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

Genetic toxicity: in vivo

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

See other information on method section for details

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

834-894-6

Cas Number:

113601-85-7

Molecular formula:

C10H8O to C82H64O5

IUPAC Name:

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

Test material form:

solid

Details on test material:

Name: CAS 113601-85-7
Chemical name: 1,4-Benzenedimethanol, polymer with 1-naphthalenol
Batch/Lot number: 0950986
Description: Brown, solid
Purity: 100%
Expiry date: 08 February 2020
Storage conditions: Controlled room temperature (15-25°C ≤ 70% relative humidity)
Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to ensure personnel health and safety. Causes skin irritation and serious eye irritation.

Specific details on test material used for the study:

No further details specified in the study report

Test animals

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.

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 000 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw (total dose)

Doses / concentrations 3

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)

Examinations

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.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.