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Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
CJ313 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 1500 μg/plate in the absence and presence of S9 metabolic activation(OECD TG471).
Read-across from supporting substance, CJ313 did not induce chromosome aberration in CHO cells in absence or presence of S9 metabolic activation (OECD TG473).
CJ313 was concluded to be negative for the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro mammalian cell forward gene mutation (CHO/HPRT) assay(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 January 17, 2017 to July 03, 2017
- 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:
- The post-mitochondrial fraction (S9) prepared from uninduced male Golden Syrian Hamster liver
- Untreated negative controls:
- yes
- Remarks:
- Sterile deionized water
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- sodium azide
- congo red
- mitomycin C
- other: Acridine mutagen ICR 191, 2-Aminofluorene, 2-Amoinoanthracene
- Evaluation criteria:
- 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:
- no cytotoxicity
- 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:
- no cytotoxicity
- 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:
- no cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 μg/plate
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 μg/plate
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: The substance was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 1500 ug/plate.
- Conclusions:
- According to OECD 471 test method, CJ313 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 1500 μg/plate.
- Executive summary:
This test using the procedures outlined in the QPS Taiwan Study Plan for T65316020-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 CJ313 show that test validity criteria was met.
Based on the preliminary assay results, 5000 μg/platewas set as the highest dose in this study. In the mutagenicity assay, five doses of CJ313 at 50, 150, 500, 1500 and 5000 μg/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.Cytotoxicity was observed in tester strains TA102 and TA1537 at 5000 μg/plate in the presence of metabolite activation. Unexpected greater than 50% reduction of negative control revertants was observed in tester strain TA1537 at 50 μg/plate but would not consider cytotoxicity in the absence of metabolite activation. Results showed that CJ313 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 in the absence of metabolite activation, three tester strains TA98, TA100 and TA1535 in the presence of metabolite activation up to 5000 μg/plate; two tester strains TA102 and TA1537 in the presence of metabolite activation up to 1500 μg/plate. Based on the data obtained from this study, it was concluded that under the test condition, CJ313 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 1500 μg/plate in the absence and presence of S9 metabolic activation.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- 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
- Justification for type of information:
- The read-across target has structure similarity to CJ313. Therefore, the data is applied.
- Reason / purpose for cross-reference:
- read-across source
- 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:
- 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)
- 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 2000μg/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 2000μg/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:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From March 31, 2016 to May 26, 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- The main structure of the read-across substance is the same of the CJ313, only the salt form is different. However, this difference does not influence the toxicity of the CJ313. CJ313 contains Li/Na salt form and the read-across substances contains only Na salt. This will not lead to difference on the toxicity results.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
- GLP compliance:
- yes
- Type of assay:
- other: in vitro mammalian cell gene mutation tests using the Hprt and xprt genes
- Specific details on test material used for the study:
- Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light. Dose formulations were adjusted to compensate for the purity (84.3%) of the test substance, using a correction factor of 1.19.
- Target gene:
- The purpose of this study is to evaluate a test article for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) in Chinese hamster ovary (CHO) cells, as assayed by colony growth in the presence of 6 thioguanine (TG resistance, TGr)
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- Arcolor 1254-induced rat liver S9
- Test concentrations with justification for top dose:
- In the preliminary toxicity assay, the concentrations tested were 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL
In the definitive mutagenicity assay, the concentrations tested were 125, 250, 500, 1000 and 2000 µg/mL with S9 and 125, 250, 500, 1000, 1200, 1500, 1800 and 2000 µg/mL without S9 - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Water (test article); DMSO (Ethylmethanesulfonate, Benzo(a)pyrene)
- Justification for choice of solvent/vehicle: Water was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 5 ± 0.5 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
SELECTION AGENT (mutation assays): 6-thioguanine
NUMBER OF REPLICATIONS: single cultures in the preliminary toxicity assay; duplicate cultures in the mutagenicity assay
NUMBER OF CELLS EVALUATED: 2.4 x 10E6 cells per culture
DETERMINATION OF CYTOTOXICITY
- Method: adjusted relative survival - Rationale for test conditions:
- None
- Evaluation criteria:
- The test substance was considered to have produced a positive response if it induced a dose-related increase in mutant frequency and an increase exceeding the 95 % historical vehicle control limits in at least one test dose level as compared with the concurrent vehicle control (p<0.01). If only one criterion was met (a statistically significant or dose-dependent increase or an increase exceeding the historical control 95% confidence interval), the results were considered equivocal. If none of these criteria were met, the results were considered to be negative.
Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations. - Statistics:
- Statistical analyses were performed using the method of Snee and Irr (1981), with significance established at the 0.05 level.
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Preliminary toxicity assay: The pH of the cultures was adjusted at all concentrations with and without S9 to maintain neutral pH; Definitive mutagenicity assay: The test substance did not have an adverse impact on the pH of the cultures (pH 7.29 at the top dose).
- Effects of osmolality: The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehicle control by more than 120 % in the preliminary toxicity assay.
- Precipitation: No visible precipitate was observed at the beginning or end of treatment in both the assays. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Under the conditions of the assay described in this report, FAT 40032/G was concluded to be negative for the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro mammalian cell forward gene mutation (CHO/HPRT) assay.
- Executive summary:
The test substance, FAT 40032/G, was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr). Water was used as the vehicle.
In the preliminary toxicity assay, the concentrations tested were 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL. The maximum concentration evaluated was the limit dose for this assay. No visible precipitate was observed at the beginning or end of treatment. Adjusted relative survival was 96.59 and 2.12% at a concentration of 2000 μg/mL with and without S9, respectively. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 125, 250, 500, 1000 and 2000 μg/mL with S9 and 125, 250, 500, 1000, 1200, 1500, 1800 and 2000 μg/mL without S9.
In the definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. The average adjusted relative survival was 90.74 and 95.36% at a concentration of 2000 μg/mL with and without S9, respectively. Cultures treated at concentrations of 125, 250, 500, 1000 and 2000 μg/mL with and without S9 were chosen for mutant selection. No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p > 0.05). All control data were within the 95% control limits except for the solvent control mutant frequency data with S9. However, these values were acceptable as they were within the observed historical control range. The positive controls induced significant increases in mutant frequency (p < 0.01).
These results indicate FAT 40032/G was negative for the ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system.
Referenceopen allclose all
Table 1. Genotype Confirmation Test 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 |
+ |
+ |
+ |
+ |
+ |
|
rfamutation |
inhibition zone of crystal violet |
+ |
+ |
+ |
+ |
+ |
ΔuvrBmutation |
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 CJ313 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 |
27 |
25 |
31 |
50 |
53 |
70 |
288 |
322 |
268 |
16 |
6 |
13 |
21 |
14 |
15 |
Mf |
28 ± 3 |
58 ± 11 |
293 ± 27 |
12 ± 5 |
17 ± 4 |
|||||||||||
50 |
Ie |
29 |
23 |
30 |
56 |
61 |
53 |
322 |
336 |
318 |
18 |
12 |
16 |
4h |
9 |
12 |
Mf |
27 ± 4 |
57 ± 4 |
325 ± 9 |
15 ± 3 |
8g± 4 |
|||||||||||
150 |
Ie |
14 |
18 |
18 |
58 |
71 |
52 |
216 |
350 |
280 |
13 |
16 |
22 |
17 |
11 |
14 |
Mf |
17 ± 2 |
60 ± 10 |
282 ± 67 |
17 ± 5 |
14 ± 3 |
|||||||||||
500 |
Ie |
18 |
17 |
26 |
57 |
49 |
80 |
332 |
256 |
204 |
12 |
15 |
16 |
6 |
12 |
16 |
Mf |
20 ± 5 |
62 ± 16 |
261 ± 59 |
14 ± 2 |
11 ± 5 |
|||||||||||
1500 |
Ie |
23 |
25 |
23 |
70 |
61 |
58 |
294 |
294 |
278 |
14 |
17 |
16 |
17 |
10 |
9 |
Mf |
24 ± 1 |
63 ± 6 |
289 ± 9 |
16 ± 2 |
12 ± 4 |
|||||||||||
5000 |
Ie |
25 |
22 |
17 |
42 |
60 |
57 |
258 |
176 |
292 |
13 |
11 |
18 |
10 |
13 |
16 |
Mf |
21 ± 4 |
53 ± 10 |
242 ± 60 |
14 ± 4 |
13 ± 3 |
|||||||||||
Positive controlb |
Ie |
181 |
255 |
266 |
374 |
374 |
430 |
1411 |
1229 |
1376 |
439 |
428 |
349 |
191 |
181 |
174 |
Mf |
234c± 46 |
393c±32 |
1339c± 97 |
405d± 49 |
182d± 9 |
a: Negative control was sterile deionized water.
b: Positive controls: 1 μg/plate 2-nitrofluorene for TA98
0.5 μg/plate sodium azide for TA100
62.5 ng/plate mitomycin C for TA102
0.1 μg/plate sodium azide for TA1535
0.3 μg/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.
g: Cytotoxicty: a > 50% reduction when compared to the mean colony number of negative control revertants.
h: Extreme low data may consider as an outlier (out of range of historical data).
Table 3. Mutagenicity Test of CJ313 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 |
38 |
41 |
38 |
86 |
75 |
89 |
515 |
486 |
562 |
15 |
11 |
13 |
29 |
33 |
32 |
Mf |
39 ± 2 |
83 ± 7 |
521 ± 38 |
13 ± 2 |
31 ± 2 |
|||||||||||
50 |
Ie |
28 |
40 |
37 |
63 |
108 |
70 |
337 |
414 |
483 |
12 |
14 |
18 |
28 |
22 |
19 |
Mf |
35 ± 6 |
80 ± 24 |
411 ± 73 |
15 ± 3 |
23 ± 5 |
|||||||||||
150 |
Ie |
43 |
38 |
47 |
61 |
76 |
88 |
422 |
416 |
131 |
7 |
15 |
11 |
26 |
24 |
31 |
Mf |
43 ± 5 |
75 ± 14 |
384 ± 61 |
11 ± 4 |
27 ± 4 |
|||||||||||
500 |
Ie |
23 |
36 |
41 |
84 |
88 |
67 |
296 |
316 |
390 |
15 |
16 |
17 |
29 |
35 |
34 |
Mf |
33 ± 9 |
80 ± 11 |
334 ± 50 |
16 ± 1 |
33 ± 3 |
|||||||||||
1500 |
Ie |
36 |
35 |
33 |
93 |
77 |
90 |
294 |
367 |
391 |
12 |
12 |
11 |
18 |
18 |
13 |
Mf |
35 ± 2 |
87 ± 9 |
351 ± 51 |
12 ± 1 |
16 ± 3 |
|||||||||||
5000 |
Ie |
27 |
27 |
17 |
65 |
54 |
80 |
343 |
179 |
136 |
10 |
11 |
7 |
10 |
8 |
16 |
Mf |
24 ± 6 |
66 ± 13 |
219g± 109 |
9 ± 2 |
11g± 4 |
|||||||||||
Positive controlb |
Ie |
263 |
329 |
312 |
254 |
298 |
346 |
1133 |
1336 |
1086 |
300 |
307 |
459 |
146 |
132 |
110 |
Mf |
301c± 34 |
299c± 46 |
1185c± 133 |
355d± 90 |
129d± 18 |
a: Negative control was sterile deionized water.
b: Positive controls: 60 μg/plate Congo Red for TA98
1 μg/plate 2-aminofluorene for TA100
2 μg/plate 2-aminoanthracene for TA102
0.5 μg/plate 2-aminoanthracene for TA1535
2 μg/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.
g: Cytotoxicty: a > 50% reduction when compared to the mean colony number of negative control revertants.
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 0.33μg/mL mitomycin C (MMC)
e Positive control was 11.2μg/mL cyclophosphamide (CPP)
f Positive control was 0.2μg/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 0.33μg/mL mitomycin C (MMC).
d: Positive control was 11.2μg/mL cyclophosphamide (CPP).
e: Positive control was 0.2μg/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. Summary Results for FAT 40032/G Preliminary Toxicity Assay ±S9
Treatment | Dose Level | S9 | Initial Survival | |||||
Cells/mL | Cloning Efficiency | Relative Survival | ||||||
(µg/mL) | (x 10^6) | (Colonies/Plate) | (%) | (adj., %) | ||||
di-H2O | 100.0a | - | 0.230 | 145 | 150 | 140 | 72.50 | 100.00 |
FAT 40032/G | 3.91 | - | 0.217 | 105 | 103 | 128 | 56.00 | 72.99 |
FAT 40032/G | 7.81 | - | 0.230 | 160 | 135 | 156 | 75.17 | 104.08 |
FAT 40032/G | 15.6 | - | 0.231 | 151 | 169 | 151 | 78.50 | 108.95 |
FAT 40032/G | 31.3 | - | 0.240 | 215 | 192 | 197 | 100.67 | 145.04 |
FAT 40032/G | 62.5 | - | 0.225 | 143 | 145 | 135 | 70.50 | 95.30 |
FAT 40032/G | 125 | - | 0.223 | 162 | 170 | 169 | 83.50 | 111.64 |
FAT 40032/G | 250 | - | 0.206 | 145 | 181 | 143 | 78.17 | 96.86 |
FAT 40032/G | 500 | - | 0.204 | 127 | 122 | 128 | 62.83 | 76.96 |
FAT 40032/G | 1000 | - | 0.116 | 146 | 168 | 140 | 75.67 | 52.73 |
FAT 40032/G | 2000 | - | 0.023 | 29 | 26 | 38 | 15.50 | 2.12 |
di-H2O | 100.0a | + | 0.196 | 150 | 150 | 160 | 76.67 | 100.00 |
FAT 40032/G | 3.91 | + | 0.223 | 107 | 113 | 134 | 59.00 | 87.82 |
FAT 40032/G | 7.81 | + | 0.219 | 168 | 183 | 162 | 85.50 | 124.84 |
FAT 40032/G | 15.6 | + | 0.232 | 137 | 137 | 126 | 66.67 | 103.08 |
FAT 40032/G | 31.3 | + | 0.222 | 171 | 135 | 169 | 79.17 | 117.13 |
FAT 40032/G | 62.5 | + | 0.216 | 154 | 147 | 146 | 74.50 | 107.43 |
FAT 40032/G | 125 | + | 0.228 | 169 | 144 | 159 | 78.67 | 119.46 |
FAT 40032/G | 250 | + | 0.245 | 125 | 95 | 120 | 56.67 | 92.49 |
FAT 40032/G | 500 | + | 0.259 | 144 | 166 | 166 | 79.33 | 137.23 |
FAT 40032/G | 1000 | + | 0.225 | 144 | 144 | 140 | 71.33 | 107.08 |
FAT 40032/G | 2000 | + | 0.173 | 158 | 158 | 185 | 83.50 | 96.59 |
a (µL/mL)
Table 2. Summary Results for FAT 40032/G Definitive Mutagenicity Assay -S9
Treatment | Dose Level | S9 | Initial Survival | TGr Mutants/Plate | Total Mutant Colonies | Selection | |||||||||||||
Cells/mL | Cloning Efficiency | Relative Survival | Cloning Efficiency | Mutant Frequency (x 10^-6) | |||||||||||||||
(µg/mL) | (x 10^6) | (Colonies/Plate) | (%) | (adj., %) | (Colonies/Plate) | (%) | Individual | Average | |||||||||||
di-H2O | 100.0a | - | 0.738 | 172 | 172 | 172 | 86.00 | 95.82 | 4 | 4 | 3 | 3 | 14 | 172 | 180 | 187 | 89.83 | 6.49 | 5.88 |
di-H2O | 100.0a | - | 0.923 | 186 | 179 | 196 | 93.50 | 104.18 | 3 | 5 | 3 | 2 | 13 | 211 | 203 | 203 | 102.83 | 5.27 | |
FAT 40032/G | 125 | - | 0.977 | 151 | 140 | 159 | 75.00 | 83.57 | 3 | 10 | 4 | 2 | 19 | 167 | 185 | 138 | 81.67 | 9.69 | 7.20 |
FAT 40032/G | 125 | - | 0.948 | 138 | 126 | 151 | 69.17 | 77.07 | 1 | 3 | 1 | 4 | 9 | 167 | 167 | 144 | 79.67 | 4.71 | |
FAT 40032/G | 250 | - | 0.644 | 189 | 147 | 162 | 83.00 | 92.48 | 3 | 3 | 1 | 0 | 7 | 151 | 194 | 155 | 83.33 | 3.50 | 6.48 |
FAT 40032/G | 250 | - | 0.934 | 144 | 153 | 186 | 80.50 | 89.69 | 7 | 3 | 8 | 1 | 19 | 182 | 150 | 170 | 83.67 | 9.46 | |
FAT 40032/G | 500 | - | 0.457 | 173 | 179 | 177 | 88.17 | 98.24 | 2 | 1 | 1 | 3 | 7 | 148 | 138 | 156 | 73.67 | 3.96 | 4.79 |
FAT 40032/G | 500 | - | 0.554 | 158 | 160 | 156 | 79.00 | 88.02 | 3 | 3 | 2 | 4 | 12 | 179 | 196 | 158 | 88.83 | 5.63 | |
FAT 40032/G | 1000 | - | 0.262 | 164 | 176 | 144 | 80.67 | 89.88 | 7 | 2 | 4 | 5 | 18 | 160 | 170 | 192 | 87.00 | 8.62 | 10.17 |
FAT 40032/G | 1000 | - | 0.492 | 180 | 159 | 185 | 87.33 | 97.31 | 5 | 4 | 9 | C | 18 | 169 | 172 | 171 | 85.33 | 11.72 | |
FAT 40032/G | 1200 | - | 0.338 | 179 | 147 | 147 | 78.83 | 87.84 | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND |
FAT 40032/G | 1200 | - | 0.284 | 203 | 171 | 197 | 95.17 | 106.04 | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | |
FAT 40032/G | 1500 | - | 0.187 | 163 | 172 | 150 | 80.83 | 90.06 | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND |
FAT 40032/G | 1500 | - | 0.131 | 146 | 151 | 159 | 76.00 | 84.68 | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | |
FAT 40032/G | 1800 | - | 0.182 | 201 | 186 | 191 | 96.33 | 107.34 | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND |
FAT 40032/G | 1800 | - | 0.138 | 170 | 179 | 160 | 84.83 | 94.52 | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | |
FAT 40032/G | 2000 | - | 0.144 | 160 | 159 | 171 | 81.67 | 90.99 | 6 | 4 | 9 | 4 | 23 | 218 | 208 | 235 | 110.17 | 8.7 | 6.90 |
FAT 40032/G | 2000 | - | 0.165 | 193 | 165 | 179 | 89.50 | 99.72 | 2 | 1 | 4 | 4 | 11 | 177 | 187 | 176 | 90 | 5.09 | |
EMS | 0.200a | - | 1.046 | 111 | 117 | 100 | 54.67 | 60.91 | 104 | 84 | 79 | 86 | 353 | 130 | 162 | 148 | 73.33 | 200.57 | 250.55** |
EMS | 0.200a | - | 1.091 | 123 | 103 | 112 | 56.33 | 62.77 | 119 | 108 | 112 | 119 | 458 | 122 | 131 | 128 | 63.50 | 300.52 |
a (µL/mL)
C = Contaminated
ND = Not determined
** p <=0.01
Table 3. Summary Results for FAT 40032/G Definitive Mutagenicity Assay +S9
Treatment | Dose Level | S9 | Initial Survival | TGr Mutants/Plate | Total Mutant Colonies | Selection | |||||||||||||
Cells/mL | Cloning Efficiency | Relative Survival | Cloning Efficiency | Mutant Frequency (x 10^-6) | |||||||||||||||
(µg/mL) | (x 10^6) | (Colonies/Plate) | (%) | (adj., %) | (Colonies/Plate) | (%) | Individual | Average | |||||||||||
di-H2O | 100.0a | + | 0.647 | 156 | 139 | 146 | 73.50 | 97.24 | 3 | 4 | 3 | 8 | 18 | 151 | 136 | 152 | 73.17 | 10.25 | 12.29 |
di-H2O | 100.0a | + | 0.707 | 151 | 170 | 145 | 77.67 | 102.76 | 4 | 14 | 7 | 7 | 32 | 159 | 191 | 208 | 93 | 14.34 | |
FAT 40032/G | 125 | + | 0.748 | 190 | 177 | 152 | 86.50 | 114.44 | 2 | 2 | 2 | 3 | 9 | 146 | 139 | 136 | 70.17 | 5.34 | 4.37 |
FAT 40032/G | 125 | + | 0.763 | 158 | 142 | 160 | 76.67 | 101.43 | 2 | 3 | 0 | 1 | 6 | 150 | 143 | 149 | 73.67 | 3.39 | |
FAT 40032/G | 250 | + | 0.767 | 157 | 168 | 137 | 77.00 | 101.87 | 2 | 6 | 3 | 7 | 18 | 178 | 139 | 152 | 78.17 | 9.59 | 9.97 |
FAT 40032/G | 250 | + | 0.730 | 139 | 163 | 176 | 79.67 | 105.40 | 12 | 4 | 2 | 2 | 20 | 172 | 159 | 152 | 80.5 | 10.35 | |
FAT 40032/G | 500 | + | 0.773 | 165 | 179 | 171 | 85.83 | 113.56 | 8 | 4 | 9 | 9 | 30 | 146 | 192 | 157 | 82.5 | 15.15 | 11.25 |
FAT 40032/G | 500 | + | 0.727 | 153 | 160 | 153 | 77.67 | 105.76 | 2 | 4 | 3 | 6 | 15 | 167 | 170 | 173 | 85 | 7.35 | |
FAT 40032/G | 1000 | + | 0.689 | 143 | 152 | 152 | 74.50 | 98.57 | 5 | 4 | 2 | 2 | 13 | 184 | 201 | 170 | 92.50 | 5.86 | 6.53 |
FAT 40032/G | 1000 | + | 0.706 | 142 | 169 | 166 | 79.50 | 105.18 | 3 | 4 | 4 | 4 | 15 | 170 | 177 | C | 86.75 | 7.2 | |
FAT 40032/G | 2000 | + | 0.501 | 155 | 130 | 145 | 71.67 | 94.82 | 2 | 8 | 2 | 7 | 19 | 164 | 165 | 165 | 82.33 | 9.62 | 7.83 |
FAT 40032/G | 2000 | + | 0.428 | 143 | 132 | 118 | 65.50 | 86.66 | 8 | 2 | 2 | 0 | 12 | 169 | 151 | 176 | 82.67 | 6.05 | |
B(a)P | 4.000 | + | 0.702 | 103 | 74 | 96 | 45.50 | 60.20 | 59 | 52 | 57 | 44 | 212 | 123 | 127 | 165 | 69.17 | 127.71 | 133.39** |
B(a)P | 4.000 | + | 0.740 | 86 | 86 | 92 | 44.00 | 58.21 | 64 | 55 | 57 | 71 | 247 | 159 | 137 | 148 | 74.00 | 139.08 |
a (µL/mL)
C = Contaminated
** p <=0.01
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
in vitro gene mutation study in bacteria
Based on the preliminary assay results, 5000 μg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of CJ313 at 50, 150, 500, 1500 and 5000 μg/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.Cytotoxicity was observed in tester strains TA102 and TA1537 at 5000 μg/plate in the presence of metabolite activation. Unexpected greater than 50% reduction of negative control revertants was observed in tester strain TA1537 at 50 μg/plate but would not consider cytotoxicity in the absence of metabolite activation. Results showed that CJ313 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 in the absence of metabolite activation, three tester strains TA98, TA100 and TA1535 in the presence of metabolite activation up to 5000 μg/plate; two tester strains TA102 and TA1537 in the presence of metabolite activation up to 1500 μg/plate. Based on the data obtained from this study, it was concluded that under the test condition, CJ313 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 1500 μg/plate in the absence and presence of S9 metabolic activation.
in vitro cytogenicity / chromosome aberration study in mammalian cells
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 2000μg/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 2000μg/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 gene mutation study in mammalian cells
In the preliminary toxicity assay, the concentrations tested were 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL. The maximum concentration evaluated was the limit dose for this assay. No visible precipitate was observed at the beginning or end of treatment. Adjusted relative survival was 96.59 and 2.12% at a concentration of 2000 μg/mL with and without S9, respectively. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 125, 250, 500, 1000 and 2000 μg/mL with S9 and 125, 250, 500, 1000, 1200, 1500, 1800 and 2000 μg/mL without S9.
In the definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. The average adjusted relative survival was 90.74 and 95.36% at a concentration of 2000 μg/mL with and without S9, respectively. Cultures treated at concentrations of 125, 250, 500, 1000 and 2000 μg/mL with and without S9 were chosen for mutant selection. No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p > 0.05). All control data were within the 95% control limits except for the solvent control mutant frequency data with S9. However, these values were acceptable as they were within the observed historical control range. The positive controls induced significant increases in mutant frequency (p < 0.01).
These results indicate FAT 40032/G was negative for the ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system.
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