Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 May 2020 to 29 June 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
This study was performed to investigate the potential of test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation method (Trial I), and the pre-incubation method (Trial II) using the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Decan-5-olide
EC Number:
211-889-1
EC Name:
Decan-5-olide
Cas Number:
705-86-2
Molecular formula:
C10H18O2
IUPAC Name:
6-pentyltetrahydro-2H-pyran-2-one
Test material form:
liquid
Remarks:
Colorless clear liquid
Details on test material:
Name of test material:
- CAS No. 705-86-2
- EC No. 211-889-1
- Molecular formula: C10H18O2
- Molecular weight: 170.25 g/mol
- Subsatnce type: Organic
- Physical state: Colorless clear liquid
- Purity: 98.4% (GC)

Method

Target gene:
Histidine Operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Not applicable
Cytokinesis block (if used):
Not applicable
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: Aroclor 1254 induced S9 was procured from Defence Research and Development Establishment, Nagpur (India)
- method of preparation of S9 mix : Appropriate quantity of S9 supernatant was mixed with S9 cofactor solution which contains D-glucose-6-phosphate 0.8 g, β-NADP 1.75 g, MgCl2 1.0 g, KCl 1.35 g, Na2HPO4 6.4 g, NaH2PO4.H2O 1.4 g in 500 ml of distilled water
- concentration or volume of S9 mix and S9 in the final culture medium : 10% v/v
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data available
Test concentrations with justification for top dose:
0,0 (NC), 0.0 (VC) 0.050, 0.158, 0.501, 1.582, and 5.000 mg/plate. Based on the results of pre-experiment, the doses were selected.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was solulble in DMSO
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Negative control : Distilled water Solvent Control : Dimethyl sulfoxide
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-Nitro-o-phenylenediamine for strains TA1537, TA98 (without metabolic activation) 2-Aminoanthracene for strains TA 1535, TA1537, TA98, TA 100 and TA102
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data
Rationale for test conditions:
No data available
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA98, TA100, and TA102) or thrice (strains TA1535 and TA1537) the colony count of the corresponding vehicle/(solvent) control is observed.
A dose-dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose-dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control and vehicle control, such an increase is not considered biologically relevant.

Statistics:
No data available

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA98, TA100, TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No data available
- Data on osmolality: No data available
- Possibility of evaporation from medium: No data available
- Water solubility: Insoluble in water
- Precipitation: No precipitation was observed in 5 mg/plate concentration

- Definition of acceptable cells for analysis: No data availble
- Other confounding effects: No data availble

RANGE-FINDING/SCREENING STUDIES (if applicable):
To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0.0 (NC), 0.0 (VC), 0.001, 0.005, 0.015, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate) were tested for cytotoxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test).
In treated concentrations, 0.001-0.501 mg/plate (T1-T6), no reduction in colony count or clearing of the background lawn, in treated concentration 1.582 mg/plate (T7) no reduction in colony count and background lawn inhibition was observed. However, at concentration 5.000 mg/plate (T8) reduction in colony and background lawn inhibition was observed both in presence (+S9) and in absence (-S9) of metabolic activation.

STUDY RESULTS
- Positive control data : Positive controls induced an unequivocal increase in revertant counts in all the five tester strains compared to respective controls used.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Please refer the table.
- Negative (solvent/vehicle) historical control data: Please refer the table
Remarks on result:
other: No mutagenic potential

Any other information on results incl. tables

TABLE 4: MEAN REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIAL-I)

Dose (mg/plate)

In the Presence (+S9) of Metabolic Activation

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

4.67

0.58

9.67

1.53

18.33

3.06

105.00

2.65

234.00

6.00

VC

(0.00)

7.00

1.00

14.33

1.53

30.67

2.52

140.00

2.65

290.67

5.51

T1

(0.050)

5.67

1.15

13.33

1.15

28.33

3.51

118.00

2.00

281.67

13.01

T2

(0.158)

6.00

1.00

13.67

2.08

26.67

2.52

123.33

3.06

254.33

15.18

T3

(0.501)

6.33

1.53

10.67

1.15

22.00

2.65

118.67

3.51

245.00

8.89

T4

(1.582)

5.33

0.58

12.33

1.53

21.33

3.51

122.33

2.52

276.33

8.62

T5

(5.000)

4.33

0.58

9.33

1.53

15.67

3.06

96.67

3.51

245.33

6.03

PC

166.67

14.05

384.00

56.00

792.00

36.66

1106.67

44.06

1696.00

81.19

Dose

(mg/plate)

In the Absence (-S9) of Metabolic Activation

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

3.33

0.58

9.00

1.00

16.00

2.65

93.67

1.53

233.67

7.09

VC

(0.00)

5.67

0.58

15.33

1.53

28.33

2.52

126.67

2.08

294.33

10.41

T1

(0.050)

4.67

0.58

12.67

1.15

26.33

2.52

99.33

3.06

261.67

7.02

T2

(0.158)

5.00

1.00

12.33

1.53

22.67

3.51

115.00

2.65

241.00

11.14

T3

(0.501)

5.33

0.58

13.67

1.53

22.00

2.00

120.00

3.00

259.67

10.69

T4

(1.582)

4.33

0.58

11.33

1.53

24.67

1.53

111.33

2.52

237.67

13.50

T5

(5.000)

3.67

1.15

8.67

1.15

14.33

2.08

84.33

3.06

240.00

11.14

PC

124.00

12.00

1040.00

36.66

510.67

16.65

964.00

52.46

1632.00

63.50

Key:-

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),R= Replicate

PC= Positive control                                                                     

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA 98, TA 100        
2- Aminoanthracene [10μg/plate]: TA 102                                           

Sodium azide [10μg/plate]: TA 1535, TA 100                                            

4-Nitro-o-phenylenediamine: TA 1537 [50μg/plate], TA 98 [10μg/plate]    

Methyl methanesulfonate [4μl/plate]: TA 102

TABLE 5 : MEAN REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL-II)

Dose

(mg/plate)

In the Presence (+S9) of Metabolic Activation

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

4.67

0.58

10.00

1.00

22.33

2.08

110.67

2.08

269.00

10.82

VC

(0.00)

6.67

0.58

16.33

1.53

28.67

1.53

123.33

3.06

289.00

6.56

T1

(0.050)

6.00

1.73

12.33

1.53

25.33

1.53

118.33

2.08

265.00

8.19

T2

(0.158)

5.67

1.53

11.67

1.15

23.33

1.53

116.33

2.52

285.00

9.54

T3

(0.501)

6.33

1.53

12.67

1.53

24.67

1.53

120.67

2.08

272.67

10.02

T4

(1.582)

5.00

1.00

14.00

1.00

23.00

2.00

115.33

1.53

284.00

6.56

T5

(5.000)

5.33

1.15

10.33

1.53

22.00

2.65

121.33

1.53

261.33

6.51

PC

134.67

6.11

264.00

28.84

880.00

58.10

1549.33

66.61

1392.00

48.00

Dose

(mg/plate)

In the Absence (-S9) of Metabolic Activation

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

4.67

0.58

9.00

1.73

18.33

2.52

107.33

2.08

262.33

7.77

VC

(0.00)

6.67

1.15

15.67

1.53

29.33

3.06

126.33

1.53

286.00

6.00

T1

(0.050)

6.00

1.00

14.33

2.08

24.00

3.00

123.00

2.00

267.33

9.45

T2

(0.158)

5.33

0.58

11.00

1.73

23.33

1.53

115.00

2.65

274.33

7.77

T3

(0.501)

6.33

1.53

13.67

0.58

25.67

2.52

121.00

2.00

272.00

9.17

T4

(1.582)

5.67

1.15

10.00

1.00

21.33

2.31

119.67

2.52

280.33

5.51

T5

(5.000)

4.33

0.58

12.33

1.53

20.33

1.53

116.33

1.53

277.00

5.57

PC

153.33

6.11

968.00

44.54

580.00

47.16

1064.00

57.69

1418.67

64.17

Key:-NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),R= Replicate

PC= Positive control                                                                     

 2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA 98, TA 100        
2- Aminoanthracene [10μg/plate]:TA 102                                            

Sodium azide [10μg/plate]: TA 1535, TA 100                                            

4-Nitro-o-phenylenediamine: TA 1537 [50μg/plate], TA 98 [10μg/plate]    

Methyl methanesulfonate [4μl/plate]: TA 102

 

Applicant's summary and conclusion

Conclusions:
The test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.
Executive summary:

The Ames study was performed to investigate the potential of test chemical to induce gene muta­tions in comparison to vehicle control according to the plate incorporation method (Trial I), and the pre-incubation method (Trial II) using the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102.

The pre-experiment was performed with TA100 and TA98 strain of Salmonella typhimurium and with eight concentrations spaced by (√10) half-log intervals in triplicates. 5.0 mg/ plate was selected as the highest dose in the pre-experiment based on the solubility and precipitation test. The following doses were selected for the pre-experiment 0.001, 0.005, 0.015, 0.050, 0.158, 0.501, 1.582 and 5.000 mg/plate. In TA98 strain and TA100 strain at concentrations of 0.001-0.501 mg/plate (T1-T6), no reduction in colony count and/or clearing of the background lawn, in treated concentration 1.582 mg/plate (T7) no reduction in colony count and background lawn inhibition was observed. However, at concentration 5.000 mg/plate (T8) reduction in colony count and background lawn inhibition was observed both in the absence (-S9) and in the presence (+S9) of metabolic activation.

Based on the pre-experiment results, the test item was tested with the following concentrations 0.050, 0.158, 0.501, 1.582, and 5.000 mg/plate for the main study, both in the presence (+S9) and absence (-S9) of metabolic activation.

Trial I :

Trial-I was performed with five concentrations of test item along with the negative, vehicle, and concurrent positive controls with the remaining three strains, i.e., TA1537, TA1535, and TA102 by the plate incorporation method. For TA98 and TAl00 the revertant colony counts were directly incorporated in the Trial-I from the pre-experiment up to the required five concentrations [T4 (0.050 mg/plate) to T8 (5.000 mg/plate)].

The concentrations 0.050, 0.158, 0.501, 1.582, and 5.000mg/plate, both in the absence (-S9) and in presence (+S9) of metabolic activation of test item were prepared with (√10) half-log interval The plates were treated and incubated at 37 °C for 48 hours (approximately). No substantial increase in the revertant count in any of the five strains was reported at any concentrations tested.

Positive controls induced an unequivocal increase in revertant counts in all the five tester strains compared to respective controls used.

Trial II :

Trial-II was performed independently with all the five tester strains along with the negative, vehicle, and positive controls by pre-incubation method for the confirmation of the Trial-I results.

The concentrations 0.050, 0.158, 0.501, 1.582, and 5.000 mg/plate both in the absence (-S9) and in presence (+S9) of metabolic activation of test item were prepared by (√10) half-log intervals.The concentration of positive controls used was the same as used in the plate incorporation method. The test item, negative, vehicle, and positive controls were pre-incubated along with 500 µL of metabolic activation mix and 100µL of bacterial culture for 60 minutes at 37°C in an incubator.

After pre-incubation, 2 mL of top agar was mixed with the pre-incubation mixture and poured on minimal glucose agar plates. The treated plates were incubated for 48 hours (approximately) in an incubator.

No substantial increase in the revertant count was observed in any of the five tester strains pre-incubated with the test item. The positive controls showed an unequivocal increase in revertant counts with all the five tester strains compared to the respective control used.

From the result obtained it can be concluded that the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

 


Categories Display