Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 August 2018 to 27 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Test material form:
solid: particulate/powder
Details on test material:
Molecular formula : C18H39NO3
Molecular weight : 317.5 g/mol
Appearance : White to off white powder
Purity : 98.1 %
Solubility : Insoluble in water, Soluble in other solvents
Stability : Not available
Storage condition : Room temperature
Specific details on test material used for the study:
Product name : DS-Phytosphingosine
CAS No. : 554-62-1
Lot No. : PYGC23
Facility code No. : 2018/00057
Manufactured date : 23 March 2018
Expired date : 22 March 2020
Received date : 23 July 2018
Received quantity : 23.09 g (including containers weight)

Method

Target gene:
Strain Target mutation (Mutation type)
TA 1535 hisG46; rfa-; uvrB- (Base-pair substitution)
TA 100 hisG46; rfa-; uvrB- (R-factor Base-pair substitution)
TA 98 hisD3052; rfa-; uvrB- (R-factor Frame shift)
TA 1537 hisC3076; rfa-; uvrB- (Frame shift)
WP2uvrA trp-, urvA- (Base-pair substition)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
N/A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 Microsomal fraction was prepared at KCL. The liver of Sprague-Dawley male rat was induced by Aroclor-1254, 500 mg/kg i.p.
Test concentrations with justification for top dose:
Preliminary range-finding test:
TA98, TA100, TA1535, TA1537, WP2uvrA: 62, 185, 556, 1667, 5000 μg/plate (S9 mix(-) and S9 mix(+))
TA1535 : 0.3, 0.8, 2, 7, 21, 62 μg/plate (S9 mix(-))

Main test:
TA98, TA1537 : 0.8, 2, 7, 21, 62, 185 μg/plate (S9 mix(-))
TA100, WP2uvrA : 7, 21, 62, 185, 556, 1667 μg/plate (S9 mix(-))
TA1535 : 0.3, 0.8, 2, 7, 21, 62 μg/plate (S9 mix(-))
TA98 : 0.8, 2, 7, 21, 62, 185 μg/plate (S9 mix(+))
TA100, WP2uvrA : 7, 21, 62, 185, 556, 1667 μg/plate (S9 mix(+))
TA1535, TA1537 : 2, 7, 21, 62, 185, 556 μg/plate (S9 mix(+))

Rational for top dose:
Cytotoxicity and test substance precipitation (see also remarks on results)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Solubility data provided by Doosan corp confirmed that the test substance (DSPhytosphingosine) was insoluble in water (Annex 2). Prior to planning of this study, the solubility of the test substance was assessed. The test substance was dissolved in DMSO at 20 % concentration.
Controls
Untreated negative controls:
yes
Remarks:
solvent
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
sodium azide
benzo(a)pyrene
other: 9-Aminoacridine hydrochloride hydrate, 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

Test condition
Composition
Bacterial suspension 0.1 ㎖
Test substance 0.05 ㎖
0.1M Phosphate buffered saline
(direct method) 0.5 ㎖
S9 mix
(metabolic activation method) 0.5 ㎖
Top agar 2.0 ㎖
Preincubation
Temperature 37 ℃
Time 20 min
Incubation
Temperature 37 ℃
Time 48 hours


NUMBER OF REPLICATIONS: 3 plates


3) Method of observation, measurement and analysis
(1) Colony counting
The number of revertants were counted only inside area of plate (diameter : 90 mm)
by manual measurement using electronic register (Model 570, SUNTEX, Taiwan).
(2) Test method of growth inhibition
All plate was observed with the naked eye.
(3) Measurement of bacterial concentration
It was performed by step-dilution method according to the Standard Operation Procedure,
KCL.
(4) Sterility test
0.1 ml of S9 mix and test substance were mixed with top agar respectively, and
then, it was added onto minimum glucose agar plate. The plate was
incubated at 37℃ for 48 hours.


DETERMINATION OF CYTOTOXICITY : method: decrease in the number of revertant colonies
Rationale for test conditions:
according to guideline
Evaluation criteria:
If no increase in mutant colonies is seen after testing several strains under several different culture conditions, the test chemical is considered to be non-mutagenic in the bacterial reverse mutation assay.
Statistics:
Mean ± Standard deviation

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
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:
valid
Positive controls validity:
valid
Additional information on results:
Range finding test
Preliminary range-finding test was performed to determine dose levels at the main test. Preliminary range-finding test was carried out on 62, 185, 556, 1667, 5000 µg/plate (three-fold serial dilutions).
As a result of preliminary range-finding test, the test substance precipitation was observed at all strains in the presence and absence of metabolic activation system (TA98, TA100, TA1535, TA1537, WP2uvrA were observed at 1667, 5000 µg/plate), which resulted in the inability to count the colony at 1667, 5000 µg/plate. Also, cytotoxicity was observed at TA98, TA1535 and TA1537 strains in the presence (TA98 : 185 ~ 556 /plate, TA1535, TA1537 : 556 /plate) and absence (TA98, TA1537 : 185 ~ 556 /plate, TA1535 : 62 ~ 556 µg /plate) of metabolic activation system.
Increase in the number of revertant colonies was not seen at TA98, TA100, TA1535, TA1537 and WP2uvrA strain compared with negative control groups in the presence and absence of metabolic activation system.
Cytotoxicity was observed at TA1535 strain at the lowest concentration in the absence of metabolic activation system, and therefore the concentrations could not be determined for the main test. For this reason, the preliminary range-finding test for TA1535 strain was carried out again on 0.3, 0.8, 5, 7, 21, 62 µg/plate in the absence of metabolic activation system (retest). As a result of preliminary range-finding test (retest), the cytotoxicity was observed at 62 µg/plate in the absence of metabolic activation system.

Main study
As a result of the main test, test substance precipitation was observed at 1667 µg/plate in the presence and absence of metabolic activation system at TA100 and WP2uvrA, which resulted in the inability to count the colony at 1667 µg/plate. Cytotoxicity was observed at TA98, TA1535 and TA1537 strains in the presence (TA98 : 185 µg/plate, TA1535, TA1537 : 556 µg/plate) and absence (TA98, TA1537 : 85 µg/plate, TA1535 : 62 µg/plate) of metabolic activation system. No significant increase in the number of revertant colonies was seen in TA98, TA100, TA1535, TA1537 and WP2uvrA strain compared with negative control groups in the presence and absence of metabolic activation system. Under the experimental conditions, Phytosphingosine did not induce bacterial reverse mutation.

Validity
The number of revertant colonies of positive control groups and negative control groups were within or close to the range of the historical data, and no contamination was observed in the sterility test.

Any other information on results incl. tables

Table. Result of bacterial reverse mutation assay with Phytosphingosine

Strain

Chemical treated

S9Mix(-)

S9Mix(+)

Dose

(/plate)

Number of colony/plate (Mean±SD)

Dose

(/plate)

Number of colony/plate (Mean±SD)

TA100

Test item

0

72 ± 1.7

0

68 ± 2.0

7

69 ± 1.0

7

70 ± 2.5

21

74 ± 1.7

21

62 ± 1.0

62

77 ± 2.3

62

74 ± 2.5

185

72 ± 1.0

185

62 ± 1.7

556

62 ± 1.5

556

56 ± 1.5

1667

U/C ± U/C

1667

U/C ± U/C

TA1535

Test item

0

8 ± 1.0

0

12 ± 1.0

0.3

8 ± 1.5

2

13 ± 1.5

0.8

7 ± 1.5

7

12 ± 0.6

2

6 ± 1.5

21

14 ± 1.0

7

6 ± 1.5

62

14 ± 0.6

21

5 ± 1.5

185

11 ± 2.5

62

P/C ± P/C*

556

5 ± 2.0*

WP2uvrA

Test item

0

47 ±2.1

0

45 ± 1.0

7

45 ±0.6

7

46 ± 2.6

21

47 ± 1.0

21

44 ± 1.7

62

46 ± 2.6

62

43 ± 1.0

185

48 ± 1.5

185

51 ± 2.1

556

58 ± 1.5

556

52 ± 2.1

1667

U/C ± U/C

1667

U/C ± U/C

TA98

Test item

0

19 ± 2.5

0

24 ± 1.0

0.8

24 ± 1.5

0.8

25 ± 2.0

2

21 ± 2.3

2

28 ± 1.0

7

21 ± 1.0

7

29 ± 2.1

21

22 ± 1.5

21

29 ± 1.0

62

14 ± 2.5

62

26 ± 1.5

185

7 ± 2.1*

185

10 ± 2.3*

TA1537

Test item

0

6 ± 1.5

0

16 ± 1.2

0.8

6 ± 0.6

2

17 ± 2.5

2

6 ± 0.6

7

18 ± 2.1

7

6 ± 0.6

21

19 ± 1.7

21

6 ± 1.2

62

16 ± 1.5

62

4 ± 0.6

185

13 ± 1.2

185

2 ± 1.2*

556

7 ± 2.0*

Positive controls

TA100

AF-2

0.01

505 ± 86.9

 

 

2-AA

 

 

1.0

337 ± 43.1

TA1535

NaN3

0.5

341 ± 77.1

 

 

2-AA

 

 

2.0

220 ± 2.1

WP2uvrA

4-NQO

0.25

433 ± 81.5

 

 

2-AA

 

 

10.0

381 ± 62.1

TA98

AF-2

0.1

376 ± 21.6

 

 

BP

 

 

10.0

485 ± 71.6

TA1537

9-AA

80.0

2818 ±81.1

 

 

2-AA

 

 

2.0

209 ±1.5

† : Test substance precipitation

P/C : Pinpoint colony

U/C : Uncountable

* ; Cytotoxicity

Applicant's summary and conclusion

Conclusions:
Phytosphingosine did not induce bacterial reverse mutation. Results for all bacteria strains were negative under the experimental conditions.
Executive summary:

The potential of Phytosphingosine to induce reverse mutation in four histidine-requiring strains of Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and a tryptophan-requiring strain of Escherichia coli WP2uvrA was evaluated according to OECD 471 (adopted 1997) and in compliance with GLP.

The test substance was dissolved in dimethyl sulfoxide (DMSO) and performed using preincubation method with and without metabolic activation system. Preliminary range-finding test was performed to determine dose levels at the main test. In the main test, substance precipitation was observed at 1667 µg/plate in the presence and absence of metabolic activation system at TA100 and WP2uvrA, which resulted in the inability to count the colony at 1667 µg/plate. Cytotoxicity was observed at TA98, TA1535 and TA1537 strains in the presence (TA98 : 185 µg/plate, TA1535, TA1537 : 556 µg/plate) and absence (TA98, TA1537 : 85 µg/plate, TA1535 : 62 µg/plate) of metabolic activation system. No significant increase in the number of revertant colonies was seen in TA98, TA100, TA1535, TA1537 and WP2uvrA strain compared with negative control groups in the presence and absence of metabolic activation system. Considering these findings, it was concluded that the test substance phytosphingosine did not induce reverse mutation in 5 strains, which were used in this study.

The study was considered valid, because the number of revertant colonies of positive control groups and negative control groups were within or close to the range of the historical data and no contamination was observed in the sterility test. Hence, the study was considered adequate and reliable for hazard assessment.

In conclusion, phytosphingosine was negative in the presence and absence of metabolic activation in all bacterial strains. Under the experimental conditions, phytosphingosine did not induce bacterial reverse mutation