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Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
CJ303 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5 mg/plate in the absence and presence of S9 metabolic activation (OECD TG471).
CJ303 induced a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. The observed effects fulfilled all the criteria of positivity. Therefore, CJ303 was considered as clastogenic in this test system (OECD TG473).
CJ303 did not induce any Gene Mutation in the L5178Y cell up to 5000 µg/mL with or without metabolic activation. The result of CJ303 in vitro Mammalian Cell Gene Mutation test was negative (OECD TG476).
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From October 4, 2018 to March 14, 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Remarks:
- CHO-K1
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Mix
- Test concentrations with justification for top dose:
- Based on the preliminary test result of cytotoxicity, the highest dose tested in this assay was 5000 ug/mL.
- Vehicle / solvent:
- Dimethylsulfoxide (DMSO)
- Untreated negative controls:
- yes
- Remarks:
- Ultrapure water
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Details on test system and experimental conditions:
- Based on the preliminary test result of cytotoxicity, the definite test were performed at 5000, 2000, 8000 and 320 µg/mL, and two replicates were prepared for each level. The groups were treated for 4 hours with metabolic activation, 4 hour without metabolic activation and 24 hours without metabolic activation.
- Evaluation criteria:
- 1. Test Validity
When the PE0 of negative control = 60%-140%, PE2 of negative control = 40%-140%; MF of the positive control ≥ 2-fold the negative control value, this study will be considered valid.
2. Result Evaluation
A test item is consider positive if it meets both of the following criteria:
(1) At least one or more concentration produces a concentration-related increase in mutant frequency.
(2) MF of the test item ≥ 2-fold the negative control value. - Species / strain:
- Chinese hamster Ovary (CHO)
- Remarks:
- CHO-K1
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- At the beginning and the end of treatment, no precipitate was observed in other dose.
- Conclusions:
- It was concluded that when tested up to 5000 µg/mL with or without metabolic activation, CJ303 did not induce any Gene Mutation in the L5178Y cell. The result of CJ303 in vitro Mammalian Cell Gene Mutation test was negative.
- Executive summary:
The mutagenic potential of CJ303 was assessed in the Mouse Lymphoma Cells (L5178Y), which can detect induced gene mutation.
L5178Y cells were exposed to CJ303 at the doses of 320µg/mL, 800µg/mL, 2000µg/mL and 5000µg/mL with metabolic activation for 4 hours, without metabolic activation for 4 hours and without metabolic activation for 24 hours, respectively. Dimethylsulfoxide (DMSO) was used as solvent control substances. Ultrapure water was used as negative control substances. Methyl methanesulfonate (MMS) and Cyclophosphamide (CP) were used as positive control substances for experiments with and without metabolic activation, respectively. After the treatment period, cytotoxicity was evaluated by the relative survival (relative to the negative control). All the culture were treated by trifluorothymidine (TF) to detect gene mutations.
When tested up to 5000µg/mL with or without metabolic activation, CJ303 did not induce any Gene Mutation in the L5178Y cell. The result of CJ303 in vitro Mammalian Cell Gene Mutation test was negative.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From July 11, 2016 to November 12, 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Mix
- Vehicle / solvent:
- 1g CJ303 was completely mixed with 4 mL DMSO and sterile water was added into solution up to 20 ml that the concentration contained 50 mg/mL.
- Untreated negative controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- sodium azide
- benzo(a)pyrene
- mitomycin C
- other:
- Details on test system and experimental conditions:
- Strains: The Salmonella typhimurium strains of TA98, TA100, TA102 TA1535and TA1537 were purchased from MOLTOX, Molecular Toxicology, Inc.
Culture medium: OXOID NB No.2
Incubation condition: 35±1℃ - Evaluation criteria:
- 1. For the groups treated with S9 Mix, the range of natural reverse mutant colony number for TA98 was 32~51 CFU/plate, TA100 was 197~386 CFU/plate, TA102 was 378~487 CFU/plate, TA1535 was 18~41 CFU/plate and TA1537 was 9~26 CFU/plate.
2. For the groups not treated with S9 Mix, the range of natural reverse mutant colony number for TA98 was 22~51 CFU/plate, TA100 was 166~326 CFU/plate, TA102 was 262~407 CFU/plate, TA1535 was 17~43 CFU/plate and TA1537 was 6~25 CFU/plate.
3. Positive response criteria: At least one of the dosages should demonstrate a reverse mutation value double or higher times than that of the negative control group, and was calculated by SPSS, p<0.05 (One-way ANOVA), or test groups should present dose-response relationships. Those are judged the positive response.
4. The negative response criterion: In the bacterial reverse mutation test, the test results for the various dosage groups failed to comply with the positive response criteria are judged the negative response. - Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Activated with or without S9 Mix each concentration of CJ303, reverse mutant colony number of five testing strains which were TA98, TA100, TA102, TA1535 and TA1537, were not twice more than negative control groups. Therefore, test article CJ303 under the condition of bacterial reverse mutation test, the test result was negative.
- Executive summary:
The bacterial reverse mutation test was conducted according to OECD 471:1997 to evaluate in vitro mutagenicity caused by CJ303. Test strains included Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537. Negative control group, positive control group and test groups of each strain needed to activate with and without S9 Mix to test. In test for toxicity of the test article, detected toxicity of 5, 2.5, 1.25, 0.625 and 0.3125 mg/plate test article to TA100. The result showed that dose of 5 mg/plate did not cause toxicity. Therefore, according to OECD 471:1997, if 5 mg/plate test article did not cause toxicity, then choose 5 mg/plate as the highest concentration of the test, and used this concentration to dilute two-fold serial as 2.5, 1.25, 0.625 and 0.3125 mg/plate. The result of bacterial reverse mutation test showed that positive control group activated with and without S9 Mix could produce significant mutagenicity. No positive criteria were observed when the reverse mutant colonies of all test groups compared with the negative control group, and all test data were within the effective data range. These results indicated that CJ303, treated or not treated with S9m would not occur mutagenicity on this test condition.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From March 22, 2017 to Octobor 09, 2017
- 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)
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Remarks:
- CHO-K1
- Metabolic activation:
- with and without
- Metabolic activation system:
- A cofactor-supplemented post-mitochondrial S9 fraction prepared from activated rat liver was used as an appropriate metabolic activation system.
- Untreated negative controls:
- yes
- Remarks:
- Distilled water
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Species / strain:
- Chinese hamster Ovary (CHO)
- Remarks:
- CHO-K1
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- CJ303 induced a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. The observed effects fulfilled all the criteria of positivity. Therefore, CJ303 was considered as clastogenic in this test system.
- Executive summary:
CJ303 was tested in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells. The test item was formulated in distilled water and it was examined up to cytotoxic concentrations according to OECD473. The treatment concentrations caused significant increases in the number of cells with structural chromosome aberrations in Assay 1 and Assay 2 with and without metabolic activation when compared with the appropriate negative (vehicle) control values. The increases were concentration-related and they were reproducible between the duplicate cultures. Based on these facts, the test item caused reproducible, concentration-related and statistically significant increases in the frequency of aberrant metaphases in V79 Chinese hamster cells, both in the absence and presence of S9-mix in this study. The negative (vehicle) control data were within the acceptable 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, CJ303 induced a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. The observed effects fulfilled all the criteria of positivity. Therefore, CJ303 was considered as clastogenic in this test system.
Referenceopen allclose all
Table 1. The Cytotoxicity Effect of CJ303 on L5178Y Cell
Treatment | Dose (µg/mL) | PE0 (%) | RS0 (%) | RSG (%) | RS2 (%) | RTG (%) |
S9- 24h |
Negative control | 63.3 | 100.0 | 100.0 | 100.0 | 100.0 |
Solvent control | 60.7 | 95.9 | 132.5 | 96.9 | 128.4 | |
5000 | 50.3 | 79.5 | 43.3 | 91.5 | 39.6 | |
2000 | 58.0 | 91.6 | 102.4 | 105.1 | 107.6 | |
800 | 63.3 | 100.0 | 111.9 | 95.1 | 106.5 | |
320 | 65.8 | 104.0 | 118.4 | 91.3 | 108.2 | |
MMS | 64.8 | 102.3 | 80.7 | 97.2 | 78.5 | |
S9- 4h |
Negative control | 68.9 | 100.0 | 100.0 | 100.0 | 100.0 |
Solvent control | 62.2 | 90.4 | 75.6 | 104.2 | 78.8 | |
5000 | 63.2 | 91.7 | 94.1 | 89.6 | 84.3 | |
2000 | 69.7 | 101.2 | 159.8 | 76.5 | 122.2 | |
800 | 61.7 | 89.6 | 163.2 | 73.0 | 119.1 | |
320 | 67.7 | 98.3 | 122.9 | 94.5 | 116.1 | |
MMS | 53.2 | 77.3 | 147.9 | 81.2 | 120.1 | |
S9+ 4h |
Negative control | 62.0 | 100.0 | 100.0 | 100.0 | 100.0 |
Solvent control | 61.5 | 99.2 | 126.7 | 99.4 | 126.0 | |
5000 | 50.9 | 82.1 | 82.2 | 99.4 | 81.8 | |
2000 | 58.0 | 93.6 | 88.8 | 112.2 | 99.6 | |
800 | 63.1 | 101.7 | 96.5 | 113.9 | 109.9 | |
320 | 67.9 | 109.5 | 85.7 | 112.2 | 96.2 | |
CP | 60.7 | 97.9 | 134.6 | 98.6 | 132.7 |
Table 2. The mutant frequencies of CJ303 on L5178Y Cell
Treatment | Dose (µg/mL) | PE2 (%) | MF (10^-6) | SC (%) |
S9- 24h |
Negative control | 62.7 | 83.4 | 58.3 |
Solvent control | 60.7 | 120.0 | - | |
5000 | 57.4 | 86.0 | - | |
2000 | 65.9 | 94.9 | - | |
800 | 59.6 | 92.5 | - | |
320 | 57.2 | 119.3 | - | |
MMS | 60.9 | 488.4 | 72.9 | |
S9- 4h |
Negative control | 67.8 | 77.2 | 67.5 |
Solvent control | 70.6 | 93.0 | - | |
5000 | 60.7 | 74.3 | - | |
2000 | 51.8 | 92.6 | - | |
800 | 49.5 | 97.1 | - | |
320 | 64.0 | 116.0 | - | |
MMS | 55.0 | 524.3 | 78.2 | |
S9+ 4h |
Negative control | 60.5 | 80.3 | 69.3 |
Solvent control | 60.2 | 82.0 | - | |
5000 | 60.2 | 77.3 | - | |
2000 | 67.9 | 85.5 | - | |
800 | 68.9 | 94.8 | - | |
320 | 67.9 | 72.7 | - | |
CP | 59.7 | 522.8 | 72.5 |
Table 3-1. The count result of PE0 and PE2 (S9- 24h)
Dose (µg/mL) | Cell number | PE0 | PE2 | ||
EW | TW | EW | TW | ||
Negative control | 011 | 33 | 96 | 36 | 96 |
012 | 32 | 96 | 33 | 96 | |
021 | 40 | 96 | 35 | 96 | |
022 | 35 | 96 | 37 | 96 | |
Solvent control | D1 | 33 | 96 | 33 | 96 |
D2 | 40 | 96 | 40 | 96 | |
5000 | 41 | 46 | 96 | 35 | 96 |
42 | 40 | 96 | 42 | 96 | |
2000 | 31 | 40 | 96 | 35 | 96 |
32 | 36 | 96 | 32 | 96 | |
800 | 21 | 32 | 96 | 38 | 96 |
22 | 38 | 96 | 36 | 96 | |
320 | 11 | 43 | 96 | 41 | 96 |
12 | 33 | 96 | 36 | 96 | |
MMS | M1 | 40 | 96 | 32 | 96 |
M2 | 29 | 96 | 41 | 96 |
Table 3-2. The count result of PE0 and PE2 (S9- 4h)
Dose (µg/mL) | Cell number | PE0 | PE2 | ||
EW | TW | EW | TW | ||
Negative control | 011 | 30 | 96 | 31 | 96 |
012 | 29 | 96 | 33 | 96 | |
021 | 34 | 96 | 35 | 96 | |
022 | 35 | 96 | 31 | 96 | |
Solvent control | D1 | 34 | 96 | 31 | 96 |
D2 | 37 | 96 | 31 | 96 | |
5000 | 41 | 37 | 96 | 40 | 96 |
42 | 33 | 96 | 33 | 96 | |
2000 | 31 | 30 | 96 | 45 | 96 |
32 | 33 | 96 | 39 | 96 | |
800 | 21 | 40 | 96 | 44 | 96 |
22 | 32 | 96 | 43 | 96 | |
320 | 11 | 33 | 96 | 33 | 96 |
12 | 32 | 96 | 36 | 96 | |
MMS | M1 | 39 | 96 | 36 | 96 |
M2 | 43 | 96 | 44 | 96 |
Table 3-3. The count result of PE0 and PE2 (S9+ 4h)
Dose (µg/mL) | Cell number | PE0 | PE2 | ||
EW | TW | EW | TW | ||
Negative control | 011 | 35 | 96 | 37 | 96 |
012 | 37 | 96 | 34 | 96 | |
021 | 31 | 96 | 36 | 96 | |
022 | 40 | 96 | 39 | 96 | |
Solvent control | D1 | 39 | 96 | 38 | 96 |
D2 | 33 | 96 | 35 | 96 | |
5000 | 41 | 43 | 96 | 42 | 96 |
42 | 42 | 96 | 32 | 96 | |
2000 | 31 | 40 | 96 | 35 | 96 |
32 | 36 | 96 | 30 | 96 | |
800 | 21 | 35 | 96 | 35 | 96 |
22 | 35 | 96 | 29 | 96 | |
320 | 11 | 35 | 96 | 35 | 96 |
12 | 30 | 96 | 30 | 96 | |
CP | C1 | 40 | 96 | 39 | 96 |
C2 | 33 | 96 | 35 | 96 |
Table 1. Characteristics of five Salmonella typhimurium strains
Test strain |
Histidine requirement |
uvrB mutation |
rfa mutation |
Ampicillin resistance |
TA98 |
+ |
+ |
+ |
+ |
TA100 |
+ |
+ |
+ |
+ |
TA102 |
+ |
ϴ |
+ |
+ |
TA1535 |
+ |
+ |
+ |
ϴ |
TA1537 |
+ |
+ |
+ |
ϴ |
+ means had the characteristic; ϴ means did not have the characteristic
Table 2. Toxicity of the test article of Salmonella typhimurium TA100
Group |
Test article (mg/plate) |
Reverse mutant colony number (CFU/plate) |
With S9 Mix |
Negative control group |
212 |
Positive control groupa |
1,277 |
|
Test group |
|
|
5 |
195 |
|
2.5 |
178 |
|
1.25 |
209 |
|
0.625 |
202 |
|
0.3125 |
253 |
|
Without S9 Mix |
Negative control group |
264 |
Positive control group |
554 |
|
Test group |
|
|
5 |
208 |
|
2.5 |
201 |
|
1.25 |
217 |
|
0.625 |
219 |
|
0.3125 |
247 |
a Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).
Without S9 Mix: Sodium azide (5μg/plate).
Table 3. Reverse mutation test of Salmonella typhimurium TA98
Group |
Test article (mg/plate) |
Reverse mutant colony number (CFU/plate) |
Average of coloniesa |
||
1 |
2 |
3 |
|||
With S9 Mix |
Negative control group |
37 |
45 |
48 |
43 ± 6 |
Positive control groupb |
398 |
408 |
415 |
407 ± 9* |
|
Test group |
|
|
|
|
|
5 |
33 |
42 |
41 |
39 ± 5 |
|
2.5 |
37 |
44 |
36 |
39 ± 4 |
|
1.25 |
43 |
40 |
49 |
44 ± 5 |
|
0.625 |
39 |
50 |
44 |
44 ± 6 |
|
0.3125 |
55 |
50 |
44 |
50 ± 6 |
|
Without S9 Mix |
Negative control group |
29 |
26 |
28 |
28 ± 2 |
Positive control group |
296 |
282 |
236 |
271 ± 31* |
|
Test group |
|
|
|
|
|
5 |
21 |
39 |
26 |
29 ± 9 |
|
2.5 |
39 |
26 |
28 |
31 ± 7 |
|
1.25 |
31 |
37 |
32 |
33 ± 3 |
|
0.625 |
35 |
37 |
39 |
37 ± 2 |
|
0.3125 |
47 |
48 |
25 |
40 ± 13 |
a Average of colonies was shown as Mean ± S.D., the data were triplicate.
b Positive control group: With S9 Mix: Benzo [a] pyrene (4.0μg/plate).
Without S9 Mix: 4-Nitroquinoline-N-oxide (0.5μg/plate).
* Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).
Table 4. Reverse mutation test of Salmonella typhimurium TA100
Group |
Test article (mg/plate) |
Reverse mutant colony number (CFU/plate) |
Average of coloniesa |
||
1 |
2 |
3 |
|||
With S9 Mix |
Negative control group |
292 |
288 |
221 |
267 ± 40 |
Positive control groupb |
1,295 |
1,358 |
1,280 |
1,311 ± 41* |
|
Test group |
|
|
|
|
|
5 |
203 |
171 |
213 |
196 ± 22 |
|
2.5 |
214 |
206 |
217 |
212 ± 6 |
|
1.25 |
193 |
230 |
262 |
228 ± 35 |
|
0.625 |
228 |
213 |
271 |
237 ± 30 |
|
0.3125 |
304 |
290 |
284 |
293 ± 10 |
|
Without S9 Mix |
Negative control group |
219 |
272 |
265 |
252 ± 29 |
Positive control group |
521 |
592 |
639 |
584 ± 59* |
|
Test group |
|
|
|
|
|
5 |
174 |
178 |
199 |
184 ± 13 |
|
2.5 |
224 |
200 |
218 |
214 ± 12 |
|
1.25 |
245 |
238 |
214 |
232 ± 16 |
|
0.625 |
216 |
202 |
200 |
206 ± 9 |
|
0.3125 |
249 |
266 |
270 |
262 ± 11 |
a Average of colonies was shown as Mean ± S.D., the data were triplicate.
b Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).
Without S9 Mix: Sodium azide (5μg/plate).
* Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).
Table 5. Reverse mutation test of Salmonella typhimurium TA102
Group |
Test article (mg/plate) |
Reverse mutant colony number (CFU/plate) |
Average of coloniesa |
||
1 |
2 |
3 |
|||
With S9 Mix |
Negative control group |
462 |
441 |
435 |
446 ± 14 |
Positive control groupb |
924 |
916 |
911 |
917 ± 7* |
|
Test group |
|
|
|
|
|
5 |
438 |
451 |
424 |
438 ± 14 |
|
2.5 |
440 |
399 |
458 |
432 ± 30 |
|
1.25 |
449 |
437 |
430 |
439 ± 10 |
|
0.625 |
448 |
425 |
455 |
443 ± 16 |
|
0.3125 |
458 |
467 |
452 |
459 ± 8 |
|
Without S9 Mix |
Negative control group |
402 |
459 |
363 |
408 ± 48 |
Positive control group |
874 |
869 |
949 |
897 ± 45* |
|
Test group |
|
|
|
|
|
5 |
346 |
390 |
370 |
369 ± 22 |
|
2.5 |
440 |
386 |
418 |
415 ± 27 |
|
1.25 |
440 |
391 |
403 |
411 ± 26 |
|
0.625 |
403 |
399 |
412 |
405 ± 7 |
|
0.3125 |
440 |
452 |
416 |
436 ± 18 |
a Average of colonies was shown as Mean ± S.D., the data were triplicate.
b Positive control group: With S9 Mix: 2-Aminoanthracene (10μg/plate).
Without S9 Mix: Mitomycin C (0.5μg/plate).
* Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).
Table 6. Reverse mutation test of Salmonella typhimurium TA1535
Group |
Test article (mg/plate) |
Reverse mutant colony number (CFU/plate) |
Average of coloniesa |
||
1 |
2 |
3 |
|||
With S9 Mix |
Negative control group |
19 |
19 |
31 |
23 ± 7 |
Positive control groupb |
192 |
259 |
263 |
238 ± 40* |
|
Test group |
|
|
|
|
|
5 |
26 |
17 |
22 |
22 ± 5 |
|
2.5 |
29 |
24 |
19 |
24 ± 5 |
|
1.25 |
31 |
26 |
21 |
26 ± 5 |
|
0.625 |
23 |
24 |
21 |
23 ± 2 |
|
0.3125 |
26 |
22 |
21 |
23 ± 3 |
|
Without S9 Mix |
Negative control group |
29 |
26 |
27 |
27 ± 2 |
Positive control group |
299 |
240 |
276 |
372 ± 30* |
|
Test group |
|
|
|
|
|
5 |
19 |
27 |
29 |
25 ± 5 |
|
2.5 |
25 |
37 |
16 |
26 ± 11 |
|
1.25 |
29 |
19 |
19 |
22 ± 6 |
|
0.625 |
25 |
22 |
25 |
24 ± 2 |
|
0.3125 |
30 |
40 |
40 |
37 ± 6 |
a Average of colonies was shown as Mean ± S.D., the data were triplicate.
b Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).
Without S9 Mix: Sodium azide (0.4μg/plate).
* Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).
Table 7. Reverse mutation test of Salmonella typhimurium TA1537
Group |
Test article (mg/plate) |
Reverse mutant colony number (CFU/plate) |
Average of coloniesa |
||
1 |
2 |
3 |
|||
With S9 Mix |
Negative control group |
20 |
15 |
23 |
19 ± 4 |
Positive control groupb |
416 |
425 |
432 |
424 ± 8* |
|
Test group |
|
|
|
|
|
5 |
24 |
15 |
12 |
17 ± 6 |
|
2.5 |
17 |
17 |
16 |
17 ± 1 |
|
1.25 |
18 |
19 |
15 |
17 ± 2 |
|
0.625 |
18 |
20 |
16 |
18 ± 2 |
|
0.3125 |
21 |
22 |
16 |
20 ± 3 |
|
Without S9 Mix |
Negative control group |
17 |
15 |
14 |
15 ± 2 |
Positive control group |
1,082 |
1,163 |
976 |
1,074 ± 94* |
|
Test group |
|
|
|
|
|
5 |
10 |
13 |
10 |
11 ± 2 |
|
2.5 |
13 |
12 |
12 |
12 ± 1 |
|
1.25 |
16 |
14 |
12 |
14 ± 2 |
|
0.625 |
25 |
15 |
16 |
19 ± 6 |
|
0.3125 |
12 |
14 |
14 |
13 ± 1 |
a Average of colonies was shown as Mean ± S.D., the data were triplicate.
b Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).
Without S9 Mix: 9-Aminoacridine (50.0μg/plate).
* Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).
Table 1. Summary table of Chromosome Aberration Assay 1 without metabolic activation
Concentration (μg/mL) [Number of analyzed cells] |
Time of Treatment / Sampling | RICC#(%) | Precipitate## | Mean % aberrant cells### |
CJ303 without metabolic activation (-S9) | ||||
Untreated control | 3h / 20h | 93 | - | NE |
Negative (vehicle) control [300] | 3h / 20h | 100 | - | 2.3 |
2000 μg/mL [300] | 3h / 20h | 61 | -a | 8.9*** |
666.7 μg/mL [300] | 3h / 20h | 80 | -a | 3.3 |
222.2 μg/mL [300] | 3h / 20h | 88 | -a | 1.3 |
74.07 μg/mL | 3h / 20h | 100 | -a | NE |
24.69 μg/mL | 3h / 20h | 93 | -a | NE |
Positive control [300] | 3h / 20h | 67 | - | 15.3*** |
Negative (vehicle) control: (Distilled water)
Positive control (-S9): Ethyl methanesulfonate, 1 μL/mL
NE: not evaluated
RICC: Relative Increase in Cell Counts
a: discoloured medium / minimally discoloured medium
#: 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
Table 2. Summary table of Chromosome Aberration Assay 1 with metabolic activation
Concentration (μg/mL) [Number of analyzed cells] |
Time of Treatment / Sampling | RICC#(%) | Precipitate## | Mean % aberrant cells### |
CJ303 with metabolic activation (+S9) | ||||
Untreated control | 3h / 20h | 105 | - | NE |
Negative (vehicle) control [300] | 3h / 20h | 100 | - | 2.7 |
2000 μg/mL [300] | 3h / 20h | 88 | -a | 9.0*** |
666.7 μg/mL [300] | 3h / 20h | 88 | -a | 4.3 |
222.2 μg/mL [300] | 3h / 20h | 92 | -a | 2.0 |
74.07 μg/mL | 3h / 20h | 101 | -a | NE |
24.69 μg/mL | 3h / 20h | 101 | -a | NE |
Positive control [70] | 3h / 20h | 66 | - | 71.4*** |
Negative (vehicle) control: (Distilled water)
Positive control (+S9): Cyclophosphamide, 6 μg/mL
NE: not evaluated
RICC: Relative Increase in Cell Counts
#: compared to the negative (vehicle) control
## : in the final treatment medium at the end of the treatment
###: excluding gaps
a: discoloured / minimally discoloured medium
***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control
Table 3. Summary table of Chromosome Aberration Assay 2 without metabolic activation
Concentration (μg/mL) [Number of analyzed cells] |
Time of Treatment / Sampling | RICC#(%) | Precipitate## | Mean % aberrant cells### |
CJ303 without metabolic activation (-S9) | ||||
Untreated control | 20h / 20h | 94 | - | NE |
Negative (vehicle) control [300] | 20h / 20h | 100 | - | 4.3 |
222.2 μg/mL | 20h / 20h | 25 | -a | NE |
74.07 μg/mL | 20h / 20h | 42 | -a | NE |
24.69 μg/mL | 20h / 20h | 41 | -a | NE |
8.23 μg/mL | 20h / 20h | 45 | -a | NE |
2.74 μg/mL [185] | 20h / 20h | 46 | -a | 27.03*** |
0.914 μg/mL [300] | 20h / 20h | 76 | - | 12.0*** |
0.305 μg/mL [300] | 20h / 20h | 78 | - | 7.3 |
0.102 μg/mL [300] | 20h / 20h | 95 | - | 6.0 |
0.0339 μg/Ml | 20h / 20h | 90 | - | NE |
Positive control [71] | 20h / 20h | 50 | - | 70.4*** |
Negative (vehicle) control: (Distilled water)
Positive control (-S9): Ethyl methanesulfonate, 0.4 μL/mL
NE: not evaluated
RICC: Relative Increase in Cell Counts
#: compared to the negative (vehicle) control
##: in the final treatment medium at the end of the treatment
###: excluding gaps
a: discoloured / minimally discoloured medium
***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control
Table 4. Summary table of Chromosome Aberration Assay 2 with metabolic activation
Concentration (μg/mL) [Number of analyzed cells] |
Time of Treatment / Sampling | RICC#(%) | Precipitate## | Mean % aberrant cells### |
CJ303 with metabolic activation (+S9) | ||||
Untreated control | 3h / 20h | 92 | - | NE |
Negative (vehicle) control [300] | 3h / 20h | 100 | - | 3.7 |
2000 μg/mL [155] | 3h / 20h | 61 | -a | 32.3*** |
666.7 μg/mL [290] | 3h / 20h | 86 | -a | 15.9*** |
222.2 μg/mL [300] | 3h / 20h | 88 | -a | 5.3 |
74.07 μg/mL | 3h / 20h | 91 | -a | NE |
24.69 μg/mL | 3h / 20h | 95 | -a | NE |
Positive control [80] | 3h / 20h | 69 | - | 62.5* |
Negative (vehicle) control: (Distilled water)
Positive control (+S9): Cyclophosphamide, 6 μg/mL
NE: not evaluated
RICC: Relative Increase in Cell Counts
#: compared to the negative (vehicle) control
##: in the final treatment medium at the end of the treatment
###: excluding gaps
a: discoloured / minimally discoloured medium
***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
CJ303 showed negative reaction for rodent peripheral blood micronucleus test under the conditions designed for this study, no mutagenic potential (OECD TG474).
Link to relevant study records
- 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
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- GLP compliance:
- yes
- Type of assay:
- mammalian erythrocyte micronucleus test
- 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. - 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.
- 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℃) - 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.
- 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). - 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.
Reference
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
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
in vitro gene mutation study in bacteria
Based on the preliminary assay results, 5 mg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of CJ303 at 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate, concurrent negative and strain-specific positive controls were tested in tester strains TA98, TA100, TA102, TA1535 and TA1537 in triplicate with or without S9 Mix activation. No cytotoxicity was observed in all five tester strains up to 5 mg/plate in the absence and presence of metabolite activations. Results showed that CJ303 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 up to 5 mg/plate either in the absence or in the presence of metabolite activation.
Based on the data obtained from this study, it was concluded that under the test condition, CJ303 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5mg/plate in the absence and presence of S9 metabolic activation.
in vitro cytogenicity / chromosome aberration study in mammalian cells
CJ303 was tested in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells. The test item was formulated in distilled water and it was examined up to cytotoxic concentrations according to OECD473. The treatment concentrations caused significant increases in the number of cells with structural chromosome aberrations in Assay 1 and Assay 2 with and without metabolic activation when compared with the appropriate negative (vehicle) control values. The increases were concentration-related and they were reproducible between the duplicate cultures. Based on these facts, the test item caused reproducible, concentration-related and statistically significant increases in the frequency of aberrant metaphases in V79 Chinese hamster cells, both in the absence and presence of S9-mix in this study. The negative (vehicle) control data were within the acceptable 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 vitro gene mutation study in mammalian cells
The mutagenic potential of CJ303 was assessed in the Mouse Lymphoma Cells (L5178Y), which can detect induced gene mutation. L5178Y cells were exposed to CJ303 at the doses of 320µg/mL, 800µg/mL, 2000µg/mL and 5000µg/mL with metabolic activation for 4 hours, without metabolic activation for 4 hours and without metabolic activation for 24 hours, respectively. Dimethylsulfoxide (DMSO) was used as solvent control substances. Ultrapure water was used as negative control substances. Methyl methanesulfonate (MMS) and Cyclophosphamide (CP) were used as positive control substances for experiments with and without metabolic activation, respectively. After the treatment period, cytotoxicity was evaluated by the relative survival (relative to the negative control). All the culture were treated by trifluorothymidine (TF) to detect gene mutations. When tested up to 5000µg/mL with or without metabolic activation, CJ303 did not induce any Gene Mutation in the L5178Y cell. The result of CJ303 in vitro Mammalian Cell Gene Mutation test was negative.
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
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.
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