), L-glutamine (2mM), penicillin G (100 units/mL) and streptomycin (100 µg/mL)
- Cultures were incubated in a humidified incubator at 37°C ± 2°C in an atmosphere of 5% ± 1.5% CO2 in air.
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: Cells have been recloned to maintain karyotypic stability
- Periodically "cleansed" against high spontaneous background: no data
- CHO cell line has an average cycle time of 12 to 14 hours with a modal chromosome number of 21.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat-liver homogenate (S9).
The in vitro metabolic activation system consisted of a rat liver post-mitochondrial fraction (S9) and an energy-producing system (NADP plus isocitric acid). Prior to collecting livers from male Sprague-Dawley rats, various P450 isoenzyme levels were increased by treatment ofthe rats with Aroclor 1254. Rats were treated once with 500 mg/kg of Aroclor 1254 and S9 prepared approximately 5 days later. The S9 fraction, prepared in sucrose or in potassium chloride, were purchased from a commercial supplier (Molecular Toxicology, Inc.; Lot #810) nd are retained frozen at ≤ -70°C until use. Aliquots of S9 were thawed immediately before use and added to the other components to form the activation system described as follows:
NADP (sodium salt) 1.5 mg/ml (1.8 mM)
Isocitric acid 2.7 mg/ml (10.5 mM)
Homogenate (S9 fraction) 15.0 nl/ml* (1.5%)
* This concenfration of rat S9, obtained from Molecular Toxicology Inc., Boone, NC, has consistently caused CP to be highly clastogenic for many different lots.

Test concentrations with justification for top dose:

without S9 mix:
3 h: 0.854, 1.22, 1.74, 2.48, 3.54, 5.05, 7.21, 10.3, 14.7, 21.0, 30.0, 42.9, 61.3, 87.5, and 125 µg/mL [42.9, 61.3, 87.5, and 125 µg/mL - analyzed for chromosomal aberrations]
Confirmatory assay: 17.8 h; 14.7, 21.0, 30.0, 42.9, 61.3, 87.5, and 125 µg/mL [42.9, 61.3, 87.5, and 125 µg/mL - analyzed for chromosomal aberrations]

with S9 mix
3 h: 0.854, 1.22, 1.74, 2.48, 3.54, 5.05, 7.21, 10.3, 14.7, 21.0, 30.0, 42.9, 61.3, 87.5, and 125 µg/mL [42.9, 61.3, 87.5, and 125 µg/mL - analyzed for chromosomal aberrations]
Confirmatory assay: 3 h: 30.0, 42.9, 61.3, 87.5, 105, and 125 µg/mL [61.3, 87.5, 105, and 125 µg/mL - analyzed for chromosomal aberrations]

Vehicle / solvent:

- Vehicle used: Ethanol was the vehicle of choice for this assay. For the chromosomal aberrations assays, a stock concentration of 12.5 mg/mL was prepared. All dosing was achieved with a 1.0% (10.0 µL/mL) dosing of the stock solutions prepared and the solvent control cultures were treated with 10.0 µL/mL of ethanol.
- Justification for choice of solvent/vehicle: The test item was soluble in ethanol based on a solubility test.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION

Initial assay (with and without S9-mix): 3 hours exposure, 20 hours harvest
Confirmatory assay (with S9-mix): 3 hours exposure, 20 hours harvest
Confirmatory assay (without S9-mix): 17.8 hours exposure, 20 hours harvest

Aberrations Assay Without Metabolic Activation
Cultures were initiated by seeding approximately 1.2E6 cells per 75 cm² flask into 10 mL of complete McCoy's 5a medium. One day after culture initiation, the cells were incubated at ca. 37°C with the test item at predetermined concentrations for approximately 3.0 hours for the initial assay or 17.8 hours in the confirmatory assay. The cultures were then washed with buffered saline. In the initial assay, the cells were refed with complete McCoy's 5a medium and incubated for the rest of the culture period up to the time of harvest with 0.1 µg/mL Colcemid present during the last 2.0 hours of incubation. In the confirmatory assay, cells were refed with complete McCoy's 5a medium with 0.1 µg/mL Colcemid® and harvested 2 hours later.

Aberrations Assay With Metabolic Activation
Cultures were initiated by seeding approximately 1.2E6 cells per 75 cm² flask into 10 mL of complete McCoy's 5a medium. One day after culture initiation, the cultures were incubated at ca. 37 °C for approximately three hours in the presence of the test item and the S9 reaction mixture in McCoy's 5a medium without FBS. After the 3-h exposure period the cells were washed twice with buffered saline and the cells were refed with complete McCoy's 5a medium. The cells were incubated for the rest of the culture period up to the time of harvest with 0.1 µg/ml Colcemid present during the last ca. 2 hours of incubation. The cultures were then harvested

Harvest Procedure
Prior to the harvest of the cultures, visual observations of toxicity were made. These observations included an assessment of the percent confluence of the cell monolayer within the culture flasks. The cultures were also evaluated for the presence of mitotic (large rounded cells) or dead cells floating in the medium. The cultures were then trypsinized to collect mitotic and interphase cells and were treated with 0.075 M KCl hypotonic solution. The cultures were then fixed with an absolute methanol: glacial acetic acid (3:1, v:v) fixative before air-dried slides were prepared. Slides were prepared by dropping the harvested cultures on clean slides. The slides were stained with 5 % Giemsa solution for the analysis of mitotic index and chromosomal aberrations. All slides were then air-dried and mounted permanently.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS / NUMBER OF CELLS EVALUATED:
100 metaphases cer culture (200 per dose level)

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition

Evaluation criteria:

Evaluation of a Positive Response:
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. Statistical evaluation of the percentage of cells with more than one aberration provided an indication of the severity of the positive response observed. The linear trend test evaluated the dose responsiveness. If a significant increase was seen at one or more concentrations, a dose-response should be observed.

Evaluation of a Negative Response:
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.

Equivocal Evaluation:
Although most assays gave clearly positive or negative results, in rare cases the data set would preclude making a definitive judgment about the activity of the test article. Results might remain equivocal or questionable regardless of the number of times the assay is repeated. Although the evaluation criteria provided here normally was sufficient, the study director might use additional considerations to obtain a final evaluation of the test article based upon the study director's scientific judgment.

Statistics:

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 item 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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the initial assay, no significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed, except for a weak increase at 125 µg/mL with metabolic activation. In a confirmatory chromosomal aberrations assay, no significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed. The weak increase observed before at 125 µg/mL, a precipitating dose level, was not observed either at 125 or at 105 µg/mL. The test item was tested up to precipitating concentrations. Each concentration was tested in duplicates. 200 metaphases were counted per concentration. There was no evidence of chromosome aberration induced over background. Positive controls induced the appropriate response. The test item was considered negative for inducing chromosome aberrations in CHO cells with and without metabolic activation.

Chromosomal Aberrations Assay without Metabolic Activation

Initial Assay

A precipitate was observed after dosing at 125 μg/ml, a slight amount of precipitate was observed after dosing at 87.5 μg/ml, and a very slight amount of precipitate was observed after dosing at 61.3 μg/ml. No visual signs of toxicity were observed in any of the test cultures. A reduction of 3% was observed in the mitotic indices of the cultures treated with 125 μg/ml as compared with the solvent control cultures. Chromosomal aberrations were analyzed from the cultures treated with 42.9, 61.3, 87.5, and 125 μg/ml (Table 1). No significant increases in cells with chromosomal aberrations, polyploidy, or endoreduplication were observed at the concentrations analyzed.

Based on the results from the initial trial, the confirmatory nonactivation aberrations assay was conducted testing concentrations of 14.7, 21.0, 30.0, 42.9, 61.3, 87.5, and 125 μg/ml. Treatment period was for 17.8 hours and cultures were harvested 20 hours from the initiation of treatment.

TABLE 1

Trial 1: Cells Fixed 20.0 Hours After Initiation of Treatment, 3.0 Hour Treatment Metabolic Activation: -S9			Number and type of aberration														# of aberrations per cell	% cells with aberrations	% cells with >1 aberrations	% PP	% E	% mitotic index
			not computed			Simple		Complex								Other
	Dose level	Cells scored	TG	SG	UC	TB	SB	ID	TR	QR	CR	D	R	CI	DF	GT
negative control (McCoy's 5a)		A 100	8	7	1	1											0.01	1.0	0.0	4.0	1.0	8.2
		B 100	9	11	2	1	2										0.03	3.0	0.0	3.0	0.0	7.5
		A+B 200	17	18	3	2	2										0.02	2.0	0.0	3.5	0.5	7.9
solvent control (ethanol)	10 µl/ml	A 100	9	8	4	4	3					3			1		0.11	10.0	1.0	4.0	0.0	7.8
		B 100	10	12	1		2										0.02	2.0	0.0	4.0	1.0	7.1
		A+B 200	19	20	5	4	5					3			1		0.07	6.0	0.5	4.0	0.5	7.5
Positive Control (MMC)	1.5 µg/ml	A 25	3	3	1	10	6		2	2	1						0.84	44.0	24.0	3.0	0.0	1.9
		B 25	8	6	1	13	6	1	8	5				1			1.36	60.0	44.0	2.0	0.0	1.5
		A+B 50	11	9	2	23	12	1	10	7	1			1			1.10	52.0*	34.0*	2.5	0.0	1.7
Test article	42.9 µg/ml	A 100	2	3	1	1	1					1					0.03	3.0	0.0	3.0	0.0	7.6
		B 100	3	1	1												0.00	0.0	0.0	3.0	0.0	7.9
		A+B 200	5	4	2	1	1					1					0.02	1.5	0.0	3.0	0.0	7.8
	61.3 µg/ml	A 100	1	2	1												0.00	0.0	0.0	3.0	1.0	7.4
		B 100	4	2												1	0.10	1.0	1.0	3.0	1.0	7.9
		A+B 200	5	4	1											1	0.05	0.5	0.5	3.0	1.0	7.7
	87.5 µg/ml	A 100	3	3													0.00	0.0	0.0	3.0	1.0	7.4
		B 100	4	3													0.00	0.0	0.0	3.0	0.0	7.8
		A+B 200	7	6													0.00	0.0	0.0	3.0	0.5	7.6
	125 µg/ml	A 100	5	6								3					0.03	2.0	1.0	2.0	1.0	7.5
		B 100	4	12	3	2	3					1					0.06	5.0	1.0	4.0	1.0	7.0
		A+B 200	9	18	3	2	3					4					0.05	3.5	1.0	3.0	1.0	7.3

* Significantly greater than the solvent controls, p ≤0.01

Confirmatory Assay

A precipitate was observed after dosing at 125 μg/ml and a very slight amount of precipitate was observed after dosing at 87.5 μg/ml. Slightly unhealthy cell monolayers were observed in the cultures treated with 125 μg/ml. No visual signs of toxicity were observed in any of the other test cultures. Reductions of 2%, 19%, 19%, and 32% were observed in the mitotic indices of the cultures treated with 42.9, 61.3, 87.5, and 125 μg/ml, respectively, as compared with the solvent control cultures. Chromosomal aberrations were analyzed from the cultures treated with 42.9, 61.3, 87.5, and 125 μg/ml (Table 2). No significant increases in cells with chromosomal aberrations, polyploidy, or endoreduplication were observed at the concentrations analyzed.

The sensitivity of the cell cultures 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 2

Trial 2: Cells Fixed 20.0 Hours After Initiation of Treatment, 17.8 Hour Treatment Metabolic Activation: -S9			Number and type of aberration														# of aberrations per cell	% cells with aberrations	% cells with >1 aberrations	% PP	% E	% mitotic index
			not computed			Simple		Complex								Other
	Dose level	Cells scored	TG	SG	UC	TB	SB	ID	TR	QR	CR	D	R	CI	DF	GT
negative control (McCoy's 5a)		A 100	4	2													0.00	0.0	0.0	2.0	0.0	9.0
		B 100	4	2			2					1					0.03	2.0	1.0	3.0	1.0	7.3
		A+B 200	8	4			2					1					0.02	1.0	0.5	2.5	0.5	8.2
solvent control (ethanol)	10 µl/ml	A 100	5	3	1		1					1			1		0.03	3.0	0.0	2.0	0.0	11.3
		B 100	13	4													0.00	0.0	0.0	1.0	1.0	10.3
		A+B 200	18	7	1		1					1			1		0.02	1.5	0.0	1.5	0.5	10.8
Positive Control (MMC)	0.08 µg/ml	A 25	3	2		3	4					1					0.32	28.0	4.0	1.0	0.0
		B 25	5	2		1	5			2		1					0.36	32.0	4.0	3.0	0.0
		A+B 50	8	4		4	9			2		2					0.34	30.0*	4.0	2.0	0.0
Test article	42.9 µg/ml	A 100	4				1						1				0.02	2.0	0.0	3.0	0.0	11.1
		B 100	10	2													0.00	0.0	0.0	2.0	0.0	10.1
		A+B 200	14	2			1						1				0.01	1.0	0.0	2.5	0.0	10.6
	61.3 µg/ml	A 100	5	2			2										0.02	1.0	1.0	2.0	1.0	8.5
		B 100	2	1								1					0.01	1.0	0.0	3.0	0.0	8.8
		A+B 200	7	3			2					1					0.02	1.0	0.5	2.5	0.5	8.7
	87.5 µg/ml	A 100	5		1							1	1				0.02	2.0	0.0	3.0	1.0	9.1
		B 100	8	2									1				0.01	1.0	0.0	3.0	0.0	8.5
		A+B 200	13	2	1							1	2				0.02	1.5	0.0	3.0	0.5	8.8
	125 µg/ml	A 100	6	2	2		1										0.01	1.0	0.0	4.0	1.0	8.0
		B 100	8	3		1						1					0.02	2.0	0.0	2.0	0.0	6.5
		A+B 200	14	5	2	1	1					1					0.02	1.5	0.0	3.0	0.5	7.3

* Significantly greater than the solvent controls, p ≤0.01

Chromosomal Aberrations Assay with Metabolic Activation

Initial Trial

A precipitate was observed after dosing at 125 μg/ml, a slight amount of precipitate was observed after dosing at 87.5 g/ml, and a very slight amount of precipitate was observed after dosing at 61.3 μg/ml. Slightly unhealthy cell monolayers, ca. 15% reduction in the cell monolayer confluence, floating debris, and a slight reduction in the number of visible mitotic cells were observed in the cultures treated with 87.5 and 125 μg/ml. No visual signs of toxicity were observed in any of the other test cultures. Reductions of 15%, 33%, and 33% were observed in the mitotic indices of the cultures treated with 61.3, 87.5, and 125 μg/ml, respectively, as compared with the solvent control cultures. Chromosomal aberrations were analyzed from the cultures treated with 42.9, 61.3, 87.5, and 125 μg/ml (Table 3). No significant increases in cells with chromosomal aberrations, polyploidy, or endoreduplication were observed at the concentrations analyzed, except for a weak increase in cells with chromosomal aberrations at 125 μg/ml.

Based on the results from the initial trial, the confirmatory aberrations assay with metabolic activation was conducted testing concentrations of 30.0, 42.9, 61.3, 87.5, 105, and 125 μg/ml. Treatment period was for 3 hours and cultures were harvested 20 hours from the initiation of treatment.

TABLE 3

Trial 1: Cells Fixed 20.0 Hours After Initiation of Treatment, 3.0 Hour Treatment Metabolic Activation: +S9			Number and type of aberration														# of aberrations per cell	% cells with aberrations	% cells with >1 aberrations	% PP	% E	% mitotic index
			not computed			Simple		Complex								Other
	Dose level	Cells scored	TG	SG	UC	TB	SB	ID	TR	QR	CR	D	R	CI	DF	GT
negative control (McCoy's 5a)		A 100	10	2													0.00	0.0	0.0	5.0	8.0	5.8
		B 100	7	3													0.00	0.0	0.0	4.0	4.0	7.3
		A+B 200	17	5													0.00	0.0	0.0	4.5	6.0	6.6
solvent control (ethanol)	10 µl/ml	A 100	5	2													0.00	0.0	0.0	6.0	7.0	6.9
		B 100	4	3	1												0.00	0.0	0.0	3.0	3.0	4.1
		A+B 200	9	5	1												0.00	0.0	0.0	4.5	5.0	5.5
Positive Control (CP)	5.0 µg/ml	A 25	10	5	1	5	5			1							0.44	36.0	8.0	3.0	1.0
		B 25	4	5		4	2	1	3	1							0.44	40.0	4.0	4.0	1.0
		A+B 50	14	10	1	9	7	1	3	2							0.44	38.0*	6.0*	3.5	1.0
Test article	42.9 µg/ml	A 100	4	5													0.00	0.0	0.0	4.0	5.0	6.8
		B 100	11	4													0.00	0.0	0.0	4.0	4.0	6.7
		A+B 200	15	9													0.00	0.0	0.0	4.0	4.5	6.8
	61.3 µg/ml	A 100	7	3													0.00	0.0	0.0	4.0	5.0	5.5
		B 100	8	7													0.00	0.0	0.0	4.0	4.0	3.9
		A+B 200	15	10													0.00	0.0	0.0	4.0	4.5	4.7
	87.5 µg/ml	A 100	12	10													0.00	0.0	0.0	5.0	6.0	4.4
		B 100	27	11	1		4		1								0.05	2.0	1.0	4.0	3.0	2.9
		A+B 200	39	21	1		4		1								0.03	1.0	0.5	4.5	4.5	3.7
	125 µg/ml	A 100	28	17	3	6			2			2					0.10	10.0	0.0	8.0	5.0	5.1
		B 100	36	18		4	1					1					0.06	6.0	0.0	9.0	7.0	2.2
		A+B 200	64	35	3	10	1		2			3					0.08	8.0*	0.0	8.5	6.0	3.7

* Significantly greater than the solvent controls, p ≤0.01

Confirmatory Assay

A precipitate was observed after dosing at 125 μg/ml, a slight amount of precipitate was observed after dosing at 105 μg/ml, and a very slight amount of precipitate was observed after dosing at 87.5 μg/ml. Floating debris was observed in the cultures treated with 125 μg/ml. No visual signs of toxicity were observed in any of the other test cultures. Reductions of 8%, 9%, 23%, and 22% were observed in the mitotic indices of the cultures treated with 61.3, 87.5,105, and 125 μg/ml, respectively, as compared with the solvent control cultures. Chromosomal aberrations were analyzed from the cultures treated with 61.3, 87.5, 105, and 125 μg/ml (Table 4). No significant increases in cells with chromosomal aberrations, polyploidy, or endoreduplication were observed at the concentrations analyzed. The weak response observed in the initial assay was not repeated at the same concentration in the confirmatory assay, nor at a very closely spaced concentration of 105 μg/ml. This indicates that the weak response observed in the first trial is probably due to the presence of a precipitate when the actual treatment is confounded by the precipitate.

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 is considered negative for inducing chromosomal aberrations, polyploidy, and endoreduplication under activation conditions.

TABLE 4

Trial 2: Cells Fixed 20.0 Hours After Initiation of Treatment, 3.0 Hour Treatment Metabolic Activation: +S9			Number and type of aberration														# of aberrations per cell	% cells with aberrations	% cells with >1 aberrations	% PP	% E	% mitotic index
			not computed			Simple		Complex								Other
	Dose level	Cells scored	TG	SG	UC	TB	SB	ID	TR	QR	CR	D	R	CI	DF	GT
negative control (McCoy's 5a)		A 100	3	4			1					1					0.02	2.0	0.0	1.0	0.0	9.9
		B 100	3	2													0.00	0.0	0.0	1.0	0.0	10.7
		A+B 200	6	6			1					1					0.01	1.0	0.0	1.0	0.0	10.3
solvent control (ethanol)	10 µl/ml	A 100	6	3													0.00	0.0	0.0	0.0	0.0	11.6
		B 100	5	6													0.00	0.0	0.0	0.0	0.0	11.1
		A+B 200	11	9													0.00	0.0	0.0	0.0	0.0	11.4
Positive Control (CP)	10.0 µg/ml	A 25	6	2		4	15		2	4					1		1.04	64.0	32.0	1.0	0.0
		B 25	6	3		17	11	2	11	7	2	1		1		1	2.12	76.0	60.0	3.0	0.0
		A+B 50	12	5		21	26	2	13	11	2	1		1	1	1	1.58	70.0*	46.0*	2.0	0.0
Test article	61.3 µg/ml	A 100	7	3													0.00	0.0	0.0	1.0	0.0	11.2
		B 100	6	3	1		2			2		1					0.05	4.0	1.0	1.0	0.0	9.8
		A+B 200	13	6	1		2			2		1					0.03	2.0	0.5	1.0	0.0	10.5
	87.5 µg/ml	A 100	7	1		1											0.01	1.0	0.0	1.0	0.0	10.9
		B 100	7	5		1											0.01	1.0	0.0	1.0	0.0	9.8
		A+B 200	14	6		2											0.01	1.0	0.0	1.0	0.0	10.4
	105 µg/ml	A 100	6	2		1	1		1								0.03	2.0	1.0	0.0	0.0	10.2
		B 100	10	2													0.00	0.0	0.0	1.0	0.0	7.3
		A+B 200	16	4		1	1		1								0.02	1.0	0.5	0.5	0.0	8.8
	125 µg/ml	A 100	10	5	2							1					0.01	1.0	0.0	1.0	0.0	8.0
		B 100	9	5	3		2										0.02	2.0	0.0	2.0	1.0	9.7
		A+B 200	19	10	5		2					1					0.02	1.5	0.0	1.5	0.5	8.9

* Significantly greater than the solvent controls, p ≤0.01

Abbreviations used for chromosome aberation types:

NOT COMPUTED

TG Chromatid Gap

SG Chromosome Gap

UC Uncoiled Chromosome

SIMPLE

TB Chromatid Break

SB Chromosome Break

COMPLEX

ID Interstitial Deletion

TR Triradia

QR Quadriradia

CR Complex Rearrangement

D Dicentric

R Ring

CI Chromosome Intrachange

T Translocation

DF Dicentric with fragment

OTHER

GT Greater than Ten (A cell which contains more than 10 aberrations)

Conclusions:

The test article was considered negative for inducing chromosome 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 article to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells with and without metabolic activation. The top concentration tested in this assay was 125 µg/ml, a concentration limited by solubility and the level of precipitate in the cultures. The test article was solubilized in ethanol at a concentration of 12.5 mg/ml for this assay. All dosing was achieved using a dosing volume of 1.0% (10.0 µl/ml) and the solvent control cultures were treated with 10.0 µl/ml of ethanol. In the initial chromosomal aberrations assay, the treatment period was for 3 hours with and without metabolic activation and cultures were harvested 20 hours from the initiation of treatment. Concentrations of 0.854,1.22, 1.74, 2.48, 3.54, 5.05, 7.21, 10.3, 14.7, 21.0, 30.0, 42.9, 61.3, 87.5, and 125 µg/ml were tested. Cultures treated with concentrations of 42.9, 61.3, 87.5, and 125 µg/ml with and without metabolic activation were analyzed for chromosomal aberrations. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed, except for a weak increase at 125 µg/ml with metabolic activation. In a confirmatory chromosomal aberrations assay, replicate cultures of CHO cells were incubated with concentrations of 14.7, 21.0, 30.0, 42.9, 61.3, 87.5, and 125 µg/ml for 17.8 hours without metabolic activation or 30.0, 42.9, 61.3, 87.5, 105, and 125 µg/ml for 3.0 hours with metabolic activation and harvested 20 hours from the initiation of treatment. Cultures treated with concentrations of 42.9, 61.3, 87.5, and 125 µg/ml without metabolic activation or 61.3, 87.5, 105, and 125 µg/ml with metabolic activation were analyzed for chromosomal aberrations. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed. The weak increase observed at 125 µg/ml, a precipitating dose level, was not observed either at 125 or at 105 µg/ml. In conclusion, the test article was considered negative for inducing chromosome 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 tests

To investigate the mutagenic potential of the test material, the Salmonella - Escherichia coli / Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay was conducted. This assay evaluated the test article and/or its metabolites for their ability to induce reverse mutations at the histidine locus in the genome of specific Salmonella typhimurium tester strains and at the tryptophan locus in an Escherichia coli tester strain both in the presence and absence of an exogenous metabolic activation system of mammalian microsomal enzymes derived from Aroclor-induced rat liver (S9). The doses tested in the mutagenicity assay were selected based on the results of a dose range-finding study. The tester strains used in the mutagenicity assay were Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537, and Escherichia coli tester strain WP2uvrA. The assay was conducted with six doses of test article, 5000, 1000, 333, 100, 33.3, and 10 µg per plate, in presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per dose. The results of the initial mutagenicity assay were confirmed in an independent experiment. The test article did not cause a positive increase in the number of revertants per plate of any of the tester strains either in the presence or absence of microsomal enzymes.

An Ames-screening assay in Salmonella typhimurium TA98, TA100, TA1535, TA1537, and Escherichia coli WP2uvrA was performed. The assay was conducted with seven doses of test article in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using two plates per dose. The doses tested were 5000, 3330, 1000, 333, 100, 33.3 and 10 µg per plate in presence and absence of S9 mix. The identity of the test material was not verified by Lot or batch number. The test material caused a positive 8.5-fold increase in the number of revertants per plate with tester strain TA98 only in the presence of S9 mix. No positive increases were observed with any of the remaining tester strain/activation condition combinations.

Mouse Lymphoma Assay

Mouse lymphoma cells were treated with the test item to determine the genotoxic potential of the substance in mammalian cells (similar to OECD guideline 476, GLP). Two nonactivation mutation assays were used in the evaluation. In the first nonactivation trial. the test article was lethal or excessively toxic above 15.0 µg/ml. The remaining three treatments from 5.00 µg/ml to 15.0 µg/ml induced weak cytotoxicity. None of the three analyzed treatments induced a mutant frequency that exceeded the minimum criterion for a positive response and a small increase in concentration from 15.0 µg/ml to 20.0 µg/ml was excessively cytotoxic. An independent repeat assay was performed to confirm these results. In the independent repeat nonactivation assay, six treatments from 5.00 µg/ml to 20.0 µg/ml were analyzed for mutant induction and a wide range of cytotoxicities was induced. Even in the presence of high cytotoxicity, there was no evidence for a mutagenic response. Two activation mutation trials were also used in the evaluation. In the first activation trial, six

treatments from 5.00 µg/ml to 40.0 µg/ml were evaluated for mutant induction. In the confirmatory trial, six treatments from 15.0 µg/ml to 40.0 µg/ml were analyzed. A wide range of cytotoxicities was induced in both trials and no evidence for a mutagenic response was observed. The test material was evaluated as negative in the mouse lymphoma forward mutation assay both with and without S9 metabolic activation over a wide range of cytotoxicity.

Chromosome aberration study

The ability of the test material to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells with and without metabolic activation was examined. The top concentration tested in this assay was 125 µg/ml, a concentration limited by solubility and the level of precipitate in the cultures; the treatment periods were 3 hours and 17.5 hours, respectively, with and without metabolic activation and cultures were harvested 20 hours from the initiation of treatment. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed at the concentrations analyzed. In conclusion, the test material did not induce chromosome aberrations in mammalian cells.

Overall conclusion

The test article did not induce chromosomal aberrations or mutations in mammallian cells. In addition, when tested in a GLP-compliant Ames test the test article did also not induce mutations in bacteria. The positive increase in revertants in the available Ames screening assay may result from impurities (substance identity and purity was not verified) or a measurement artefact; a confirmatory experiment was not performed. The test article is therefore considered to have no genotoxic potential.

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.