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Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 7, 2019 to Decenber 13, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction products of cuprate(4-), [C-(aminosulfonyl)-C-[[[2-[[4-chloro-6-[(2,5-disulfophenyl)amino]-1,3,5-triazin-2-yl]amino]ethyl]amino]sulfonyl]-29H,31H-phthalocyanine-C,C-disulfonato(6-)-N29,N30,N31,N32]-, tetrasodium and lithium chloride
EC Number:
942-810-5
Molecular formula:
C43H24ClCuN15O16S6.xLi.yNa, (x + y) = 4; 0 < (x,y) < 4 with 1341.9 < MW < 1374 g/mol (UVCB substance), and traces of NaCl and NaSO4
IUPAC Name:
Reaction products of cuprate(4-), [C-(aminosulfonyl)-C-[[[2-[[4-chloro-6-[(2,5-disulfophenyl)amino]-1,3,5-triazin-2-yl]amino]ethyl]amino]sulfonyl]-29H,31H-phthalocyanine-C,C-disulfonato(6-)-N29,N30,N31,N32]-, tetrasodium and lithium chloride
Test material form:
solid: particulate/powder

Test animals

Species:
mouse
Strain:
ICR
Sex:
male
Details on test animals or test system and environmental conditions:
- Animal species: 25 of 6 weeks male ICR mice.
- Source: BioLASCO Taiwan Co., Ltd
- Temperature: 22±3℃
- Relative humidity: 55±15%
- Light cycle: 12 hours light and 12 hours dark
- Animal feeding situation: 5 mice per cage. Frequency of ventilation: 10~15 times/hour.
- Drinking water: Reverse osmosis water provided with water bottle.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Test article was prepared with reverse osmosis water to 200.0000, 100.0000 and 50.0000 mg/mL, respectively.
Details on exposure:
The dosing volume of negative control and test article was 10 mL/kg BW by intraperitoneal injections.
Duration of treatment / exposure:
72 hours
Frequency of treatment:
During study period, the clinical observation of the mice was conducted every day. The body weights of the mice were determined before getting started with administration (Day 1) and 72 hours after the administration (Day 4).
Post exposure period:
48 and 72 hours after the test article administration, 5 µL of blood were collected from submandibular. The blood specimens were placed on slides that pre-dyed with acridine orange (1 mg/mL) and covered with coverslips. The blood were uniformly spread to a one layer blood cell thickness, and placed under room temperature for 3 hours protecting from light. Subsequently, fluorescence microscope was employed to observe the number of reticulocytes and micronucleus.
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Positive control group
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Low dose group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Medium dose group
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
High dose group
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Article name: Cyclophosphamide monohydrate (CPP)
Major ingredients: CPP, Form: Powder
Storage condition: Refrigeration (5±3℃)

Examinations

Evaluation criteria:
1. Clinical observations were conducted during the study period and animal deaths should be documented.
2. The ratio of reticulocytes: Fluorescence microscope was employed to calculate the number of reticulocytes, stained with orange-red color, per 2,000 erythrocytes.
3. The micronucleus incidence: Fluorescence microscope was employed to calculate the number of micronucleated reticulocytes, micronuclei was stained with yellow-green color, per 4,000 reticulocytes.
4. The body weight of test and control groups were analyzed by using One-Way ANOVA of Duncan’s multiple range test in SPSS software. The ratio of reticulocyte and the micronucleus incidence of test and control groups were analyzed by using median and Mann-Whitney U test in SOSS software. A significant difference was defined as p< 0.05.
5. Positive criteria: The micronucleus incidence had statistic difference compared with negative control group, and showed dose-response relation.
1. Clinical observations were conducted during the study period and animal deaths should be documented.
2. The ratio of reticulocytes: Fluorescence microscope was employed to calculate the number of reticulocytes, stained with orange-red color, per 2,000 erythrocytes.
3. The micronucleus incidence: Fluorescence microscope was employed to calculate the number of micronucleated reticulocytes, micronuclei was stained with yellow-green color, per 4,000 reticulocytes.
4. The body weight of test and control groups were analyzed by using One-Way ANOVA of Duncan’s multiple range test in SPSS software. The ratio of reticulocyte and the micronucleus incidence of test and control groups were analyzed by using median and Mann-Whitney U test in SOSS software. A significant difference was defined as p< 0.05.
5. Positive criteria: The micronucleus incidence had statistic difference compared with negative control group, and showed dose-response relation.
Statistics:
Quality control: The ratio of reticulocyte and micronucleus in the negative control group at 48 hour and 72 hour should be in the range of laboratory historical data. The ratio of reticulocytes in the positive control group was significantly lower than negative control group and the micronucleus incidence was significantly higher than negative control group, then considered as effective data.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
1. During study duration, results of body weight in all test groups showed no significantly different with negative control group (p>0.05) (Table 1). It indicated that test article had no influence on animal body weight.
2. The clinical observation results showed that testing mice of all groups were survival till the end of the study and no abnormal clinical sign were observed (Table 2).
3. The results of reticulocytes observation in 48 hours showed that the reticulocytes ratio median of negative control group was 56.0‰. The positive control group was 20.5‰ and significantly lower than negative control group (p<0.05). The low dose and medium dose group were 53.5‰ and 54.0‰ in reticulocytes ratio median, respectively. There were no significant difference compared with negative control group (p>0.05). The high dose group was 45.5‰ in reticulocytes ratio median and significantly lower than negative control group (p<0.05) (Table 3).
4. The results of micronucleus observation in 48 hours showed that the micronucleus incidence median of negative control group was 0.3‰. The positive control group was 10.5‰ and significantly higher than negative control group (p<0.05). The low dose, medium dose, and high dose group were 0.5‰, 0.5‰ and 0.5‰ in micronucleus incidence median respectively. There were no significant difference compared with negative control group (p>0.05) (Table 3).
5. The results of reticulocytes observation in 72 hours showed that the reticulocytes ratio median of negative control group was 56.0‰. The positive control group was 19.0‰ and significantly lower than negative control group (p<0.05). The low dose and medium dose group were 52.5‰ and 52.5‰ in reticulocytes ratio median, respectively. There were significantly lower than negative control group (p<0.05). The high dose group was 51.0‰ in reticulocytes ratio median. Tjere was no significant difference compared with negative control group (p<0.05) (Table 3).
6. The results of micronucleus observation in 72 hours showed that the micronucleus incidence median of negative control group was 0.5‰. The positive control group was 10.8‰ and significantly higher than negative control group (p<0.05). The low dose, medium dose, and high dose group were 0.3‰, 0.3‰ and 0.3‰ in micronucleus incidence median, respectively. There were no significant difference compared with negative control group (p>0.05) (Table 3).

Any other information on results incl. tables

Table 1. The body weight of testing mice

Group Dose (mg/kg BW) Body weight (g)a
Day 1 Day 4
Negative control group - 28.2±0.8 29.6±0.8
Positive control group 50 28.5±0.8 30.0±0.9
Test group
Low dose group 500 28.6±1.4 30.1±1.0
Medium dose group 1000 29.3±1.0 30.9±1.7
High dose group 2000 28.5±1.2 30.0±1.4

aAll data were shown as mean±SD, n=5.

Table 2. Clinical observation of testing mice

Group Dose (mg/kg BW) Daily Clinical Observation
Day 1 Day 2 Day 3 Day 4
Negative control group - N N N N
Positive control group 50 N N N N
Test group
Low dose group 500 N N N N
Medium dose group 1000 N N N N
High dose group 2000 N N N N

aN: Normal.

Table 3. Reticulocytes ratio and micronucleus incidence in peripheral blood of all groups mice

Group Dose (mg/kg BW) Reticulocytes ratio
RETs/2000 RBCs (‰)a
Micronucleus incidence
Mn-RETs/4000 RBCs (‰)b
Medianc
(Q1~Q3)
p valued Median
(Q1~Q3)
p valued
48 hours
Negative control group - 56.0(51.8~58.8) - 0.3 (0.2~0.7) -
Positive control group 50 20.5(14.5~21.8) 0.008* 10.5(10.2~11.1) 0.008*
Test group
Low dose group 500 53.5(49.8~57.3) 0.413 0.5(0.0~0.9) 0.833
Medium dose group 1000 54.0(51.5~56.8) 0.548 0.5(0.2~08) 0.810
High dose group 2000 45.5(44.5~48.3) 0.008* 0.5(0.0~0.9) 0.833
72 hours
Negative control group - 56.0(54.3~59.5) - 0.5(0.3~0.8) -
Positive control group 50 19.0(16.8~21.3) 0.008* 10.8(10.4~13.4) 0.008*
Test group
Low dose group 500 52.5(50.8~53.8) 0.016* 0.3(0.2~0.7) 0.468
Medium dose group 1000 52.5(50.8~53.5) 0.016* 0.3(0.2~0.7) 0.468
High dose group 2000 51.0(50.3~59.5) 0.381 0.3(0.2~0.7) 0.468

aReticulocytes ratio: Reticulocytes (RETs)/2000 red blood cells (RBCs), n=5

bMicronucleus incidence: Reticulocytes with micronucleus (Mn-RETs) / 4000 reticulocytes (RETs), n=5.

c Q1: first quartile; Q3: third quartile.

d Analysis by Mann-Whitney U test.

*Significantly different compared with negative control group, p<0.05

Applicant's summary and conclusion

Conclusions:
The reticulocytes ratio of high dose group at 48 hours and low and medium dose groups at 72 hours were significantly lower than negative control group, but the average value were in the range of laboratory historical data. Therefore, it did not affect the determination of genotoxicity in this test. The micronucleus incidence of low, medium and high dose groups were no significantly different with negative control group. In conclusion, the test article CJ303 showed negative reaction for rodent peripheral blood micronucleus test under the conditions designed for this study, no mutagenic potential.
Executive summary:

This study was conducted according to the OECD474:2016 to evaluate the mutagenicity of CJ303 by rodent peripheral blood micronucleus test. Testing system was male ICR mice. The study included negative control group, positive control group and three test groups including 500, 1000 and 2000 mg/kg BW. Five testing animal were used per group. In this study, blood specimens of all group mice were collected and prepared for blood slide after 48 and 72 hours of test article or control article administration. Using fluorescence microscope observed the reticulocytes and number of micronucleus. Results showed that the reticulocytes ratio of high dose group at 48 hours and low and medium dose groups at 72 hours were significantly lower than negative control group, but the average value were in the range of laboratory historical data. Therefore, it did not affect the determination of genotoxicity in this test. The micronucleus incidence of low, medium and high dose groups were no statistical different compared with negative control group. The reticulocytes ratio of positive group was significantly lower than negative control group and the micronucleus incidence of positive control group was significantly higher than negative control group. In conclusion, CJ303 showed negative reaction for rodent peripheral blood micronucleus test under the conditions designed for this study, no mutagenic potential.