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Description of key information
CJ309 was not mutagenic in the reverse mutation analysis of Salmonella typhimuriumup to 5000μg/plate in the absence and presence of S9 metabolic activation.(OECD TG471).
CJ309 did not induce chromosome aberration in CHO cells in absence or presence of S9 metabolic activation (OECD TG473).
Based on the read across result, the in vitro mammalian cell gene mutation test was negative (OECD TG476).
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From February 18, 2016 to August 29, 2016
- 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
- Target gene:
- Bacterial gene reverse mutation
- 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:
- The post-mitochondrial fraction (S9) prepared from Aroclor 1254-induced Sprague-Dawley rats
- Untreated negative controls:
- yes
- Remarks:
- sterile deionized water
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- sodium azide
- mitomycin C
- other: Acridine mutagen ICR 191 2-Aminofluorene 2-Aminoanthracene
- Evaluation criteria:
- Acceptable ranges of background revertants for five tester strains are:
Tester Strain Revertants
TA98 10-60
TA100 50-240
TA102 180-480
TA1535 5-45
TA1537 2-25 - 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:
- According to OECD 471 test method, CJ309 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5000 μg/plate.
- Executive summary:
This test using the procedures outlined in the QPS Taiwan Study Plan for T65315029-GT which is based on the SOP for the OECD 471 (CTPS-TE00201) and OECD 471 (OECD,1997). The results of this OECD 471 test for CJ309 show that test validity criteria was met.
Based on the preliminary assay results, 5000μg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of CJ309 at 50, 150, 500, 1500 and 5000μg/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 5000μg/plate in the absence and presence of metabolite activations. Results showed that CJ309 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 up to 5000μg/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, CJ309 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5000μg/plate in the absence and presence of S9 metabolic activation.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From January 11, 2017 to June 07, 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome 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:
- The post-mitochondrial fraction (S9) of liver from Aroclor 1254 induced Sprague- Dawley rats
- Untreated negative controls:
- yes
- Remarks:
- Sterile deionized water
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- 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:
- According to OECD 473 test method, CJ309 did not induce chromosome aberration in CHO cells in absence or presence of S9 metabolic activation.
- Executive summary:
This test using the procedures outlined in the QPS Taiwan Study Plan for T65316027-GT and OECD 473 (OECD, 2016). The results of this OECD 473 test for CJ309 show that test validity criteria was met.
A preliminary concentration-range finding cytotoxicity test was performed in three test schemes: 3-hour treatment in the absence and presence of S9 metabolic activation (Schemes I and II) and 20-hour continuous treatment in the absence of S9 metabolic activation (Scheme III), and the results suggested that a concentration of 2000 μg/mL in all three schemes would be used in the chromosome aberration assay. Based on the cytotoxicity result, five concentrations of 51.2, 128, 320, 800 and 2000 μg/mL were used for all three schemes in the chromosome aberration test. In test group, the cell proportions with abnormal chromosome in three Schemes were not higher than 3%. Based on the data obtained from this study, it was concluded that under the test condition,CJ309 did not induce chromosome aberration in CHO cells in absence or presence of S9 metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- From September 11, 2018 to December 25, 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: The study was conducted according to OECD guideline and in accordance with GLP
- Justification for type of information:
- Demonstrates that the hypothesis is supported by referring to a data set: all claims must be supported by data.
- Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- GLP compliance:
- yes
- Type of assay:
- other: in vitro mammalian cell gene mutation tests using Chinese Hamster ovary cell (CHO-K1)
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- Cell line and incubation condition:
1. Cell line: Chinese hamster ovary cells (CHO-K1, BCRC 60006) is purchased from the Food Industry Research and Development Institute, Taiwan.
2. Culture medium Ham’s F-12 medium (including 2.0 mM L-Glutamine) with 10% fetal bovine serum (FBS).
3. Incubation condition: 37±1℃ incubator with 5±1% CO2. - Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- 1. Cell viability test:
The cell viability for each test group after treating with or without S9 Mix was analyzed (Table 1). When the dosage of test article was 2.0 mg/mL with or without S9 Mix for 3 hours, the cell viability was 60.85% and 74.63%. Therefore, 2.0 mg/L was used as the highest dosage for the gene mutation test.
2. Gene mutation test:
The colonies formation frequency and mutation frequency for each test group after treating with or without S9 Mix were analyzed (Table 2 &3). The results were as below:
2.1 After treating with S9 Mix for 3 hours, the negative control group mutation frequency was 19.0 x 10^-6, the positive control group (Benzo [a] pyrene) mutation frequency was 51.8 x 10^-6, that was significantly different from the negative control group (p<0.05). At test article concentrations of 2.0 mg/mL, 1.0 mg/mL, 0.5 mg/mL and 0.25 mg/mL, the mutation frequency were 37.3 x 10^-6, 36.7 x 10^-6, 39.9 x 10^-6 and 25.5 x 10^-6, was no significantly different from the negative control group (p> 0.05).
2.2 After treating without S9 Mix for 3 hours, the negative control group mutation frequency was 17.1 x 10^-6, the positive control group (4- Nitroquinoline-1-oxide) mutation frequency was 44.8 x 10^-6, that was significantly different from the negative control group (p<0.05). At test article concentrations of 2.0 mg/mL, 1.0mg/mL, 0.5mg/mL and 0.25 mg/mL, the mutation frequency were 21.1 x 10^-6, 21.4 x 10^-6, 20.9 x 10^-6 and 18.0 x 10^-6, was no significantly different from the negative control group (P>0.05) (Table3). - Remarks on result:
- no mutagenic potential (based on QSAR/QSPR prediction)
- Conclusions:
- This in vitro mammalian cell gene mutation test was performed in accordance with OECD 476:2015 guideline. The results indicated that the positive control group (Benzo[a]pyrene and 4-Nitroquinoline-1-oxide) mutation frequency was significantly different from the negative control group (p<0.05), meaning that this experiment was effective. The results by statistical analysis indicated that the mutation frequency of the test article for 3 hours treatment with S9 Mix and without S9 Mix was no significantly different from the negative control group. According to the above results, the in vitro mammalian cell gene mutation test was negative.
- Executive summary:
This in vitro mammalian cell gene mutation test was conducted according to OECD 476:2015 guideline to evaluate the mutagenicity of CJ302. Chinese Hamster ovary cell (CHO-K1) was used in the experiment. The cytotoxicity of the test article for CHO-K1 was used to set the highest dosage and number of seeding cells in the gene mutation test. The gene mutation test processing conditions were treating with S9 Mix and without S9 Mix for 3 hours. The test article highest concentration was 2.0 mg/mL as determined by a cell viability test. By statistical analysis, the mutation frequency of each test group had no significant difference from the negative control group after the test article for 3 hours treatment with S9 Mix and without S9 Mix. Therefore, under this test conditions, the in vitro mammalian cell gene mutation test was negative.
Referenceopen allclose all
Table 1. Genotype Confirmation Tests of Salmonella typhimurium Tester Strains
Genotype character |
Phenotypic observation |
Tester Strains |
||||
TA98 |
TA100 |
TA102 |
TA1535 |
TA1537 |
||
Histidine requirement |
Growing on biotin plate |
- |
- |
- |
- |
- |
Growing on histidine/biotin plate |
+ |
+ |
+ |
+ |
+ |
|
rfa mutation |
Inhibition zone of crystal violet |
+ |
+ |
+ |
+ |
+ |
△uvrB mutation |
Growing on non UV-irradiated plate |
+ |
+ |
+ |
+ |
+ |
Growing on UV-irradiated plate |
- |
- |
+ |
- |
- |
|
R-factor |
Ampicillin resistance |
+ |
+ |
+ |
- |
- |
Genotype confirmed |
Passed |
Passed |
Passed |
Passed |
Passed |
+: the presence
-: the absence
Table 2. Mutagenicity Test of CJ309 in Salmonella typhimurium Strains without S9 Metabolic Activation
Treatment (μg/plate) |
Number of Revertant Colonies in Salmonella typhimurium |
|||||||||||||||
TA98 |
TA100 |
TA102 |
TA1535 |
TA1537 |
||||||||||||
replicate |
1 |
2 |
3 |
1 |
2 |
3 |
1 |
2 |
3 |
1 |
2 |
3 |
1 |
2 |
3 |
|
Negative controla |
Ie |
18 |
23 |
25 |
82 |
79 |
60 |
380 |
407 |
433 |
16 |
13 |
23 |
9 |
25 |
8 |
Mf |
22 ± 4 |
74 ± 12 |
407 ± 27 |
17 ± 5 |
14 ± 10 |
|||||||||||
50 |
Ie |
35 |
25 |
17 |
68 |
81 |
71 |
402 |
314 |
345 |
20 |
10 |
16 |
9 |
16 |
14 |
Mf |
26 ± 9 |
73 ± 7 |
354 ± 45 |
15 ± 5 |
13 ± 4 |
|||||||||||
150 |
Ie |
25 |
14 |
25 |
70 |
71 |
67 |
335 |
343 |
336 |
14 |
24 |
12 |
15 |
16 |
22 |
Mf |
21 ± 6 |
69 ± 2 |
338 ± 4 |
17 ± 6 |
18 ± 4 |
|||||||||||
500 |
Ie |
26 |
23 |
22 |
86 |
66 |
66 |
364 |
315 |
317 |
24 |
25 |
23 |
13 |
13 |
16 |
Mf |
24 ± 2 |
73 ± 12 |
332 ± 28 |
24 ± 1 |
14 ± 2 |
|||||||||||
1500 |
Ie |
29 |
24 |
24 |
71 |
67 |
70 |
399 |
324 |
364 |
22 |
16 |
14 |
19 |
17 |
8 |
Mf |
26 ± 3 |
69 ± 2 |
362 ± 38 |
17 ± 4 |
15 ± 6 |
|||||||||||
5000 |
Ie |
17 |
24 |
16 |
58 |
65 |
67 |
384 |
282 |
326 |
14 |
15 |
11 |
19 |
14 |
16 |
Mf |
19 ± 4 |
63±5 |
331 ± 51 |
13 ± 2 |
16 ± 3 |
|||||||||||
Positive controlb |
Ie |
224 |
217 |
196 |
575 |
587 |
591 |
1218 |
1018 |
1182 |
380 |
355 |
416 |
171 |
143 |
140 |
Mf |
212c± 15 |
584c± 8 |
1139c± 107 |
384d± 31 |
151d± 17 |
a: Negative control was sterile deionized water.
b: Positive controls: 1μg/plate 2-nitrofluorene for TA98 0.5 μg/plate sodium azide for TA100
62.5μg/plate mitomycin C for TA102 0.1 μg/plate sodium azide for TA1535
0.5μg/plate acridine mutagen ICR 191 for TA1537
c: Greater than 2-fold negative control spontaneous revertants
d: Greater than 3-fold negative control spontaneous revertants
e: I: Number of revertants/plate is shown for each individual plate
f: M: The value of mean ± S.D. from triplicate plates of each treatment was calculated
Table 3. Mutagenicity Test of CJ309 in Salmonella typhimurium Strains with S9 Metabolic Activation
Treatment (μg/plate) |
Number of Revertant Colonies in Salmonella typhimurium |
|||||||||||||||
TA98 |
TA100 |
TA102 |
TA1535 |
TA1537 |
||||||||||||
replicate |
1 |
2 |
3 |
1 |
2 |
3 |
1 |
2 |
3 |
1 |
2 |
3 |
1 |
2 |
3 |
|
Negative controla |
Ie |
46 |
49 |
44 |
73 |
87 |
78 |
344 |
427 |
377 |
10 |
8 |
10 |
13 |
10 |
13 |
Mf |
46 ± 3 |
79 ± 7 |
383 ± 42 |
9 ± 1 |
12 ± 2 |
|||||||||||
50 |
Ie |
39 |
40 |
51 |
77 |
70 |
67 |
342 |
298 |
284 |
9 |
8 |
8 |
11 |
22 |
18 |
Mf |
43 ± 7 |
71 ± 5 |
308 ± 30 |
8 ± 1 |
17 ± 6 |
|||||||||||
150 |
Ie |
49 |
45 |
41 |
86 |
86 |
75 |
281 |
310 |
232 |
6 |
7 |
9 |
9 |
16 |
10 |
Mf |
45 ± 4 |
82 ± 6 |
274 ± 39 |
7 ± 2 |
12 ± 4 |
|||||||||||
500 |
Ie |
44 |
36 |
38 |
76 |
70 |
76 |
266 |
343 |
316 |
5 |
5 |
8 |
6 |
11 |
12 |
Mf |
39 ± 4 |
74 ± 3 |
308 ± 39 |
6 ± 2 |
10 ± 3 |
|||||||||||
1500 |
Ie |
48 |
37 |
35 |
80 |
65 |
64 |
324 |
454 |
364 |
8 |
12 |
10 |
13 |
8 |
19 |
Mf |
40 ± 7 |
70 ± 9 |
381 ± 67 |
10 ± 2 |
13 ± 6 |
|||||||||||
5000 |
Ie |
23 |
30 |
45 |
64 |
70 |
64 |
263 |
431 |
382 |
7 |
9 |
1 |
7 |
9 |
10 |
Mf |
33 ± 11 |
66 ± 3 |
359 ± 86 |
6 ± 4 |
9 ± 2 |
|||||||||||
Positive controlb |
Ie |
248 |
229 |
225 |
610 |
651 |
642 |
1424 |
1350 |
1688 |
272 |
287 |
249 |
270 |
316 |
307 |
Mf |
234c± 12 |
634c± 22 |
1487c± 178 |
269d± 19 |
298d± 24 |
a: Negative control was sterile deionized water.
b: Positive controls: 0.5μg/plate 2-aminofluorene for TA98 4 μg/plate 2-aminofluorene for TA100
4μg/plate 2-aminoanthracene for TA102 1 μg/plate 2-aminoanthracene for TA1535
2μg/plate 2-aminoanthracene for TA1537
c: Greater than 2-fold negative control spontaneous revertants
d: Greater than 3-fold negative control spontaneous revertants
e: I: Number of revertants/plate is shown for each individual plate
f: M: The value of mean ± S.D. from triplicate plates of each treatment was calculated
Table 1. Karyology Analysis of Chinese Hamster Ovary Cells
No. of chromosome |
<18 |
18 |
19 |
20 |
21 |
22 |
>22 |
No. of cells |
0 |
0 |
9 |
35 |
5 |
1 |
0 |
Table 2. Concurrent Cytotoxicity Analysis of CJ309 in Chinese Hamster Ovary Cells
Concentration (μg/mL) |
Cell Number (× 105cells) |
ICCa N-N0 |
RICCb (%) |
Cytotoxicityc (%) |
Before Treatment |
||||
Untreated (-S9) |
30.4 |
|
|
|
Untreated (+S9) |
26.4 |
|
|
|
After Treatment |
||||
Scheme I (-S9, 3h) |
||||
Negative Control |
63.6 |
33.2 |
100.0 |
0.0 |
51.2 |
62.2 |
31.8 |
95.8 |
4.2 |
128 |
67.0 |
36.6 |
110.2 |
0.0 |
320 |
65.0 |
34.6 |
104.2 |
0.0 |
800 |
62.6 |
32.2 |
97.0 |
3.0 |
2000 |
61.2 |
30.8 |
92.8 |
7.2 |
Positive Controld |
48.6 |
18.2 |
54.8 |
45.2 |
Scheme II (+S9, 3h) |
||||
Negative Control |
55.8 |
29.4 |
100.0 |
0.0 |
51.2 |
54.4 |
28.0 |
95.2 |
4.8 |
128 |
54.6 |
28.2 |
95.9 |
4.1 |
320 |
55.6 |
29.2 |
99.3 |
0.7 |
800 |
49.0 |
22.6 |
76.9 |
23.1 |
2000 |
52.8 |
26.4 |
89.8 |
10.2 |
Positive Controle |
44.6 |
18.2 |
61.9 |
38.1 |
Scheme III (-S9, 20h) |
||||
Negative Control |
63.8 |
33.4 |
100.0 |
0.0 |
51.2 |
57.6 |
27.2 |
81.4 |
18.6 |
128 |
110.2 |
79.8 |
238.9 |
0.0 |
320 |
108.0 |
77.6 |
232.3 |
0.0 |
800 |
54.4 |
24.0 |
71.9 |
28.1 |
2000 |
58.8 |
28.4 |
85.0 |
15.0 |
Positive Controlf |
56.8 |
26.4 |
79.0 |
21.0 |
a ICC: increased in cell counts = Cell No.After treatment (N) - Cell No.Before treatment (N0)
b RICC: relative increase in cell counts; RICC = (ICCtreatment/ICCcontrol) × 100
c Cytotoxicity (%) = 100 – RICC
d Positive control was 0.33 μg/mL mitomycin C (MMC)
e Positive control was 11.2 μg/mL cyclophosphamide (CPP)
f Positive control was 0.2 μg/mL mitomycin C (MMC)
Table 3. Summary of Chromosome Aberrations in Chinese Hamster Ovary Cells for CJ309
Treatment |
Concentration (μg/mL) |
Treating Hour |
S9 (-/+) |
Aberrant Cells (%) |
Scheme I (-S9, 3h) |
||||
Negative Controla |
0 |
3 |
- |
0.33 |
Test Article |
51.2 |
3 |
- |
0.33 |
128 |
3 |
- |
0.67 |
|
320 |
3 |
- |
0.33 |
|
800 |
3 |
- |
0 |
|
2000 |
3 |
- |
1 |
|
Positive Controlc |
0.33 |
3 |
- |
19.33* |
Scheme II (+S9, 3h) |
||||
Negative Controlb |
0 |
3 |
+ |
0 |
Test Article |
51.2 |
3 |
+ |
0.67 |
128 |
3 |
+ |
0.67 |
|
320 |
3 |
+ |
0.67 |
|
800 |
3 |
+ |
1 |
|
2000 |
3 |
+ |
0.33 |
|
Positive Controld |
11.2 |
3 |
+ |
24* |
Scheme III (-S9, 20h) |
||||
Negative Controla |
0 |
20 |
- |
0.33 |
Test Article |
51.2 |
20 |
- |
0.33 |
128 |
20 |
- |
0.67 |
|
320 |
20 |
- |
1 |
|
800 |
20 |
- |
1.33 |
|
2000 |
20 |
- |
− |
|
Positive Controle |
0.2 |
20 |
- |
29* |
All data were scored from 300 metaphase cells of each treatment (duplicate cultures).
a: Negative control was 10% sterile deionized water in McCoy’s 5A medium.
b: Negative control was 10% sterile deionized water in S-9 mixture medium.
c: Positive control was 0.33 μg/mL mitomycin C (MMC).
d: Positive control was 11.2 μg/mL cyclophosphamide (CPP).
e: Positive control was 0.2 μg/mL mitomycin C (MMC).
*: The frequency of aberrant cells was significantly higher than that of the negative control (One-tailed binomial test, α = 0.01).
−: Too few metaphases
Table 1. Cell viability analysis
Group | Test article | Average colony numbers a | Relative survival (%)b |
With S9 Mix | Negative control c | 74.5±12.0 | 100.00±0.00 |
Positive control d | 65.0±5.7 | 85.98±0.07 | |
Test groups (mg/mL) | |||
2.0 | 46.0±9.9 | 60.85±0.13 | |
1.0 | 46.0±2.8 | 60.85±0.04 | |
0.5 | 50.5±9.2 | 66.80±0.12 | |
0.25 | 57.5±7.8 | 76.06±0.10 | |
Without S9 Mix | Negative control | 60.0±7.1 | 100.00±0.00 |
Positive control | 42.5±13.4 | 63.43±0.20 | |
Test groups (mg/mL) | |||
2.0 | 50.0±8.5 | 74.63±0.13 | |
1.0 | 45.5±4.9 | 67.91±0.07 | |
0.5 | 53.5±6.4 | 79.85±0.09 | |
0.25 | 56.0±4.2 | 83.58±0.06 |
a Values were expressed as Mean±SD, and tests were repeated two times.
b Relative survival = each colony numbers of the positive control or test groups / the average of colony numbers in the negative control x 100%, then calcultated the Mean±SD.
c Negative control: Ham's F-12 medium with 10% FBS (S9 Mix or not)
d Positive control: 4 µg/mL B[a]P for the cells treated with S9 Mix, and 0.25 µg/mL 4-NQD for the cells treated without S9 Mix.
Table 2. Colonies formation frequency analysis
Group | Test article | Average colony numbers a | Colonies formation frequency b |
With S9 Mix | Negative control c | 141.3±9.0 | 0.71 |
Positive control d | 115.7±13.7 | 0.58 | |
Test groups (mg/mL) | |||
2.0 | 157.7±25.5 | 0.79 | |
1.0 | 128.0±39.4 | 0.64 | |
0.5 | 103.7±62.9 | 0.52 | |
0.25 | 101.7±37.5 | 0.51 | |
Without S9 Mix | Negative control | 176.3±20.2 | 0.88 |
Positive control | 104.3±22.9 | 0.52 | |
Test groups (mg/mL) | |||
2.0 | 138.3±41.2 | 0.69 | |
1.0 | 122.7±17.4 | 0.61 | |
0.5 | 117.0±34.7 | 0.59 | |
0.25 | 115.3±46.9 | 0.58 |
a Values were expressed as Mean±SD, and tests were repeated three times.
b Colonies formation frequency = numbers of colonies / the number of seeding.
c Negative control: Ham's F-12 medium with 10% FBS (S9 Mix or not)
d Positive control: 4 µg/mL B[a]P for the cells treated with S9 Mix, and 0.25 µg/mL 4-NQD for the cells treated without S9 Mix.
Table 3. Mutation frequency analysis
Group | Test article | Average colony numbers a | Mutationfrequency (x 10^-6) b |
With S9 Mix | Negative control c | 13.3±7.0 | 19.0 |
Positive control d | 30.0±4.4 | 51.8 | |
Test groups (mg/mL) | |||
2.0 | 28.7±4.5 | 37.3 | |
1.0 | 22.7±6.5 | 36.7 | |
0.5 | 18.3±7.4 | 39.9 | |
0.25 | 12.3±4.7 | 25.5 | |
Without S9 Mix | Negative control | 15.3±7.4 | 17.1 |
Positive control | 25.0±15.1 | 44.8 | |
Test groups (mg/mL) | |||
2.0 | 13.7±3.5 | 21.1 | |
1.0 | 13.0±2.6 | 21.4 | |
0.5 | 11.0±2.0 | 20.9 | |
0.25 | 9.7±2.5 | 18.0 |
a Values were expressed as Mean±SD, and tests were repeated three times.
b Mutation frequency = (numbers of colonies / number of seeding) x (1/colonies formation frequency).
c Negative control: Ham's F-12 medium with 10% FBS (S9 Mix or not)
d Positive control: 4 µg/mL B[a]P for the cells treated with S9 Mix, and 0.25 µg/mL 4-NQD for the cells treated without S9 Mix.
e Significantly different from the negative control group (p<0.05)
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Bacterial reverse mutation test (OECD TG471)
Based on the preliminary assay results, 5000μg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of CJ309 at 50, 150, 500, 1500 and 5000μg/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. The results of concurrent positive and negative controls and three non-cytotoxic dose levels obtained supported the validity of the assay.
No cytotoxicity was observed in all five tester strains up to 5000 μg/plate in the absence and presence of metabolite activations. Results showed that CJ309 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 up to 5000 μg/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, CJ309 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5000μg/plate in the absence and presence of S9 metabolic activation.
Mammalian chromosomal aberration test (OECD TG473)
A preliminary concentration-range finding cytotoxicity test was performed in three test schemes: 3-hour treatment in the absence and presence of S9 metabolic activation (Schemes I and II) and 20-hour continuous treatment in the absence of S9 metabolic activation (Scheme III), and the results suggested that a concentration of 2000 μg/mL in all three schemes would be used in the chromosome aberration assay. Based on the cytotoxicity result, five concentrations of 51.2, 128, 320, 800 and 2000 μg/mL were used for all three schemes in the chromosome aberration test. In test group, the cell proportions with abnormal chromosome in three Schemes were not higher than 3%. Based on the data obtained from this study, it was concluded that under the test condition, CJ309 did not induce chromosome aberration in CHO cells in absence or presence of S9 metabolic activation.
In vitro mammalian cell gene mutation test (OECD 476)
This in vitro mammalian cell gene mutation test was performed in accordance with OECD 476:2015 guideline. The results indicated that the positive control group (Benzo[a]pyrene and 4-Nitroquinoline-1-oxide) mutation frequency was significantly different from the negative control group (p<0.05), meaning that this experiment was effective. The results by statistical analysis indicated that the mutation frequency of the test article for 3 hours treatment with S9 Mix and without S9 Mix was no significantly different from the negative control group. According to the above results, the in vitro mammalian cell gene mutation test was negative.
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