Reaction products of 1,4-Benzenedimethanol and 1-naphthol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test:

The test item SN-475N had no mutagenic activity in the bacterium tester strains under the test conditions used in this study.

Chromosome Aberration Test:

SN-475N did not induce a significant level of chromosome aberrations in the performed experiments with or without metabolic activation. Therefore, SN-475N is considered not clastogenic in this test system.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 October 2012 to 18 October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

No further details specified in the study report

Target gene:

Histidine and tryptophan

Species / strain / cell type:

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

Cytokinesis block (if used):

Not specified

Metabolic activation:

with and without

Metabolic activation system:

an exogenous metabolic activation system is added in the form of a mammalian microsomal enzyme activation mixture (liver extract, S9 fraction). The activation system uses nicotinamide-adenine dinucleotide phosphate (NADP+)-cytochrome P450 dependent mixed function oxidase enzymes of the liver. The liver extract was obtained from rats, which were pre-treated with phenobarbital and β-naphthoflavone, two inducers of several drug-metabolizing enzymes.

Test concentrations with justification for top dose:

Concentrations were selected on the basis of the Preliminary Solubility Test and Preliminary Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test and Confirmatory Mutation Test the same concentrations were used.

Preliminary Solubility Test
Based on the results of the preliminary solubility test, the test item is insoluble in Distilled water. However, it was soluble in Dimethyl sulfoxide (DMSO) and Acetone at 100 mg/mL concentration. Due to the better biocompatibility to the test system, DMSO was selected for solvent of the study.
The obtained stock formulation (50 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension.

Preliminary Concentration Range Finding Test (Informatory Toxicity Test)
Based on the solubility test, 100 mg/mL stock formulation was prepared in DMSO which was diluted in 6 steps by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98 and TA100) were determined at the concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item. In the Preliminary Concentration Range Finding Test the plate incorporation method was used.

Test Item Concentrations in the Mutagenicity Tests (Initial Mutation Test and Confirmatory Mutation Test)
Based on the results of the preliminary tests, 100 mg/mL stock formulation was prepared from the test item with DMSO, which was diluted by serial dilutions in seven steps to obtain eight dosing formulations. The maximum test concentration was 5000 μg test item/plate in the main tests.
Examined concentrations in the main tests were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate.

Vehicle / solvent:

DMSO and Distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSOand Distilled water

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylene-diamine, 2-aminoanthracene

Details on test system and experimental conditions:

Experimental Method
The experimental methods were conducted according to the methods described Ames et al. [1] and Maron and Ames [2], Kier et al. [3], Venitt and Parry [4], OECD Guideline No. 471, 1997 [5], Commission Regulation (EC) No. 440/2008, 2008 [6], EPA Guidelines, OPPTS 870.5100, 1998, 1996 [7][8], and according to the relevant SOPs of CiToxLAB Hungary Ltd.

Procedure for Exposure in the Initial Mutation Test
A standard plate incorporation procedure was performed, as an Initial Mutation Test. Bacteria were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).

The content of the tubes:
top agar 2000 μL
vehicle (solvent) or test item formulation (or reference controls) 50 μL
overnight culture of test strain 100 μL
phosphate buffer (pH 7.4) or S9 mix 500 μL
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48 hours.

Procedure for Exposure in the Confirmatory Mutation Test
A pre-incubation procedure was performed as a Confirmatory Mutation Test since in the Initial Mutation Test no positive effect was observed.
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent).
The tubes (3 tubes per control or concentration level) were gently mixed and incubated for 20 min at 37ºC in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes; the content was mixed up and poured onto minimal glucose agar plates as described for the standard plate incorporation method. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48 hours.

Rationale for test conditions:

A pre-incubation procedure was performed as a Confirmatory Mutation Test since in the Initial Mutation Test no positive effect was observed

Evaluation criteria:

The colony numbers on the untreated / negative (vehicle/solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

Statistics:

Not specified

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY TEST)
In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (vehicle/solvent) and positive controls. In the test each sample (including the controls) was tested in triplicate.
In the Range Finding Test the concentrations examined were: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.
The observed numbers of revertant colonies were mostly in the normal range. Minor differences were observed in some sporadic cases, but the numbers of revertant had no biological relevance and they were within the historical control range in all cases.
Precipitate / slight precipitate was observed in the preliminary experiment in both tester strains at 5000, 2500, 1000 and 316 μg/plate concentration with or without metabolic concentration.
The experimental results (revertant colony numbers per plate, mutation factors and standard deviations) are detailed in Table 7 (Appendix 2) and in Appendix 3.

INITIAL AND CONFIRMATORY MUTATION TESTS
In the Initial Mutation Test, the plate incorporation method; in the Confirmatory Mutation Test, the pre-incubation method was used. The Initial Mutation Test and Confirmatory Mutation Test were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA strain. The Initial Mutation Test and Confirmatory Mutation Test were performed in the presence and absence of metabolic activation system (±S9 mix). Each test was performed with appropriate untreated, negative (vehicle/solvent) and positive controls. In the main tests each sample (including the controls) was tested in triplicate.*

*Note: In the Confirmatory Mutation Test in Escherichia coli WP2 uvrA strain at 5000 μg/plate concentration without metabolic activation, one of the plates was infected. In this case, the mutation factor value was calculated using the data of the two remaining replicates.

Examined concentrations in the main tests were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate.
In the Initial Mutation Test and Confirmatory Mutation Test none of the observed revertant colony numbers were above the respective biological threshold value. There were no reproducible dose-related trends and no indication of any treatment effect.
Using the plate incorporation method (Initial Mutation Test), the highest revertant rates were observed in Salmonella typhimurium TA1535 bacterial strain with metabolic activation at the concentration of 50 and 1.581 μg/plate. The mutation factor values were 1.43. However, there was no dose response, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
Using the pre-incubation method (Confirmatory Mutation Test), the highest revertant rate was observed in Escherichia coli WP2 uvrA bacterial strain without metabolic activation at 1.581 μg/plate concentration. The mutation factor value was 1.30. However, there was no dose response, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
Lower revertant counts compared to the vehicle control were observed in the Initial Mutation Test and Confirmatory Mutation Test at some non-cytotoxic concentrations. However, they had no biological significance and were considered as reflecting the variability of the test system.

Note: Lower or slightly reduced numbers of revertant colonies compared to the vehicle control plates were observed in the Initial Mutation Test in Salmonella typhimurium TA100 strain with metabolic activation in the whole tested concentration range. However, no effect of the background lawn was observed, and lower numbers of revertants compared to the DMSO solvent control were observed for untreated control and Distilled water control also in this strain. Therefore, this fact was not considered as sign of cytotoxicity and had no adverse effect on the results or integrity of the study.
Precipitate / slight precipitate was observed in the Initial Mutation in all examined bacterial strains at 5000, 1581 and 500 μg/plate concentrations with and without metabolic activation, in the Confirmatory Mutation Test in all tester strains at 5000, 1581 and 500 μg/plate concentrations without metabolic activation and at 5000 μg/plate concentration with metabolic activation.
Inhibitory, cytotoxic effect of the test item (reduced numbers of revertant colonies) was observed in the Initial Mutation Test in Salmonella typhimurium TA1535 strain at 5000 and 1581 μg/plate concentrations without metabolic activation and in Salmonella typhimurium TA1537 strain at 5000 μg/plate concentration without metabolic activation.
Stronger inhibitory, cytotoxic effect of the test item (reduced / slightly reduced background lawn development and / or reduced numbers of revertant colonies; in some cases pinpoint colonies were also detected) was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA98, TA100 and TA1537 strains at 5000, 1581 and 500 μg/plate concentrations without metabolic activation, in Salmonella typhimurium TA1535 strain at 5000 and 1581 μg/plate concentrations without metabolic activation, in Escherichia coli WP2 uvrA bacterial strain at 5000 μg/plate concentration without metabolic activation and in Salmonella typhimurium TA1537 strain at 5000 μg/plate concentration with metabolic activation.

Note: Slightly reduced numbers of revertant colonies compared to the vehicle control plates were observed in some other cases in the Confirmatory Mutation Test, although no effect on the background lawn was observed. However, these facts were not considered as sign of cytotoxicity in these cases and they had no adverse effect on the results or integrity of the study.

Genotypes of the Strains Used for Mutagenicity Testing

Strain	Genotype		Mutations		Main DNA target	Plasmid
	trp/his mutation	Type of mutation	Cell wall	DNA-repair
S.typhimuriumTA98	hisD3052	Frameshift	rfa	uvrB	GC	pKM101
S.typhimuriumTA100	hisG46	Base pair substitution	rfa	uvrB	GC	pKM101
S.typhimuriumTA1535	hisG46	Base pair substitution	rfa	uvrB	GC	No
S.typhimurium. TA1537	hisC3076	Frameshift	rfa	uvrB	GC	No
E.coliWP2uvrA	trpE	Base pair substitution	+	uvrA	AT	No

The Solubility of the Test Item in DMSO

Concentration of test item in DMSO (mg/mL)	Solubility in DMSO	Solubility in the top solution (test item formulation 50 μL + phosphate buffer 500 μL +top agar 2 mL)	Test item concentration in the test tube (μg/tube)
100	Clear solution	Clear solution	5000

Summary of Controls

Type of control	Activation with S9 Mix	Vehicle/ Solvent	Top Agar	Strain-Specific Positive Chemical Solutions	Phosphate Buffer
Untreated	-	-	+	-	+
Untreated	+	-	+	-	-
Vehicle/Solvent	-	+	+	-	+
Vehicle/Solvent	+	+	+	-	-
Positive Reference Control	-	-	+	+	+
Positive Reference Control	+	-	+	+	-

Note: if the solvent of the positive control substance differed from the vehicle (solvent) of the test item, both solvents were run in the assay.

Summary of Range Finding Test

Concentrations (μg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains
		TA98		TA100
		-S9	+S9	-S9	+S9
Untreated control	Mean	17.0	20.3	121.0	125.3
	MF	0.86	1.03	0.96	0.82
DMSO control	Mean	19.7	19.7	126.7	153.0
	MF	1.00	1.00	1.00	1.00
Distilled water control	Mean	-	-	-	-
	MF	-	-	0.91	-
5000	Mean	17.3	26.0	134.3	143.3
	MF	0.88	1.32	1.06	0.94
2500	Mean	17.3	18.7	124.7	158.0
	MF	0.88	0.95	0.98	1.03
1000	Mean	17.3	26.0	121.7	145.3
	MF	0.88	1.32	0.96	0.95
316	Mean	16.7	24.3	111.7	165.3
	MF	0.85	1.24	0.88	1.08
100	Mean	21.7	27.3	109.0	142.3
	MF	1.10	1.39	0.86	0.93
31.6	Mean	19.7	26.0	116.3	128.0
	MF	1.00	1.32	0.92	0.95
10	Mean	22.0	27.0	116.3	128.0
	MF	1.12	1.37	0.92	0.84
NPD (4μg)	Mean	235.3	-	-	-
	MF	11.97	-	-	-
2AA (2μg)	Mean	-	2444.0	-	2794.7
	MF	-	124.27	-	18.27
SAZ (2μg)	Mean	-	-	1273.3	-
	MF	-	-	11.10	-

Summary of Initial Mutation Test

Concentrations (μg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	25.7	29.3	111.0	127.0	9.0	10.0	8.0	9.7	25.7	39.0
	MF	1.22	0.81	1.03	0.60	1.08	1.30	1.00	1.16	1.01	0.84
DMSO control	Mean	21.0	36.3	108.0	210.7	8.3	7.7	8.0	8.3	25.3	46.7
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
Distilled water control	Mean	-	-	124.0	-	7.3	-	-	-	25.3	-
	MF	-	-	1.15	-	0.88	-	-	-	1.00	-
5000	Mean	16.3	29.3	128.3	96.0	2.3	7.3	4.0	5.3	20.7	33.0
	MF	0.78	0.81	1.19	0.46	0.28	0.96	0.50	0.64	0.82	0.71
1581	Mean	21.0	18.7	109.3	130.7	3.3	9.3	7.7	9.3	26.0	41.7
	MF	1.00	0.51	1.01	0.62	0.40	1.22	0.96	1.12	1.03	0.89
500	Mean	22.7	29.3	102.3	119.3	7.3	10.3	7.0	7.3	27.0	31.3
	MF	1.08	0.81	0.95	0.57	0.88	1.35	0.88	0.88	1.07	0.67
158.1	Mean	20.3	30.0	111.7	138.3	8.3	5.3	5.7	6.7	22.7	35.3
	MF	0.97	0.83	1.03	0.66	1.00	0.70	0.71	0.80	0.89	0.76
50	Mean	21.0	29.3	120.3	140.0	8.7	11.0	6.7	6.7	26.3	38.7
	MF	1.00	0.81	1.11	0.66	1.04	1.43	0.83	0.80	1.04	0.83
15.81	Mean	26.0	34.3	103.0	126.7	7.0	8.0	7.7	7.7	22.7	31.7
	MF	1.24	0.94	0.95	0.60	0.84	1.04	0.96	0.92	0.89	0.68
5	Mean	25.3	30.3	109.3	130.7	7.0	10.3	5.7	9.3	25.3	33.0
	MF	1.21	0.83	1.01	0.62	0.84	1.35	0.71	1.12	1.00	0.71
1.581	Mean	21.3	31.7	114.3	123.7	6.7	11.0	7.0	10.3	30.3	30.3
	MF	1.02	0.87	1.06	0.59	0.80	1.43	0.88	1.24	1.20	0.65
NPD (4μg)	Mean	289.3	-	-	-	-	-	-	-	-	-
	MF	13.78	-	-	-	-	-	-	-	-	-
2AA (2μg)	Mean	-	2400.0	-	2273.3	-	214.0	-	125.3	-	-
	MF	-	66.06	-	10.79	-	27.91	-	15.04	-	-
2AA (50μg)	Mean	-	-	-	-	-	-	-	-	-	253.3
	MF	-	-	-	-	-	-	-	-	-	5.43
SAZ (2μg)	Mean	-	-	1244.0	-	1101.3	-	-	-	-	-
	MF	-	-	10.03	-	150.18	-	-	-	-	-
9AA (50μg)	Mean	-	-	-	-	-	-	509.3	-	-	-
	MF	-	-	-	-	-	-	63.67	-	-	-
MMS (2μL)	Mean	-	-	-	-	-	-	-	-	1037.3	-
	MF	-	-	-	-	-	-	-	-	40.95	-

 

Summary of Confirmatory Mutation Test

Concentrations (μg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	22.0	26.7	110.0	122.3	7.0	11.0	5.7	12.0	34.0	38.3
	MF	1.02	0.91	1.08	0.93	0.68	0.87	0.71	1.50	1.23	0.91
DMSO control	Mean	21.7	29.3	102.0	132.0	10.3	12.7	8.0	8.0	27.7	42.3
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
Distilled water control	Mean	-	-	119.0	-	7.0	-	-	-	44.3	-
	MF	-	-	1.17	-	0.68	-	-	-	1.60	-
5000	Mean	2.0	20.3	18.7	96.0	3.3	8.7	1.0	1.0	11.0	34.0
	MF	0.09	0.69	0.18	0.73	0.32	0.68	0.13	0.13	0.40	0.80
1581	Mean	4.0	25.3	25.0	103.7	1.7	10.0	9.3	6.7	26.0	41.7
	MF	0.18	0.86	0.25	0.79	0.16	0.79	1.17	0.83	0.94	0.98
500	Mean	14.0	27.0	66.0	98.3	7.0	5.7	3.7	6.0	31.0	37.0
	MF	0.65	0.92	0.65	0.74	0.68	0.45	0.46	0.75	1.12	0.87
158.1	Mean	20.7	27.0	97.0	107.7	8.7	8.7	5.3	7.0	27.3	44.0
	MF	0.95	0.92	0.95	0.82	0.84	0.68	0.67	0.88	0.99	1.04
50	Mean	17.0	21.7	93.7	111.7	4.7	12.7	7.0	9.7	31.3	49.3
	MF	0.78	0.74	0.92	0.85	0.45	1.00	0.88	1.21	1.13	1.17
15.81	Mean	16.0	21.0	91.0	119.3	7.7	10.0	4.3	7.7	28.0	38.7
	MF	0.74	0.72	0.89	0.90	0.74	0.79	0.54	0.96	1.01	0.91
5	Mean	18.7	32.7	101.0	132.	7.3	12.7	6.0	7.7	24.3	33.3
	MF	0.86	1.11	0.99	1.00	0.71	1.00	0.75	0.96	0.88	0.79
1.581	Mean	15.0	34.3	100.3	119.3	9.3	11.7	7.7	6.0	36.0	40.7
	MF	0.69	1.17	0.98	0.90	0.90	0.92	0.96	0.75	1.30	0.96
NPD (4μg)	Mean	298.7	-	-	-	-	-	-	-	-	-
	MF	13.78	-	-	-	-	-	-	-	-	-
2AA (2μg)	Mean	-	2374.7	-	2468.0	-	192.3	-	207.3	-	-
	MF	-	80.95	-	18.7	-	15.18	-	25.92	-	-
2AA (50μg)	Mean	-	-	-	-	-	-	-	-	-	235.3
	MF	-	-	-	-	-	-	-	-	-	5.56
SAZ (2μg)	Mean	-	-	1202.7	-	1134.7	-	-	-	-	-
	MF	-	-	10.11	-	162.1	-	-	-	-	-
9AA (50μg)	Mean	-	-	-	-	-	-	364	-	-	-
	MF	-	-	-	-	-	-	45.5	-	-	-
MMS (2μL)	Mean	-	-	-	-	-	-	-	-	1102.7	-
	MF	-	-	-	-	-	-	-	-	24.87	-

Conclusions:

The test item SN-475N had no mutagenic activity in the bacterium tester strains under the test conditions used in this study

Executive summary:

The test item SN-475N was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of the Solubility Test and available information, the test item was dissolved in DMSO. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the main tests were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate.

Inhibitory, cytotoxic effect of the test item was observed in the Initial Mutation Test and/or Confirmatory Mutation Test in all tester strains without metabolic activation and in Salmonella typhimurium TA1537 strain with metabolic activation.

Precipitate / slight precipitate was observed in all tester strains in the main tests in the higher concentration range (1-3 concentrations).

The mean values of revertant colonies of the solvent control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests. The tests were considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item SN-475N had no mutagenic activity in the bacterium tester strains under the test conditions used in this study.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 March 2013 to 09 May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

No further details specified in the study report

Target gene:

V79: Chinese hamster lung, male
ECACC Cat. No.: 86041102
Lot No.: 10H016
Supplier: ECACC (European Collection of Cells Cultures)
Morphology: Fibroblast

The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for genetic toxicity assays with low background aberrations. These cells are chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12-14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male). This cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks were kept in a freezer at -80 ± 10°C. The stock was checked for mycoplasma infection. No infection of mycoplasma was noted.

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Remarks:

male

Metabolic activation:

with and without

Metabolic activation system:

An advantage of using in vitro cell cultures is the accurate control of the concentration and exposure time of cells to the test item under the study. However, due to the limited capacity of cells growing in vitro for metabolic activation of potential mutagens, an exogenous metabolic activation system is necessary. Many substances only develop mutagenic potential after they are metabolised. Metabolic activation of substances can be achieved by supplementing the cell cultures with liver microsome preparations (S9 mix).
In the experiments with metabolic activation in this study, a cofactor-supplemented post-mitochondrial S9 fraction prepared from activated rat liver was used as an appropriate metabolic activation system.
The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of CiToxLAB Hungary Ltd. according to Ames et al. [3] and Maron and Ames [4]. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly. The composition of solution refers to 1000 mL.

Induction of Rat Liver Enzymes
Male Wistar rats (281-321 g, animals were 8-9 weeks old at the initiation of E11498; and 313-367 g, animals were 8-9 weeks old at the initiation of E11580) were treated with Phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 hour before sacrifice when food was removed. Initiations of the induction of liver enzymes used for preparation S9 used in this study were 06 November 2012 (E11498) and 03 March 2013 (E11580).

Preparation of Rat Liver Homogenate S9 Fraction
On Day 4, the rats were euthanized (sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels) and the livers were removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized.
Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-3 mL portions, frozen quickly and stored at -80 ± 10ºC.
The protein concentration of the preparation was determined by a chemical analyzer at 540 nm in the Clinical Chemistry Laboratory of CiToxLAB Hungary Ltd. The protein concentration of the S9 fractions used in the study was determined to be 35.1 g/L (E11498) and 35.1 g/L (E11580). The dates of preparation of S9 fraction used in this study were 09 November 2012 (CiToxLAB code: E11498) and 06 March 2013 (CiToxLAB code: 11580)*. The sterility of the preparation was confirmed in both cases.

*Note: Lot E11498 was used in the preliminary experiment and Assays 1-2; lot E11580 was used in Assay 3.
The biological activity in the Salmonella assay of S9 was characterized using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. Each batch of S9 used in this study functioned appropriately.

Preparation of S9-mix
The complete S9-mix was freshly prepared on the day of use according to the following ratio:
S9 fraction 3 mL
HEPES 20 mM 2 mL
KCl 330 mM 1 mL
MgCl2 50 mM 1 mL
NADP 40 mM 1 mL
Glucose-6-phosphate 50 mM 1 mL
DME medium 1 mL
Prior to addition to the culture medium the S9-mix was kept in an ice bath.
For all cultures treated in the presence of S9-mix, a 0.5 mL aliquot of the mix was added to each cell culture (final volume: 10 mL). The final concentration of the liver homogenate in the test system was 1.5%.

Test concentrations with justification for top dose:

Due to detected insolubility of the test substance the following concentrations were used for each of the assays:

Assay 1:
Concentrations of 6, 4 and 2 μg/mL (a total of three) were chosen for evaluation in the experiments without metabolic activation; and concentrations of 15, 10 and 5 μg/mL (a total of three) were chosen for evaluation in the experiments with metabolic activation.

Assay 2:
Based on the elevated aberration frequency of the negative (vehicle) control samples, Assay 2 was considered to be invalid. An additional experiment (Assay 3) was performed using the same experimental conditions to complete data.

Assay 3:
Concentrations of 2, 1 and 0.5 μg/mL (a total of three) were chosen for evaluation in the experiment without metabolic activation, and concentrations of 10, 7.5 and 5 μg/mL (a total of three) were chosen for evaluation in the experiment with metabolic activation.

Vehicle / solvent:

Negative (solvent) control: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Toxicity and Concentration Selection
Treatment concentrations for the mutation assay were selected based on the results of a short preliminary test.
In this Preliminary Toxicity Test, two separate assays were performed. In Assay A, cells were treated for 3-hours in the presence and absence of S9-mix with a 20-hour harvesting time. In Assay B, cells were treated for 3 hours in the presence of S9-mix and for 20 hours in the absence of S9-mix with a 28-hour harvesting time.

The assays were performed with a range of test item concentrations to determine cytotoxicity. Treatment was performed as described for the main test. However, single cultures were used and positive controls were not included. Visual examination of the final culture medium was conducted at the beginning and end of the treatments. Measurement of pH and osmolality was also performed at the end of the treatment period.
At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (vehicle) control as % relative survival.

Chromosome Aberration Assays
The Chromosome Aberration Assays were conducted as three independent experiments (Assay 1, Assay 2 and Assay 3) in the presence and in the absence of metabolic activation.

Treatment of the Cells
For the cytogenetic experiments, 1-3 day old cultures (more than 50 % confluency) were used. Cells were seeded into 92 x 17 mm tissue culture dishes at 5 x 105 cells/dish concentration and incubated for approximately 24 hours at 37°C in 10 mL of culture medium (DMEM-10). Duplicate cultures were used for each test item concentration or controls. After the seeding period, the medium was replaced with 9.9 mL treatment medium (DMEM-5) in case of experiments without metabolic activation or with 9.4 mL treatment medium (DMEM-5) + 0.5 mL S9-mix in case of experiments with metabolic activation.
Cells were treated with different concentration test item solutions, negative (solvent) or positive control solution (treatment volume: 100 μL/dish in all cases) for the given period of time at 37°C in the absence or presence of S9-mix. After the exposure period, the cultures were washed with DMEM-0 medium (Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine and 1 v/v% Antibiotic-antimycotic solution). Then, 10 mL of fresh culture medium were added into the dishes and cells were incubated further until the scheduled harvesting time.
Harvesting was performed after 20 hours (approximately 1.5 normal cell cycles) or 28 hours (approximately 2 normal cell cycles) from the beginning of treatment.

Solubility of the test item in the final treatment medium was visually examined at the beginning and end of the treatment in each case. Measurement of pH and osmolality can was also performed at the end of the treatment period in both main tests.
For concurrent measurement of cytotoxicity an extra dish was plated for each sample and treated in the same manner. At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (vehicle) control as % relative survival.

Preparation of Chromosomes
2-2.5 hours prior to harvesting, cell cultures were treated with Colchicine (0.2 μg/mL). The cells were swollen with 0.075 M KCl hypotonic solution, then were washed in fixative (Methanol : Acetic-acid 3 : 1 (v : v) mixture) until the preparation became plasma free (4 washes). Then, a suspension of the fixed cells was dropped onto clean microscope slides and air-dried. The slides were stained with 5 % Giemsa solution, air-dried and coverslips were mounted. At least three slides were prepared for each culture.

Examination of Slides
The stained slides were given random unique code numbers at the Test Facility by a person who was not involved in the metaphase analysis. The code labels covered all unique identification markings on the slides to ensure that they were scored without bias.
The coded slides were sent for evaluation to the attention of the Principal Investigator to:
Natalie Danford, B.Sc., MPH, PhD.
Principal Investigator
Microptic Cytogenetics
2 Langland Close
Mumbles
Swansea, SA3 4LY, United Kingdom

or

Dr. Maureen King
(on behalf of Microptic Cytogenetics)
20 Charles St,
Annan, DG12 5AJ, United Kingdom
Claire Birkinshaw
(on behalf of Microptic Cytogenetics)
25 Musgraves Orchard,
Welton, Lincoln, LN2 3NP, United Kingdom

The metaphase analysis was conducted under the control of the Principal Investigator. When the metaphase analysis was completed for each test, the slide codes were broken and the number of metaphases with aberrations (excluding gaps) and the types of aberrations for each culture were presented in tables.

At least one hundred metaphases* with 22±2 chromosomes (dicentric chromosomes were counted as two chromosomes) from each culture were examined for the presence or absence of chromosomal aberrations (approximately 1000x magnification), where possible. Where there were insufficient metaphases in one replicate, the total was made up to 200 cells examined per concentration using the other replicate. Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.
*Note: The examination of slides from a culture was halted when 15 or more metaphases with aberrations (excluding gaps) have been recorded for that culture.

The aberrations are defined in the following way:

Gap: small unstained lesion smaller than the width of a chromatid and with minimal misalignment of the chromatid(s)
Break: unstained lesion larger than the width of a chromatid, or with clear misalignment
Exchange: breakage and reunion of chromatids within a chromosome, or between chromosomes
Chromatid-type: structural chromosome damage expressed as breakage of single chromatids or breakage and reunion between chromatids
Chromosome-type: structural chromosome damage expressed as breakage, or breakage and reunion, of both chromatids at an identical site.

Fragments could arise from breakage and exchange events. When the origin of a fragment was clear, it was recorded under that category (e.g. a dicentric chromosome with a fragment was recorded as one chromosome exchange event). When the origin of the fragment was not clear, it was recorded as a chromatid break. Metaphases with more than five aberrations (excluding gaps) were recorded as showing multiple damage.

Rationale for test conditions:

The tested concentrations in the chromosome aberration assays were selected based on the results of the preliminary experiments. Insolubility and cytotoxicity were detected in the performed main experiments. The evaluated concentration ranges of Assay 1 and Assay 3 were considered to be adequate, as they covered the range from cytotoxicity to little or no cytotoxicity. The lowest examined concentrations contained no insolubility either.

Evaluation criteria:

The assay is considered valid, if the following criteria are met:
The negative (vehicle) control data are within the laboratory’s normal range for the spontaneous aberration frequency.
The positive controls induce increases in the aberration frequency, which are significant.
The test item is considered to have shown clastogenic activity in this study if all of the following criteria are met:
Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
The increases are statistically significant.
The increases are not associated with large changes in pH or osmolarity of the treated cultures.

The historical control data for this laboratory were also considered in the evaluation. Evidence of a dose-response relationship (if any) was considered to support the conclusion.
The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed.

Statistics:

For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

VEHICLE AND CONCENTRATION SELECTION
Based on the results of the preliminary solubility test and available information (CiToxLAB study code: 12/356-007M), the test item was insoluble in Distilled water, but the formulation at 500 mg/mL concentration using Dimethyl sulfoxide (DMSO) as vehicle was suitable for the test. As DMSO is compatible to the test system, it was selected as vehicle for the study. The highest examined concentration in the preliminary test was 5000 μg/mL.
Two Concentration Selection Cytotoxicity Assays (Assay A: 3-hour treatment with and without metabolic activation, 20-hour harvesting time; and Assay B: 3-hour treatment with metabolic activation or 20-hour treatment without metabolic activation, 28-hour harvesting time) were performed as part of the study to establish an appropriate concentration range for the Chromosome Aberration Assays, both in the absence and in the presence of a metabolic activation system.

In the preliminary toxicity test, single cultures were tested and positive controls were not included. Following treatments, cell concentrations were determined using a haemocytometer. Precipitation of the test item in the final culture medium was visually examined at the beginning and end of the treatments. Measurement of pH and osmolality was also performed at the end of the treatment period.
A total of eight test concentrations between 5000 and 2.29 μg/mL were used to evaluate toxicity in the presence and absence of metabolic activation in each cytotoxicity assay. Treatment concentrations for the chromosome aberration assays were selected on the basis of results of the performed Concentration Selection Cytotoxicity Assays according to the OECD guideline instructions.

CHROMOSOME ABERRATION ASSAYS
In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 20, 10, 8, 6, 5, 4, 3, 2, 1 and 0.5 μg/mL (experiment without metabolic activation); and 40, 20, 15, 10, 7.5, 5, 2.5 and 1.25 μg/mL (experiment with metabolic activation).

In Assay 1, insolubility was detected at the end of the treatment period in the final treatment medium in the 40-15 μg/mL concentration range with metabolic activation. There were no large changes in the pH and osmolality. Cytotoxicity was observed in this assay at 20, 10, 8 and 6 μg/mL concentration without metabolic activation (relative survival values were 20, 30, 39 and 43%, respectively); and at 40, 20 and 15 μg/mL concentration with metabolic activation (relative survival values were 4, 6 and 31%, respectively). Therefore, concentrations of 6, 4 and 2 μg/mL (a total of three) were chosen for evaluation in the experiments without metabolic activation; and concentrations of 15, 10 and 5 μg/mL (a total of three) were chosen for evaluation in the experiments with metabolic activation. None of the treatment doses caused a significant increase in the number of cells with structural chromosome aberrations, therefore this test was considered to be negative.

In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 28 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 8, 6, 4, 3, 2, 1.5, 1, 0.75, 0.5, 0.25 and 0.125 μg/mL (experiment without metabolic activation), and 40, 20, 15, 10, 7.5, 5, 2.5 and 1.25 μg/mL (experiment with metabolic activation).

In Assay 2, insolubility was detected at the end of the treatment period in the final treatment medium in the 40-7.5 μg/mL concentration range with metabolic activation. No large changes in the pH and osmolality were detected. Marked cytotoxicity was observed in this assay at 8, 6, 4, 3 and 2 μg/mL concentrations without metabolic activation (relative survival values of 0, 9, 22, 30 and 47%, respectively) and at 40, 20, 15 and 10 μg/mL concentrations with metabolic activation (relative survival values of 4, 8, 29 and 47%, respectively) as detailed in Tables 8 and 9 of Appendix 4. Therefore, concentrations of 2, 1 and 0.5 μg/mL (a total of three) were chosen for evaluation in the experiment without metabolic activation, and concentrations of 10, 7.5 and 5 μg/mL (a total of three) were chosen for evaluation in the experiment with metabolic activation.

Based on the elevated aberration frequency of the negative (vehicle) control samples (three out of the four negative control cultures resulted in an increased aberration frequency over of the upper limit of the historical control range), Assay 2 was considered to be invalid. An additional experiment (Assay 3) was performed using the same experimental conditions to complete data. The metaphase analysis results of the invalid experiment are not reported; however, all the data will be kept and archived in the raw data binder.

In Chromosome Aberration Assay 3, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 28 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 8, 6, 4, 3, 2, 1.5, 1, 0.75, 0.5, 0.25 and 0.125 μg/mL (experiment without metabolic activation), and 40, 20, 15, 10, 7.5, 5, 2.5 and 1.25 μg/mL (experiment with metabolic activation).

In Assay 3, similarly to the second experiment, insolubility was detected at the end of the treatment period in the final treatment medium in the 40-7.5 μg/mL concentration range with metabolic activation. No large changes in the pH and osmolality were detected. Marked cytotoxicity was observed in this assay at 8, 6, 4, 3 and 2 μg/mL concentrations without metabolic activation (relative survival values of 0, 6, 27, 31 and 46%, respectively) and at 40, 20, 15 and 10 μg/mL concentrations with metabolic activation (relative survival values of 4, 10, 29 and 42%, respectively). Therefore, concentrations of 2, 1 and 0.5 μg/mL (a total of three) were chosen for evaluation in the experiment without metabolic activation, and concentrations of 10, 7.5 and 5 μg/mL (a total of three) were chosen for evaluation in the experiment with metabolic activation. None of the treatment doses caused a significant increase in the number of cells with structural chromosome aberrations either with or without metabolic activation. This assay was considered to confirm the negative results of the first main test.

Polyploid metaphases (1-7) were found in some cases in the negative (vehicle) control, positive control or test item treated samples in the performed experiments. No endoreduplicated metaphases were observed in the performed experiments except of one endoreduplicated metaphase observed in Assay 3 at 0.5 μg/mL concentration without metabolic activation.

Summaryof Chromosome Aberration Assay 1 experiment without metabolic activation

Concentration (μg/mL)	Time of Treatment / Sampling	Relative Survival# (%)	Insolubility##	Mean % aberrant cells###
SN-475N without metabolic activation (-S9)
Negative (vehicle) control	3h / 20h	100	-	1.5
20 μg/mL	3h / 20h	20	-	NE
10 μg/mL	3h / 20h	30	-	NE
8 μg/mL	3h / 20h	39	-	NE
6 μg/mL	3h / 20h	43	-	3.5
5 μg/mL	3h / 20h	58	-	NE
4 μg/mL	3h / 20h	76	-	2.5
3 μg/mL	3h / 20h	89	-	NE
2 μg/mL	3h / 20h	90	-	1.0
1 μg/mL	3h / 20h	86	-	NE
0.5 μg/mL	3h / 20h	100	-	NE
Positive control	3h / 20h	91	-	3.5

Negative (vehicle) control: 1% (v/v) DMSO

Positive control (-S9): Ethyl methanesulfonate, 1 μL/mL

NE: not evaluated

#: compared to the negative (vehicle) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

Summaryof Chromosome Aberration Assay 1 experiment with metabolic activation

Concentration (μg/mL)	Time of Treatment / Sampling	Relative Survival# (%)	Insolubility##	Mean % aberrant cells###
SN-475N without metabolic activation (-S9)
Negative (vehicle) control	3h / 20h	100	-	3.0
40 μg/mL	3h / 20h	4	+	NE
20 μg/mL	3h / 20h	6	+^a	NE
15μg/mL	3h / 20h	31	+^a	3.0
10 μg/mL	3h / 20h	50	-	2.0
7.5 μg/mL	3h / 20h	66	-	NE
5 μg/mL	3h / 20h	84	-	4.0
2.5 μg/mL	3h / 20h	88	-	NE
1.25 μg/mL	3h / 20h	77	-	NE
Positive control	3h / 20h	67	-	62.5***

Negative (vehicle) control: 1% (v/v) DMSO

Positive control (-S9): Ethyl methanesulfonate, 1 μL/mL

NE: not evaluated

#: compared to the negative (vehicle) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

a: minimal amount of insolubility was detected

 

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

 

Summary of Chromosome Aberration Assay 3 experiment without metabolic activation

Concentration (μg/mL)	Time of Treatment / Sampling	Relative Survival# (%)	Insolubility##	Mean % aberrant cells###
SN-475N without metabolic activation (-S9)
Negative (vehicle) control	3h / 28h	100	-	2.5
8 μg/mL	3h / 28h	0	-	NE
6 μg/mL	3h / 28h	6	-	NE
4 μg/mL	3h / 28h	27	-	NE
3 μg/mL	3h / 28h	31	-	NE
2 μg/mL	3h / 28h	46	-	3.0
1.5 μg/mL	3h / 28h	52	-	NE
1 μg/mL	3h / 28h	63	-	2.5
0.75 μg/mL	3h / 28h	90	-	NE
0.5 μg/mL	3h / 28h	98	-	4.0
0.25 μg/mL	3h / 28h	98	-	NE
0.125 μg/mL	3h / 28h	102	-	NE
Positive control	3h / 28h	78	-	40.0***

Negative (vehicle) control: 1% (v/v) DMSO

Positive control (-S9): Ethyl methanesulfonate, 1 μL/mL

NE: not evaluated

#: compared to the negative (vehicle) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

Summary of Chromosome Aberration Assay 3 experiment with metabolic activation

Concentration (μg/mL)	Time of Treatment / Sampling	Relative Survival# (%)	Insolubility##	Mean % aberrant cells###
SN-475N without metabolic activation (-S9)
Negative (vehicle) control	3h / 28h	100	-	3.5
40 μg/mL	3h / 28h	4	+^a	NE
20 μg/mL	3h / 28h	10	+^a	NE
15 μg/mL	3h / 28h	29	+^a	NE
10 μg/mL	3h / 28h	42	+^a	4.0
7.5 μg/mL	3h / 28h	63	+^a	3.5
5 μg/mL	3h / 28h	88	-	2.5
2.5 μg/mL	3h / 28h	91	-	NE
1.25 μg/mL	3h / 28h	94	-	NE
Positive control	3h / 28h	47	-	49.2***

Negative (vehicle) control: 1% (v/v) DMSO

Positive control (-S9): Ethyl methanesulfonate, 1 μL/mL

NE: not evaluated

#: compared to the negative (vehicle) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

a: minimal amount of insolubility was detected

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

Conclusions:

SN-475N did not induce a significant level of chromosome aberrations in the performed experiments with or without metabolic activation. Therefore, SN-475N is considered not clastogenic in this test system.

Executive summary:

SN-475N was tested in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells. The test item was formulated in DMSO and it was examined up to the cytotoxic concentrations according to the OECD guideline recommendations. In the performed independent Chromosome Aberration Assays using duplicate cultures at least 200 well-spread metaphase cells (or until a clear positive response was detected) were analysed for each test item treated, negative (vehicle) and positive control sample.

In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 20, 10, 8, 6, 5, 4, 3, 2, 1 and 0.5 μg/mL (experiment without metabolic activation); and 40, 20, 15, 10, 7.5, 5, 2.5 and 1.25 μg/mL (experiment with metabolic activation).

In Assay 1, insolubility was detected at the end of the treatment period in the final treatment medium in the 40-15 μg/mL concentration range with metabolic activation. There were no large changes in the pH and osmolality. Cytotoxicity was observed in this assay at 20, 10, 8 and 6 μg/mL concentration without metabolic activation (relative survival values were 20, 30, 39 and 43%, respectively); and at 40, 20 and 15 μg/mL concentration with metabolic activation (relative survival values were 4, 6 and 31%, respectively). Therefore, concentrations of 6, 4 and 2 μg/mL (a total of three) were chosen for evaluation in the experiments without metabolic activation; and concentrations of 15, 10 and 5 μg/mL (a total of three) were chosen for evaluation in the experiments with metabolic activation. None of the treatment doses caused a significant increase in the number of cells with structural chromosome aberrations, therefore this test was considered to be negative.

In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 28 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 8, 6, 4, 3, 2, 1.5, 1, 0.75, 0.5, 0.25 and 0.125 μg/mL (experiment without metabolic activation), and 40, 20, 15, 10, 7.5, 5, 2.5 and 1.25 μg/mL (experiment with metabolic activation).

Based on the elevated aberration frequency of the negative (vehicle) control samples, Assay 2 was considered to be invalid. An additional experiment (Assay 3) was performed using the same experimental conditions to complete data. The metaphase analysis results of the invalid experiment are not reported; however, all the data will be kept and archived in the raw data binder.

In Chromosome Aberration Assay 3, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 28 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 8, 6, 4, 3, 2, 1.5, 1, 0.75, 0.5, 0.25 and 0.125 μg/mL (experiment without metabolic activation), and 40, 20, 15, 10, 7.5, 5, 2.5 and 1.25 μg/mL (experiment with metabolic activation).

In Assay 3, insolubility was detected at the end of the treatment period in the final treatment medium in the 40-7.5 μg/mL concentration range with metabolic activation. No large changes in the pH and osmolality were detected. Marked cytotoxicity was observed in this assay at 8, 6, 4, 3 and 2 μg/mL concentrations without metabolic activation (relative survival values of 0, 6, 27, 31 and 46%, respectively) and at 40, 20, 15 and 10 μg/mL concentrations with metabolic activation (relative survival values of 4, 10, 29 and 42%, respectively). Therefore, concentrations of 2, 1 and 0.5 μg/mL (a total of three) were chosen for evaluation in the experiment without metabolic activation, and concentrations of 10, 7.5 and 5 μg/mL (a total of three) were chosen for evaluation in the experiment with metabolic activation. None of the treatment doses caused a significant increase in the number of cells with structural chromosome aberrations either with or without metabolic activation. This assay was considered to confirm the negative results of the first main test.

The occurrence of polyploid and endoreduplicated metaphases was recorded in the main tests. Polyploid metaphases (1-7) were found in some cases in the negative (vehicle) control, positive control or test item treated samples in the performed experiments. No endoreduplicated metaphases were observed in the performed experiments except of one endoreduplicated cell in Assay 3.

The negative (vehicle) control data were within the laboratory’s normal range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system. The evaluated concentration range was considered to be adequate; at least three test item treated concentrations were evaluated in each assay. The tests were considered to be valid.

In conclusion, SN-475N did not induce a significant level of chromosome aberrations in the performed experiments with or without metabolic activation. Therefore, SN-475N is considered not clastogenic in this test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Mouse Micronucleus Test:

No induction of micronuclei in bone marrow erythrocytes was observed following administration of SN-475N to mice at up to and including 2000 mg/kg bw/day; thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 May 2013 to 13 May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In-Vivo study carried out as substance is intended for global registration where In-Vivo data is required.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

See other information on method section for details

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

See other information on method section for details

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

No further details specified in the study report

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The NMRI mouse is one of the standard animals used internationally in this type of mutagenicity testing

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and strain: RjHan: NMRI mice
Source: Elevage Janvier
Route des Chènes Secs B.P. 4105
53940 LE GENEST-ST-ISLE, France
Justification of species/strain: The NMRI mouse is one of the standard animals used internationally in this type of mutagenicity testing
Hygienic level: SPF at arrival; standard laboratory conditions during the study
Number of animals: Preliminary experiment: 8 males + 8 females
4 groups, 2 animals/sex/group
Main test: 35+2 males
Positive control group: 5 mice
Negative control group: 10 mice
High-dose group: 10+2 mice
Low- and mid-dose groups: 5 mice
Age of animals: approximately 7 weeks (at the treatment)
Body weight: 35.2 – 37.5 g (males, preliminary experiment)
25.0 - 26.5 g (females, preliminary experiment)
32.7 – 35.5 g (males, main test)
Acclimatisation period: at least 5 days
Note: The weight variation did not exceed ± 20 percent of the mean weight/sex at the start of the treatment.

Husbandry
Animal health: Only animals in acceptable health condition were used for the test. Health status was certified by the veterinarian.
Housing/Enrichment: Group caging (5 animals/cage or 2 animals/cage) to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities.
Cage type: II. type polypropylene/polycarbonate
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.4 – 24.3°C
Relative humidity: 30 – 70 %
Ventilation: 15 – 20 air exchanges/hour
Animal room: 244/2 (preliminary experiment), 240 (main test)

The environmental parameters were recorded twice daily during the acclimatisation period and experimental phases of the study.

Food and Water Supply
Animals received ssniff® SM R/M-Z+H "Autoclavable complete diet for mice and rats – breeding and maintenance" (Batch number: 445 8440 / 175 8935, Expiry date: May 2013 / August 2013) produced by ssniff Spezialdiäten GmbH (D-59494, Soest, Germany) and tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum.
The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The contents of the standard diet are detailed in Appendix 8. The supplier provided an analytical certificate for the batch used.
Water quality control analysis is performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary). The quality control results are retained in the archives at CiToxLAB Hungary Ltd.

Bedding
Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. Lignocel 3/4-FASERN Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH & Co.KG (Holzmühle 1, 73494 Rosenberg, Germany) was available to animals during the study.

Identification
Animals were identified by a number unique within the study, written with indelible ink on the tail and cross-referenced to the Animal Master File at CiToxLAB Hungary Ltd. The cages were marked with identification cards, with information about study code, sex, dose group and individual animal numbers.

Randomisation
The animals were assigned to their respective treatment groups by randomization based on body weights. Animals were randomly allocated to the negative and positive control groups based on the most recent actual body weight; SPSS/PC+ software was used in order to verify homogeneity/variation among/within groups. Females and males were randomized separately.

Route of administration:

oral: gavage

Vehicle:

PEG-400 was used for vehicle of the study.

Details on exposure:

Preliminary toxicity test:
A preliminary toxicity test was performed to identify the appropriate maximum dose level for the main test. The preliminary toxicity test also determined whether there are large differences in toxicity between the sexes or not. Groups of two male and female mice were treated at one occasion by oral gavage at the dose levels of 2000, 1000, 500 and 250 mg/kg body weight.
The treatment volume was 10 mL/kg body weight. Animals were examined regularly for toxic signs and mortalities. The surviving mice were euthanized 48 hours after treatment. No bone marrow smears were prepared in the preliminary experiment.

Main test:
Based on the results of the preliminary toxicity test, 2000 mg/kg body weight (the maximum recommended dose) was selected for the highest dose level of the test item in the main micronucleus test. Two lower dose levels separated by a factor of two (1000 and 500 mg/kg body weight) were also included in the main test. The dose levels were expressed in terms of the test item as received.
The main test was performed using male animals only because the toxic effect of the test item was similar in both sexes in the preliminary toxicity test.

Duration of treatment / exposure:

48 hours

Frequency of treatment:

Dose of 2000, 1000 and 500 mg/kg body weight by oral gavage administered at the start of the study. No further treatment was administered for the duration of the study.

Post exposure period:

In the low and mid dose groups, furthermore in the positive control group the sampling was made once at 24 hours after treatment. In the high dose group and vehicle control group, sampling was made 24 and 48 hours after treatment. Five male animals per dose group were used for sampling on each one occasion.

Dose / conc.:

2 000 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

500 mg/kg bw (total dose)

No. of animals per sex per dose:

5 animals per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide monohydrate was used as positive control material for the study. It was dissolved in sterile physiological saline solution for treatment. Routine safety precautions (lab coat, gloves, safety glasses and face mask) for the positive control material were applied to assure personnel health and safety. The positive control formulations were prepared immediately before treatment in the Central Dispensary Unit of CiToxLAB Hungary Ltd.

Data of the chemical used as positive control substance are shown below:

Name: Cyclophosphamide monohydrate
Abbreviation: CP
Supplier: Sigma-Aldrich Co.
Lot No.: 120M1253V
Appearance: White powder
Expiry date: 31 December 2013
Storage condition: Refrigerated (2-8 °C)

Tissues and cell types examined:

Erythrocytes obtained from the bone marrow of the femur.

Details of tissue and slide preparation:

The bone marrow was flushed out of each pair of femurs with foetal bovine serum (5 mL) using a syringe and needle into a sterile centrifuge tube. After mixing, the cell suspension was concentrated by a gentle centrifugation and the supernatant was discarded. Smears of the cell pellet were made on standard microscope slides (at least 2 slides / animal). Slides were air-dried at room temperature for approximately 24 hours.
Dried slides were fixed in methanol for a minimum of 5 minutes and allowed to air-dry. Two slides per animal were stained with 10 % Giemsa solution for 20 minutes then thoroughly rinsed with distilled water, and then air-dried at room temperature for at least 12 hours. After staining, coverslips were mounted on them.

Evaluation criteria:

Criteria for a positive response:
The test item is considered to have shown genotoxic activity if statistically significant increases in the frequency of micronucleated polychromatic erythrocytes are observed in treated animals compared to the corresponding negative controls, and the increases are dose-related. Historical control data are taken into consideration when evaluating the biological significance of small increases.
Criteria for a negative response:
The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed above the concurrent and historical negative control values.
Equivocal response:
Results which do not meet the criteria for a positive or negative response are considered to be equivocal. Further investigations or scoring of additional cells may be necessary in case of an equivocal result.

Statistics:

Not specified.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

PRELIMINARY TOXICITY TEST

According to the observation in a short preliminary solubility test, no proper formulation could be made at 200 mg/mL concentration using physiological saline as vehicle. However, the formulation at the same concentration using PEG 400 (Poly(ethylene glycol) 400) as vehicle was suitable for treatment of the animals. Therefore, PEG 400 was selected for vehicle of the study and the following dose groups were examined in the preliminary toxicity test: 2000, 1000, 500 and 250 mg/kg body weight (2 animals/sex/group).

There was no treatment related effect on the body weight in the preliminary experiment. The individual body weights of the animals in the preliminary experiment are shown in Table 3 of Appendix 3. The observed clinical signs are listed in Table 4 of Appendix 3. All animals were free of clinical signs in the preliminary experiment except of one male in the 1000 mg/kg body weight dose group showing piloerection at one time point, but this observation was not considered to represent a dose-related toxicity.

Based on the results of the preliminary toxicity test, dose levels of 2000, 1000 and 500 mg/kg body weight were selected for the micronucleus test. As there were no differences between male and female animals in the preliminary experiment, the main experiment was performed using male mice only.

MOUSE MICRONUCLEUS TEST

Groups of five male mice were treated with the test item at 2000, 1000 and 500 mg/kg body weight or with the vehicle (PEG 400) in the main experiment (two replacement animals were also treated in the high dose group). All mice in the negative (vehicle) control and test item groups were dosed by oral gavage using a dose volume of 10 mL/kg body weight. Animals of the positive control group were treated by intraperitoneal injection with Cyclophosphamide at 60 mg/kg body weight using a dose volume of 10 mL/kg body weight.

No marked effect of test item treatment on the body weight of the mice was observed in the main test. Marked body weight loss (>10%) was detected for one negative control animal but since it had normal bone marrow results, this was not considered to be a significant finding

No mortality or signs of systemic toxicity were observed during the study. Piloerection and wheal at the left thorax ventral area was observed for one animal in the negative control group, since it had normal bone marrow results, this was not considered to be a significant finding (Appendix 5). The animals in the test item treated and positive control groups were symptom-free during the whole observation period (Appendix 5).

Two thousand polychromatic erythrocytes (PCEs) were scored per animal* to assess the micronucleated cells. The frequency of micronucleated cells was expressed as percent of micronucleated cells based on the first 2000 PCEs counted in the optic field. The proportion of immature among total erythrocytes was determined for each animal by also counting mature erythrocytes (NCEs) until a total of 1000 PCEs+NCEs had been observed.

*Note: During the initial analysis, two animals in the group treated with 2000 mg/kg bw at the 48-hour sampling point had less than 2000 PCEs on the slides. Although sufficient cells were subsequently found on further slides which were sent later, the slides of additional 2 replacement animals were then also analysed. Thus, there are 7 animals in this treatment group.

The group treated with 2000 mg/kg bw, which gave the highest number of micronuclei at the 24-hour sampling point, were compared with the relevant vehicle control group using the Kruskal Wallis test. This gave a value of H = 2.635, which is non-significant. The average number of micronuclei observed at 2000 mg/kg bw at the 48-hour sampling point was lower than the corresponding negative control group. Therefore there was a negative response at both times.

The positive and negative control results were also compared, and gave a value of H = 6.990 (p<0.01). The positive control treatment therefore caused a very substantial increase, demonstrating the sensitivity of the test system.

The positive and negative control* data are considered to give adequate data to confirm the validity of the study.

*Note: For one animal in the vehicle control group at the 48-hour sampling point, the number of micronuclei was slightly higher than the upper limit of the historical control range. However, the difference was minor and the mean value of this group was within the historical control range, so this fact was considered to be acceptable.

The frequency of micronucleated polychromatic erythrocytes of the negative (solvent) control group was within the range of historical laboratory control data; the positive control item produced a biologically and statistically relevant increase in the number of micronucleated polychromatic erythrocytes; each treated and control group included at least 5 analysable animals; therefore, the test was considered to be valid.

Results of the Preliminary Experiment

Individual Body Weights for all Animals with Group Means

Animal Number	Gender	Dose (mg/kg/bw	Body weight (%)			Change (%)
			Day 1	Day 2	Day 3
1980	M	2000	36.3	34.6	36.3	0.0
1984	M	2000	36.7	36.1	37.2	1.4
		Mean	36.5	35.35	36.75	0.7
1978	M	1000	36.8	36.1	36.4	-1.1
1985	M	1000	36.2	35.1	36.0	-0.6
		Mean	36.5	35.60	36.20	-0.8
1979	M	500	36.8	36.4	36.7	-0.3
1986	M	500	35.8	34.2	34.6	-3.4
		Mean	36.30	35.30	35.65	-1.8
1981	M	250	37.5	36.7	36.5	-2.7
1989	M	250	35.2	34.7	35.0	-0.6
		Mean	36.35	35.7	35.75	-1.7
1995	F	2000	26.0	25.5	26.3	1.2
1997	F	2000	26.2	25.7	27.4	4.6
		Mean	26.10	25.6	26.85	2.9
1991	F	1000	26.3	27.1	27.5	4.6
1996	F	1000	25.9	25.6	26.1	0.8
		Mean	26.1	26.35	26.8	2.7
1993	F	500	26.5	25.7	25.9	-2.3
2001	F	500	25.0	25.0	25.0	0.0
		Mean	25.75	25.35	25.45	-1.2
1999	F	250	25.0	24.4	24.8	-0.8
2000	F	250	26.5	26.3	27.0	1.9
		Mean	25.75	25.35	25.9	0.6

Notes:

1. M: male, F: female

2. Change: [Body Weight (Day 3) – Body Weight (Day 1)] / Body Weight (Day 1) x 100

 

Clinical observations

DOSE LEVEL: 2000 mg/kg body weight/day SEX: MALE

ID Number	Animal Number	Observations	Time points								Frequency
			After treatment
			30’	1h	2h	3h	4h	5h	24h	48h
1980	3	Symptom free	+	+	+	+	+	+	+	+	8/8
1984	7	Symptom free	+	+	+	+	+	+	+	+	8/8

 

DOSE LEVEL: 1000 mg/kg body weight/day SEX: MALE

ID Number	Animal Number	Observations	Time points								Frequency
			After treatment
			30’	1h	2h	3h	4h	5h	24h	48h
1978	1	Symptom free	+	+	+	+	+	+	+	-	7/8
		Piloerection	-	-	-	-	-	-	-	+	1/8
1985	8	Symptom free	+	+	+	+	+	+	+	+	8/8

 

DOSE LEVEL: 500 mg/kg body weight/day SEX: MALE

ID Number	Animal Number	Observations	Time points								Frequency
			After treatment
			30’	1h	2h	3h	4h	5h	24h	48h
1979	2	Symptom free	+	+	+	+	+	+	+	+	8/8
1986	9	Symptom free	+	+	+	+	+	+	+	+	8/8

  

DOSE LEVEL: 250 mg/kg body weight/day SEX: MALE

ID Number	Animal Number	Observations	Time points								Frequency
			After treatment
			30’	1h	2h	3h	4h	5h	24h	48h
1981	4	Symptom free	+	+	+	+	+	+	+	+	8/8
1989	12	Symptom free	+	+	+	+	+	+	+	+	8/8

DOSE LEVEL: 2000 mg/kg body weight/day SEX: FEMALE

ID Number	Animal Number	Observations	Time points								Frequency
			After treatment
			30’	1h	2h	3h	4h	5h	24h	48h
1995	18	Symptom free	+	+	+	+	+	+	+	+	8/8
1997	20	Symptom free	+	+	+	+	+	+	+	+	8/8

  

DOSE LEVEL: 1000 mg/kg body weight/day SEX: FEMALE

ID Number	Animal Number	Observations	Time points								Frequency
			After treatment
			30’	1h	2h	3h	4h	5h	24h	48h
1991	14	Symptom free	+	+	+	+	+	+	+	+	8/8
1996	19	Symptom free	+	+	+	+	+	+	+	+	8/8

 

 DOSE LEVEL: 500 mg/kg body weight/day SEX: FEMALE

ID Number	Animal Number	Observations	Time points								Frequency
			After treatment
			30’	1h	2h	3h	4h	5h	24h	48h
1993	16	Symptom free	+	+	+	+	+	+	+	+	8/8
2001	24	Symptom free	+	+	+	+	+	+	+	+	8/8

  

DOSE LEVEL: 250 mg/kg body weight/day SEX: FEMALE

ID Number	Animal Number	Observations	Time points								Frequency
			After treatment
			30’	1h	2h	3h	4h	5h	24h	48h
1999	22	Symptom free	+	+	+	+	+	+	+	+	8/8
2000	23	Symptom free	+	+	+	+	+	+	+	+	8/8

Individual Body Weight Data

Mice sacrificed 24 hours after dosing

Treatment	ID Number	Animal number	Body weight (g)
			Day 1	Day 2	Change
Group 1 Negative (vehicle) control (PEG 400)	2327	2	34.2	33.3	-2.6
	2343	18	32.7	28.3	-13.5
	2354	29	34.6	33.3	-3.8
	2356	31	34.1	33.1	-2.9
	2370	45	34.7	34.7	0.0
	Mean		34.1	32.5	-4.5
Group 2 SN-475N (500 mg/kg bw)	2334	9	34.9	33.5	-4.0
	2337	12	34.1	34.4	0.9
	2338	13	34.2	33.7	-1.5
	2340	15	34.6	33.5	-3.2
	2360	35	32.7	31.7	-3.1
	Mean		34.1	33.4	-2.2
Group 3 SN-475N (1000 mg/kg bw)	2333	8	33.4	33.1	-0.9
	2341	16	34.9	34.8	-0.3
	2358	33	34.0	34.1	0.3
	23361	36	34.2	33.8	-1.2
	2366	41	34.5	34.5	0.0
	Mean		34.2	34.1	-0.4
Group 4 SN-475N (2000 mg/kg bw)	2331	6	34.5	32.9	-4.6
	2332	7	34.3	32.8	-4.4
	2346	21	34.0	32.7	-3.8
	2349	24	35.0	35.6	1.7
	2365	40	33.4	32.4	-3.0
	Mean		34.2	33.3	-2.8
Group 5 Positive control (Cyclophosphamide 60 mg/kg bw)	2330	5	34.5	33.7	-2.3
	2342	17	34.0	32.5	-4.4
	2350	25	33.5	31.8	-5.1
	2351	26	35.1	33.0	-6.0
	2363	38	34.3	32.5	-5.2
	Mean		34.3	32.7	-2.6

Note:

Change = [Terminal Body Weight (Day 2) – Initial Body Weight (Day 1)] / Initial Body Weight (Day 1) x 100

Individual Body Weight Data

Mice sacrificed 48 hours after dosing

Treatment	ID Number	Animal number	Body weight (g)
			Day 1	Day 2	Change
Group 1 Negative (vehicle) control (PEG 400)	2326	1	34.5	34.2	-0.9
	2329	4	32.9	32.5	-1.2
	2348	23	35.3	35.3	0.0
	2359	34	33.9	32.9	-2.9
	2364	39	34.3	34.2	-0.3
	Mean		34.2	33.8	-1.1
Group 4 SN-475N (2000 mg/kg bw)	2344	19	35.5	34.1	-3.9
	2347	22	33.6	33.8	0.6
	2353	28	34.4	32.5	-5.5
	2355	30	33.6	33.2	-1.2
	2369	44	34.3	33.3	-2.9
	2328*	3*	35.2	34.7	-1.4
	2367*	42*	33.5	33.5	0.0
	Mean		34.3	33.6	-2.1

Notes:

Change = [Terminal Body Weight (Day 3) – Initial Body Weight (Day 1)] / Initial Body Weight (Day 1) x 100

*: replacement animal

Clinical Observations

Mice sacrificed 24 hours after dosing

DOSE LEVEL: Negative(vehicle) control  SEX: MAL

ID Number	Animal Number	Observations	Time points							Frequency
			After treatment
			30’	1h	2h	3h	4h	5h	24h
2327	2	Symptom free	+	+	+	+	+	+	+	7/7
2343	18	Symptom free	+	+	+	-	-	-	-	3/7
		Piloerection	-	-	-	-	-	-	+	1/7
		Wheal (left thorax ventral)	-	-		+	+	+	+	4/7
2354	29	Symptom free	+	+	+	+	+	+	+	7/7
2356	31	Symptom free	+	+	+	+	+	+	+	7/7
2370	45	Symptom free	+	+	+	+	+	+	+	7/7

DOSE LEVEL: 500 mg/kg body weight  SEX: MALE

ID Number	Animal Number	Observations	Time points							Frequency
			After treatment
			30’	1h	2h	3h	4h	5h	24h
2334	9	Symptom free	+	+	+	+	+	+	+	7/7
2337	12	Symptom free	+	+	+	+	+	+	+	7/7
2338	13	Symptom free	+	+	+	+	+	+	+	7/7
2340	15	Symptom free	+	+	+	+	+	+	+	7/7
2360	35	Symptom free	+	+	+	+	+	+	+	7/7

DOSE LEVEL: 1000 mg/kg body weight  SEX: MALE

ID Number	Animal Number	Observations	Time points							Frequency
			After treatment
			30’	1h	2h	3h	4h	5h	24h
2333	8	Symptom free	+	+	+	+	+	+	+	7/7
2341	16	Symptom free	+	+	+	+	+	+	+	7/7
2358	33	Symptom free	+	+	+	+	+	+	+	7/7
2361	36	Symptom free	+	+	+	+	+	+	+	7/7
2366	41	Symptom free	+	+	+	+	+	+	+	7/7

 

 DOSE LEVEL: 2000 mg/kg body weight  SEX: MALE

ID Number	Animal Number	Observations	Time points							Frequency
			After treatment
			30’	1h	2h	3h	4h	5h	24h
2331	6	Symptom free	+	+	+	+	+	+	+	7/7
2332	7	Symptom free	+	+	+	+	+	+	+	7/7
2346	21	Symptom free	+	+	+	+	+	+	+	7/7
2349	24	Symptom free	+	+	+	+	+	+	+	7/7
23655	40	Symptom free	+	+	+	+	+	+	+	7/7

 

DOSE LEVEL: Positive control (CP, 60 mg/kg body weight) SEX: MALE

ID Number	Animal Number	Observations	Time points							Frequency
			After treatment
			30’	1h	2h	3h	4h	5h	24h
2330	5	Symptom free	+	+	+	+	+	+	+	7/7
2342	17	Symptom free	+	+	+	+	+	+	+	7/7
23250	25	Symptom free	+	+	+	+	+	+	+	7/7
2351	26	Symptom free	+	+	+	+	+	+	+	7/7
2363	38	Symptom free	+	+	+	+	+	+	+	7/7

Clinical Observations

Mice sacrificed 48 hours after dosing

 

DOSE LEVEL: Negative (vehicle) control SEX: MALE

ID Number	Animal Number	Observations	Time points								Frequency
			After treatment
			30’	1h	2h	3h	4h	5h	24h	48h
2326	1	Symptom free	+	+	+	+	+	+	+	+	8/8
2329	4	Symptom free	+	+	+	+	+	+	+	+	8/8
2348	23	Symptom free	+	+	+	+	+	+	+	+	8/8
2359	34	Symptom free	+	+	+	+	+	+	+	+	8/8
2364	39	Symptom free	+	+	+	+	+	+	+	+	8/8

DOSE LEVEL: 2000 mg/kg body weight/day SEX: MALE

ID Number	Animal Number	Observations	Time points								Frequency
			After treatment
			30’	1h	2h	3h	4h	5h	24h	48h
2344	19	Symptom free	+	+	+	+	+	+	+	+	8/8
2347	22	Symptom free	+	+	+	+	+	+	+	+	8/8
2353	28	Symptom free	+	+	+	+	+	+	+	+	8/8
2355	3	Symptom free	+	+	+	+	+	+	+	+	8/8
2369	44	Symptom free	+	+	+	+	+	+	+	+	8/8
2328*	3*	Symptom free	+	+	+	+	+	+	+	+	8/8
2367*	42*	Symptom free	+	+	+	+	+	+	+	+	8/8

*: replacement animal

Micronucleus Data

Mice sacrificed 24 hours after dosing

Treatment	ID Number	Animal number	MNPCE/ 2000 PCE	PCE/1000 PCE + NCE
Group 1 Negative (vehicle) control (PEG 400)	2327	2	4	346
	2343	18	1	238
	2354	29	4	289
	2356	31	3	591
	2370	45	4	321
	Mean		3.2	357.0
	SD		1.30	136.89
Group 2 SN-475N (500 mg/kg bw)	2334	9	9	344
	2337	12	2	221
	2338	13	1	431
	2340	15	5	450
	2360	35	3	283
	Mean		4.0	345.8
	SD		3.16	97.00
Group 3 SN-475N (1000 mg/kg bw)	2333	8	5	225
	2341	16	8	245
	2358	33	2	513
	23361	36	3	300
	2366	41	5	459
	Mean		4.6	348.4
	SD		2.30	129.99
Group 4 SN-475N (2000 mg/kg bw)	2331	6	5	278
	2332	7	3	371
	2346	21	8	209
	2349	24	9	268
	2365	40	4	313
	Mean		5.8	287.8
	SD		2.59	59.70
Group 5 Positive control (Cyclophosphamide 60 mg/kg bw)	2330	5	54	182
	2342	17	66	266
	2350	25	68	450
	2351	26	120	330
	2363	38	62	353
	Mean		74.0	316.2
	SD		26.27	99.97

MNPCE: Number of Micronucleated Polychromatic Erythrocytes referring to counts of 2000 PCE.

PCE: Polychromatic Erythrocyte

NCE: Normochromatic Erythrocyte

Micronucleus Data

Mice sacrificed 48 hours after dosing

Treatment	ID Number	Animal number	MNPCE/ 2000 PCE	PCE/1000 PCE + NCE
Group 1 Negative (vehicle) control (PEG 400)	2326	1	3	126
	2329	4	8	291
	2348	23	5	393
	2359	34	7	195
	2364	39	5	324
	Mean		5.6	265.8
	SD		1.95	105.78
Group 4 SN-475N (2000 mg/kg bw)	2344	19	4	303
	2347	22	2	351
	2353	28	8	178
	2355	30	5	161
	2369	44	4	208
	2328*	3*	1	232
	2367*	42*	3	217
	Mean		3.9	235.7
	SD		2.27	68.15

MNPCE: Number of Micronucleated Polychromatic Erythrocytes referring to counts of 2000 PCE.

PCE: Polychromatic Erythrocyte

NCE: Normochromatic Erythrocyte

*: replacement animal

Conclusions:

No induction of micronuclei in bone marrow erythrocytes was observed following administration of SN-475N to mice at up to and including 2000 mg/kg bw/day; thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.

Executive summary:

The objective of the study was to determine whether SN-475N test item caused genotoxic effects resulting in the formation of micronuclei in erythrocytes of treated mice in accordance with the OECD guideline “Mammalian Erythrocyte Micronucleus Test”, No. 474 (1997).

In the preliminary toxicity test, groups of two male and two female mice were treated with the test item formulated in PEG 400 at 2000, 1000, 500 and 250 mg/kg body weight by oral gavage. No treatment related effect was observed in the preliminary experiment. Therefore, the highest dose level selected for the main test was 2000 mg/kg body weight which is the maximum recommended dose level for materials of low toxicity. Only male mice were used in the main test as observations in the preliminary test showed that there was no substantial difference in the toxicity of the test item between the sexes.

In the main test, groups of male mice were treated with the vehicle (PEG 400) or the test item at 2000, 1000 and 500 mg/kg body weight by oral gavage or the positive control item (Cyclophosphamide dissolved in physiological saline) at 60 mg/kg body weight administered by intraperitoneal injection (two replacement animals were also treated in the high dose group). Five mice from each group were examined 24 hours after dosing, and a further five mice dosed with the vehicle or test item at 2000 mg/kg body weight were examined 48 hours after dosing. Bone marrow smears were prepared on glass slides for each of the mice, stained, and scored. Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells.

Test item treatment showed no marked effect on body weight in the main test. No mortality or signs of systemic toxicity were observed during the study. The animals in the test item treated and positive control groups were symptom-free during the whole observation period.

No biologically or statistically significant increases in the frequency of micronucleated polychromatic erythrocytes were seen in mice treated with the test item, compared to the negative (vehicle) control values at any of the sampling time points. The positive control treatment caused a large, clearly positive response demonstrating the sensitivity of the test system.

In conclusion, no induction of micronuclei in bone marrow erythrocytes was observed following administration of SN-475N to mice at up to and including 2000 mg/kg bw/day; thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)