Registration Dossier

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

Referenceopen allclose all

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.

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.

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

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

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'.

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

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.

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.

Justification for classification or non-classification