2-Naphthalenesulfonic acid, 6-amino-5-[[4-[[4-chloro-6-[ethyl[3-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-2-sulfophenyl]azo]-4-hydroxy-, sodiumsalt

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct. 28, 2008 ~ Nov. 19, 2008

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

other company data

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): Everzol Red LF-B
- Molecular formula (if other than submission substance): C29H24ClN8O13S4 · 3Na
- Molecular weight (if other than submission substance): 925.21
- Smiles notation (if other than submission substance): not applicable
- InChl (if other than submission substance): not applicable
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type: mono constituent substance
- Physical state: Dark Purple Powder
- Analytical purity: 68.6%
- Impurities (identity and concentrations): see section 1.2
- Composition of test material, percentage of components: see section 1.2
- Isomers composition: see section 1.2
- Lot/batch No.: R4505504
- Expiration date of the lot/batch: Aug. 12, 2009
- Stability under test conditions: stable
- Storage condition of test material: 2~8 ℃

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: National Laboratory Animal Center, Taipei, Taiwan
- Age at study initiation: about 6 ~ 7 weeks
- Weight at study initiation: 22.0 ~ 25.3g
- Assigned to test groups randomly: yes, under the body weight basis
- Fasting period before study: no
- Housing: Male mice were housed up to five per cage in polycarbonate autoclavable cages with hardwood chip bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2℃
- Humidity (%): 50 ± 20%
- Air changes (per hr): 22
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light


IN-LIFE DATES: From: Oct. 28, 2008 To: Nov. 14, 2008

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water for injection
- Justification for choice of solvent/vehicle: high solubility in water
- Concentration of test material in vehicle: 25, 50, 100 mg/mL
- Amount of vehicle (if gavage or dermal): 20 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On each dosing day, appropriate weights of test article will be taken and dissolved in water for injection to make the concentrations of 25, 50 and
100 mg/kg

Duration of treatment / exposure:

Nov. 10, 11, and 12, 2008

Frequency of treatment:

once daily

No. of animals per sex per dose:

5 males/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C;
- Justification for choice of positive control(s): laboratory historical data
- Route of administration: intraperitoneal injection
- Doses / concentrations: 1.0 mg/kg

Examinations

Tissues and cell types examined:

peripheral blood will be collected once in each animal of test article treated and negative control groups between 36 hours and 48 hours after the last treatment. Blood in the positive control will be sampled once approximately 48 hours after administration.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: dose-ranging finding test


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): between 36 hours and 48 hours after the last treatment


DETAILS OF SLIDE PREPARATION: The blood samples will be collected by the tail trimming and smeared on acridine orange-coated slides. The slides will be wrapped to protect from light and stored at 0 ~ 6℃ for at least 4 hours before scoring. A minimum of two slide will be prepared from each animal.


METHOD OF ANALYSIS: Fluorenscence microscope observation

Evaluation criteria:

Cytotoxicity evaluation: The quantification of the effect of the test article on erythropoiesis will be used as indicator of bone marrow toxicity, the proportion of erythropoiesis to total erythrocytes will be presented in each animal and every group. This ratio in the treatemnt groups should no be less than 20% of negative control.
The incidence of micronucleated reticulocytes per 1000 reticulocytes (MN/1000 RETs) will be presented for each animal and the mean ± SD will be expressed for each group.

Statistics:

One-way ANOVA for comparisons of all data collected on the body weight for each treatment group and negative control.
U-test (Mann-Whitney Rank Sum test) for comparing treated group and negative control of frequencies of micronucleated reticulocytes.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

 Table 1. Dose Levels of Everzol Red LF-B in Mice Micronucleus Assay

Group	Dose Level	Concentration	Dose Volume	No. of Mice
Number	(mg/kg)	(mg/mL)	(mL/kg)
1	0 (Vehicle)	0	20	5
2	500	25	20	5
3	1000	50	20	5
4	2000	100	20	5
5	1.0*	0.05	20	5

*: Mitomycin C        Vehicle: Water for injection

Table 2. The Effect of Everzol Red LF-B Treatment on Body Weights of BALB/c Mice

Treatment	Dose	No. of Mice	Body Weight (g) Group Mean ± S.D.
	(mg/kg)		Day 1^a	Day 2^a	Day 3^a	Day 4	Day 5
Water for Injection	0	5	23.5 ± 1.0	24.2 ± 0.9	24.5 ± 0.9	24.4 ± 1.1	24.7 ± 0.9
Test Article	500	5	23.7 ± 0.9	24.4 ± 1.0	24.7 ± 1.2	24.8 ± 1.1	24.7 ± 1.1
	1000	5	24.2 ± 1.1	25.1 ± 0.9	25.5 ± 0.9	25.5 ± 0.9	25.9 ± 1.3
	2000	5	23.2 ± 0.8	23.8 ± 0.9	24.3 ± 0.9	24.6 ± 0.9	24.5 ± 0.9
Mitomycin C	1.0	5	24.4 ± 0.3	25.2 ± 0.2	25.4 ± 0.3	25.3 ± 0.4	25.4 ± 0.4

^a: Day 1, Day 2, and Day 3 were the days of dosing; all data were recorded immediately prior to dosing.

Table 3. Clinical Observations of Micronucleus Assay in BALB/c Mice for Everzol Red LF-B

Dose Level (mg/kg)	Animal ID	Day 1 ^a	Day 2 ^a	Day 3^a	Day 4	Day 5
0 (Negative control)	2080231019 2080231006 2080231007 2080231016 2080231015	all A01	all A01	all A01	all A01	all A01
500	2080231027 2080231012 2080231002 2080231011 2080231029	all X01	all X01	all X01	all A01	all A01
1000	2080231003 2080231005 2080231013 2080231020 2080231022	all X01	all X01	all X01	X01 A01 X01 A01 A01	all A01
2000	2080231028 2080231025 2080231030 2080231008 2080231009	all X01	all X01	all X01	X01 A01 A01 X01 A01	all A01
1^b (Positive control)	2080231014 2080231024 2080231023 2080231026 2080231004	all A01	all A01	all A01	all A01	all A01

^a: Day 1, day 2 and  Day 3 were the days of dosing, clinical observation was conducted after 5 hours dosing.

^b: Mitomycin C 1 mg/kg

Key: A01 = Normal

X01 = Discolored feces (The color was similar to that of the test article)

Table 4. The Effect on the Percentages of Reticulocytes to Total Erythrocytes in BALB/c Mice Treated with Everzol Red LF-B

Dose (mg/kg)	Individual Animal Data (% reticulocytes of 50,000 erythrocytes)					Group Mean ± S.D. (% reticulocytes)
0 (Negative control)	3.75	3.36	3.78	4.11	4.10	3.82 ± 0.31
500	3.38	4.11	4.33	4.05	3.27	3.83 ± 0.47
1000	3.79	3.73	5.03	3.67	5.09	4.26 ± 0.73
2000	3.21	3.70	3.58	3.41	3.45	3.47 ± 0.18
MMC (Positive control)	1.74	2.26	1.80	1.72	1.61	1.83 ± 0.25

Table 5. The Effect on the Frequencies of Micronucleated Reticulocytes of BALB/c Mice Treated with Everzol Red LF-B

Dose (mg/kg)	Individual Animal Data (MN/1000 RETs)					Group Mean ± S.D. (MN/1000 RETs)
0 (Negative control)	3, 0	3, 1	1, 0	1, 1	1, 2	1.3 ± 1.1
500	4, 0	0, 1	1, 1	0, 1	2, 1	1.1 ± 1.2
1000	0, 1	1, 2	0, 3	4, 1	2,1	1.5 ± 1.3
2000	1, 1	4, 0	1, 1	1, 2	0, 0	1.1 ± 1.2
MMC (Positive control)*	36, 33	32, 29	42, 27	34, 33	41, 34	34.1 ± 4.7

*: The individual frequency of MN/1000 RETs was significantly higher than that of the negative control (p <= 0.05, U-test).

Table 6. Concurrent and Historical Control Values of the Frequencies of Micronucleated  Reticulocytes in Mice

	No. of  MN/1000 RETs
	Negative Control*	1.0 mg/kg Mitomycin C**
Concurrent controls	1.3 ± 1.1	34.1 ± 4.7
Historical control
Mean ± S.D.	1.5 ± 0.8	30.8 ± 6.4
Maximum	4.0	45.5
Minimum	0	15.0

*: Negative control: 0.5% CMC, water for injection, 1% CMC, sesame oil, PBS, 2% MC, 20% PEG in saline, 2% Tween 80 and soybean oil; concurrent negative control: water for injection

**:  Mitomycin C was administered by i.p.

Historical control data recording period: Mar. 2003 to Oct. 2008

Negative control sample size = 261

Positive control sample size = 255

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
All criteria for a valid study as described in the protol were met. The results of the micronucleus ssay in BALB/c mice indicated that , under the conditions of this study, Everzol Red LF-B casused a negative response in mouse peripheral blood micronucleus assay.