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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

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.

In vitro mammalian chromosome aberration study:

The test chemical did not induce gene mutation by chromosomal aberration in human peripheral blood lymphocyte both in presence (1% and 2%) and in the absence of metabolic activation under the specified conditions.Hence it is not likely to be classified as a gene mutant as per the criteria mentioned in CLP regulation.

In vitro mammalian cell gene mutation assay:

The given test chemical does not give rise to gene mutations when exposed at ≤ 5.0 mM for 3 hrs or more, and it does not give rise to cytotoxicity in CHO cells at concentrations of ≤ 5.0 mM.

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:
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
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
Target gene:
Histidine Operon
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
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
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

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

 

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.

 


Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 December 2019 to 10 March 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
Principles of method if other than guideline:
This in vitro assay was performed to assess the potential of test chemical to induce structural / numerical chromosomal aberrations in one experiment (Phase I). The induction of cytogenetic damage in human lymphocytes was assessed with and without metabolic activation. Due to the negative result in Phase I, a second experiment (Phase II) was performed.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: Human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Human blood
- Suitability of cells: No data

For lymphocytes:
- Sex, age and number of blood donors: 24 to 33 years of age range
- Whether whole blood or separated lymphocytes were used: Seperated lymphocytes
- Whether blood from different donors were pooled or not: No data

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: No data
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically 'cleansed' against high spontaneous background: No data
Cytokinesis block (if used):
No data available
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
- method of preparation of S9 mix : Appropriate quantity of S9 supernatant was mixed with S9 cofactor solution, which contains 0.80 g of D-glucose-6-phosphate, 1.00 g of MgCl2, 1.35 g of KCl, 6.40 g of Na2HPO4, 1.40 g of NaH2PO4.H2O, 1.75 g of β-NADP in 500 mL of RO water. During the experiment, S9 mix was freshly prepared.
- concentration or volume of S9 mix and S9 in the final culture medium: 1 % v/v for Phase I of experiment and 2 % v/v for Phase II of experiment.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data
Test concentrations with justification for top dose:
0.250, 0.5 and 1.0 mg/mL. On the basis of cytotoxicity study the doses were selected. (Both in presence and in absence of metabolic activation system).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Negative control : Distilled water Vechile control : DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: Cyclophosphamide monohydrate
Details on test system and experimental conditions:
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure.: Colcemide (0.3 µg/mL) was added 3 hour prior to harvesting and kept under incubation at 37 ± 2 °C

- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried on the slide warmer and labelled. Two slides were made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX. All slides, including those of positive, vehicle and negative controls, were independently coded before microscopic analysis.

- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 1000 cells per slides

- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverization, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46  2 centromere regions were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) were determined.

- Determination of polyploidy: Yes

- Determination of endoreplication: Yes


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: Mitotic Index

- Any supplementary information relevant to cytotoxicity: To evaluate the toxicity of the test item a cytotoxicity assay was performed both in the presence and absence of metabolic activation system. Test concentrations (0.0 (NC), 0.0 (VC), 0.5 (T1), 1.0 (T2) and 2.0 (T3) mg/mL of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with vehicle control.


- OTHER: No data
Rationale for test conditions:
No data available
Evaluation criteria:
A test item can be classified as clastogenic if:
 At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
 If the increase is dose-related
 Any of the results are outside the historical vehicle control range
A test item can be classified as non - clastogenic if:
 None of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
 If there is no dose-related increase
 All results are inside the historical vehicle control range
Statistical significance was confirmed by means of the non-parametric Mann-Whitney Test. However, both biological and statistical significance were considered together.
If the above-mentioned criteria for the test item were not clearly met, the classification with regard to the historical data and the biological relevance was discussed and/or a confirmatory experiment was performed.
Statistics:
Statistical significance at the p < 0.05 was evaluated by means of the non-parametric Mann-Whitney test.
Species / strain:
lymphocytes: Human peripheral lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At concentrations of 2 (T3) mg/mL both in the absence and in the presence of metabolic activation (1%).
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: The pH of test item in culture medium was assessed at 0 h and 4 h after incubation at 37 ± 2 °C. No significant change in pH was observed at 0 h and 4 h when compared with negative controls.
- Data on osmolality: No data
- Possibility of evaporation from medium: No data
- Water solubility: Insoluble in water
- Precipitation and time of the determination: Since, Precipitation was not observed at 2 mg/ml it was taken as high concentration for the treatment in cytotoxicity experiment.
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES (if applicable):
To evaluate the toxicity of test item cytotoxicity was performed both in presence and in absence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.0 (NC), 0.0 (VC), 0.5 (T1), 1.0 (T2) and 2.0 (T3) mg/mL of culture media. Cytotoxicity was observed in the treated concentrations of 2 (T3) mg/mL both, in the absence and in the presence of metabolic activation (1%). In the absence of S9 mix, the mean mitotic index observed was 10.05 (NC), 9.93 (VC), 5.43 (T1), 4.19 (T2), 3.58 (T3) and 8.20 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.19 (NC), 9.92 (VC), 5.49(T1), 4.47 (T2), 3.03 (T3) and 8.49 (PC). In the cytotoxicity experiment, the test concentration 2 (T3) mg/ mL of culture media showed more than 50% reduction in mitotic index when compared to the respective vehicle control both in the presence or absence of metaboli activation. Hence these concentrations was not selected for the main study due to extensive cytotoxicity evoked.
Hence, 1.0 mg/mL of culture media was selected as the highest concentration for main study both in the presence and in the absence of metabolic activation.


Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For lymphocytres in primary cultures: mitotic index (MI) : In the absence of S9 mix, the mean mitotic index observed was 10.05 (NC), 9.93 (VC), 5.43, (T1), 419 (T2), 3.58 (T3) and 8.20 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.195 (NC), 9.92 (VC), 5.49 (T1), 4.47 (T2), 3.03 (T3) and 8.49 (PC).
o For cell lines: relative population doubling (RPD), relative Increase in cell count (RICC), number of cells treated and cells harvested for each culture, information on cell cycle length, doubling time or proliferation index : No data

- Genotoxicity results (for both cell lines and lymphocytes): Genotoxicity negative

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 in remark section
- Negative (solvent/vehicle) historical control data: Please refer the table in remarks section.
Remarks on result:
other: No mutagenic potential

Cytotoxicity was assessed at the concentrations of 0.0 (NC), 0.0 (VC), 0.5 (T1),1.0 (T2) and N2.0 (T3) mg/mL of culture media. Cytotoxicity was observed in treated concentrations of 2 (T3) mg/mL both in the absence and in the presence of metabolic activation (1%). In the absence of S9 mix, the mean mitotic index observed was 10.05 (NC), 9.93 (VC), 5.43 (T1), 4.19 (T2), 3.58 (T3) and 8.20 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.19 (NC), 9.92 (VC), 5.49 (T1), 4.47 (T2), 3.03 (T3) and 8.49 (PC).

In the cytotoxicity experiment, the test concentrations 2 (T3) mg/ mLof culture media showed more than 50% reduction the mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation confirms the cytotoxicity effect. Hence, this concentration was not selected for the main study due to the extensive cytotoxicity evoked.

Thereby, 1.0 mg/mL of culture media was selected as the highest concentration for main study both in the presence and in the absence of metabolic activation.

The main study was performed in two independentphases as described below.

Phase I:      

In the experiment, the cultures were exposed to Decan-5-olide (CAS No. 705-86-2) for 4 hours both in the absence and in the presence of metabolic activation system

(1%).The mean percentage of aberrant cells was 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.000 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 11.000 (PC) in the presence of metabolic activation at the concentration of 0.0 (NC), 0.0 (VC) 0.062 (T1), 0.125 (T2) and 0.250 (T3) mg/mL and positive controls.

Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamide monohydrate at the concentration of 30 µg/mL in the presence of metabolic activation (1%) caused significant increases in percent aberrant cells (≥10.33% aberrant cells). Even though the analysis did not reveal any statistical significance, the increases were biologically significant.

During the treatment with the test item both in the absence and presence of S9 mix, there was slight to moderate reductions of mitotic index observed at low (0.250 mg/mL) and middle (0.5 mg/mL) dosesThe observed mean mitotic index in the absence of metabolic activation were 10.03, 9.79,6.64,5.11,4.30and 8.73and in the presence of metabolic activation were 10.18, 9.89,6.80,5.59,4.10and 8.69for 0.0 (NC), 0.0 (VC) 0.250(T1), 0.5 (T2) and 1.0(T3) mg/mL and  30 µg/mL(+S9), 600 ug/ml(-S9) of PC concentration.

Phase II:

The phase II experiment was performed to confirm the negative results obtained in the absence and in the presence of metabolic activation in Phase I. In the absence of metabolic activation, test items concentrations used were 0.0 (NC), 0.0 (VC) 0.250 (T1), 0.5 (T2) and 1.0 (T3) mg/mL and 600 µg/mL (PC; ethylmethanesulfonate). In the presence of metabolic activation (2%),test items concentrations used were 0.0 (NC), 0.0 (VC) 0.250 (T1), 0.5 (T2) and 1.0 (T3) mg/mL and 30 µg/mL(PC; cyclophosphamidemonohydrate).The duration of exposure to the test item in presence of metabolic activation system was 4 hours and in absence of metabolic activation was 24 hours. The mean percent aberrant cells were 0.333 (NC), 0.333 (VC) 0.333 (T1), 0.667 (T2), 0.667 (T3) and 10.333 (PC) in the absence of metabolic activation and 0.333 (NC), 0.667 (VC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.000 (PC) in the presence of metabolic activation at the concentration of 0.0 (NC), 0.0 (VC) 0.250 (T1), 0.5 (T2) and 1.0 (T3) mg/mL of culture and positive control.

Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamide monohydrate at the concentration of 30 µg/mL in the presence of metabolic activation (2%) caused significant increases in percent aberrant cells (≥10.00% aberrant cells) .Though the analysis did not reveal any statistical significance, the increases were biologically significant.

The increased frequency of aberrations observed in the concurrent positive control groups (Phase I and II) demonstrated the sensitivity of the test system, suitability of the methods and conditions employed in the experiment.

Conclusions:
The test chemical did not induce gene mutation by chromosomal aberration in human peripheral blood lymphocyte both in presence (1% and 2%) and in the absence of metabolic activation under the specified conditions. Hence it is not likely to be classified as a gene mutant as per the criteria mentioned in CLP regulation.
Executive summary:

The in vitro mammalian chromosomal aberration study was conducted to determine the chromosomal aberration induction potential of test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on29thJuly 2016.

The experiment was conducted using human peripheral blood lymphocytes. Blood was drawn from a healthy volunteer, by venous puncture using heparinised syringe. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 hours mitogenic stimulation. The cells were treated with metaphase arresting substance (colcemid) 3 hours prior to harvesting and stained. The slides were analysed with the aid of microscope and a minimum number of 1000 of cells per culture were counted and number of metaphases were recorded in different fields to determine the mitotic index. In total, 300 well-spread metaphases per concentration and control were scored for chromosomal aberrations.

Based on the solubility and precipitation test, Dimethyl sulfoxide (DMSO) was selected as the vehicle for treatment. The pH of test item in culture medium was assessed at 0 hour and 4 hours after incubation at 37°C. No significant change in pH was observed at 0 hour and 4 hours when compared with negative control. Hence, 2.0 mg/mL was selected as the highest concentration for cytotoxicity test.

Before conducting the chromosomal aberration study, test chemical was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.0 (NC), 0.0 (VC), 0.5 (T1),1.0 (T2) and 2.0 (T3) mg/mL of culture media. Cytotoxicity was observed in the treated concentration of 2.0(T3) mg/mL both in the absence and in the presence of metabolic activation (1%).

In the absence of S9 mix, the mean mitotic index observed was 10.05 (NC), 9.93 (VC), 5.43 (T1), 4.19 (T2), 3.58 (T3) and 8.20 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.19 (NC), 9.92 (VC), 5.49 (T1), 4.47 (T2), 3.03 (T3) and 8.49 (PC).

The test concentration 2.0(T3) mg/ mL showed more than 60% reduction on the mitotic index in treated culture both in the presence or absence of metabolic activation when it was compared to the respective vehicle control, which confirms extensive cytotoxicity effect. Thereby, this concentration was not selected for the main study.

Hence, 1.0 mg/mL of culture media was selected as the highest concentration for main study both in the presence and in the absence of metabolic activation.

The main study was performed in two independent phases:

In Phase I experiment, the cultures were exposed to the test chemical for a short period of time (4 hours) both in the absence and in the presence of metabolic activation system (1%).The mean percentage of aberrant cells was 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.667 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.333 (PC)in the presence of metabolic activation at the concentration of 0.0 (NC), 0.0 (VC) 0.250 (T1), 0.5 (T2) and 1.0 (T3) mg/mL and positive controls, respectively.

Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamide monohydrate at the concentration of 30 µg/mL in the presence of metabolic activation (1%) caused significant increases in percent aberrant cells (≥10.33% aberrant cells). Even though the analysis did not reveal any statistical significance, the increases were biologically significant.

During thetreatment with the test item both in the absence and presence of S9 mix, there were slight to moderate reductions of mitotic index observed at low (0.250 mg/mL) and middle (0.5 mg/mL) doses. The observed mean mitotic indexin the absence of metabolic activation were 10.03, 9.79,6.64,5.11,4.30and 8.73 and in the presence of metabolic activation were 10.18, 9.89,6.80,5.59,4.10 and 8.69 for 0.0 (NC), 0.0 (VC) 0.250 (T1), 0.5 (T2) and 1.0 (T3) mg/mL and 30 µg/mL (+S9), 600 ug/ml (-S9) of PC concentrations.

The Phase II experiment was performed to confirm the negative results obtained in the absence and in the presence of metabolic activation in Phase I. In the absence of metabolic activation, test items concentrations used were 0.0 (NC), 0.0 (VC) 0.250 (T1), 0.5 (T2) and 1.0 (T3) mg/mL and 600 µg/mL (PC; ethylmethanesulfonate). In the presence of metabolic activation (2%), test items concentrations used were 0.0 (NC), 0.0 (VC) 0.250 (T1), 0.5 (T2) and 1.0 (T3) mg/mL and 30 µg/mL (PC; cyclophosphamide monohydrate).The duration of exposure to the test item in presence of metabolic activation system was 4 hours and in absence of metabolic activation was 24 hours. The mean percent aberrant cells were 0.333 (NC), 0.333 (VC) 0.333 (T1), 0.667 (T2), 0.667 (T3) and 10.333 (PC) in the absence of metabolic activation and 0.333 (NC),0.667 (VC),0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.000 (PC) in the presence of metabolic activation at the concentration of 0.0 (NC), 0.0 (VC) 0.250 (T1), 0.5 (T2) and 1.0 (T3) mg/mL of culture and positive control.

Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamide monohydrate at the concentration of 30 µg/mL in the presence of metabolic activation (2%) caused significant increases in percent aberrant cells (≥10.00% aberrant cells) .Though the analysis did not reveal any statistical significance, the increases were biologically significant.

The increased frequency of aberrations observed in the concurrent positive control groups (Phase I and II) validated the sensitivity of the test system, suitability of the methods and conditions employed in the experiment.

Treatment with test item both in the absence and presence of S9 mix induced slight to moderate reductions of mitotic index at low (0.250 mg/mL) and middle (0.5 mg/mL) doses. The observed mean mitotic index in the absence of metabolic activation were 10.13, 9.90,6.40,5.05,4.20and 8.29 andin the presence of metabolic activation were 10.03, 9.90,6.14,5.35,4.37and 8.68 for 0.0 (NC), 0.0 (VC) 0.250 (T1), 0.5 (T2) and 1.0 (T3) and 30 µg/mL(+S9), 600 ug/ml(-S9) of PC concentrations, respectively.

Hence from the above data it can be concluded that, the test chemical was found to be not mutagenic for chromosomal aberration in human peripheral blood lymphocyte both in presence (1% and 2%) and in the absence of metabolic activation under the specified conditions.Hence it is not likely to be classified as a gene mutant as per the criteria mentioned in CLP regulation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
The purpose of this study was to assess toxic and genotoxic effects of test chemical on Chinese Hamster Ovary (CHO) cells by using several different in vitro-based assays, including genotoxicity tests based on the OECD Guideline No. 476 “In Vitro Mammalian Cell Gene Mutation Test”.
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot. This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.HPRT catalyses the transformation of the purine analogue 6-thioguanine (TG) and thus rendering the analogue cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham’s F12K (Kaighn’s) Medium including 2 mM Glutamine supplemented with 10% Foetal Bovine Serum (FBS; qualified, EU-approved, South America origin), penicillin (50 U/ml) and streptomycin (50 µg/ml).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No detection for mycoplasma was performed
- Periodically checked for karyotype stability: No detection for karyotype stability was performed
Additional strain / cell type characteristics:
other: Hypodiploid, modal No. 20
Cytokinesis block (if used):
Not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany)
Test concentrations with justification for top dose:
0, 0.5, 1.0, 2.5 or 5.0 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle:Phenethyl phenylacetate was easily dissolved in ethanol.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
other: N-ethyl-N-nitrosourea (ENU)
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium with pre-incubation
DURATION- Preincubation period:One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.
- Exposure duration:3 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days (harvest of cells)
SELECTION AGENT (mutation assays): 6-thioguanine (TG)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Crystal violet
NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.
NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.
DETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.
- OTHER:
Rationale for test conditions:
No data
Evaluation criteria:
The plates were scored for total number of colonies by manual counting. As a result, the mutation frequency could be calculated
Statistics:
Mean were observed.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No data
Remarks on result:
other: No mutagenic effect were observed.

GENE MUTATION TEST

 

Table 1A.Effect of 5-Decanolide exposure on gene toxicity in CHO cells. After being exposed to the test chemical for 3 hrs, cells was washed with sterile PBS and then incubated for 7 days at 37°C, 5% CO2. After 7 days, cells were re-seeded in new 6-well plates in the absence or presence of 10mM TG as a selection agent and returned to the incubator for 14 days at 37°C, 5% CO2. On day 15, all 6-well plates were stained with crystal violet and the number of colonies were counted manually. The results are presented as the total number of colonies found in the number of independent wells analyzed (e.g. 0 colonies in 4 wells will give 0/4) (n = 2 samples from 2 independent cultures).

 

 

With S9

Without S9

 

with TG

without TG

with TG

without TG

Neg. control

0/4

773/4

0/4

856/4

Pos. control

0/4

788/4

36/4

769/4

0.5 mM

0/4

773/4

0/4

720/4

1.0 mM

0/4

603/4

0/4

747/4

2.5 mM

0/4

666/4

0/4

725/4

5.0 mM

0/4

611/4

0/4

403/4

 

 

 

Table 1B.Mutation frequency in CHO cells after 3 hrs of exposure to 5-Decanolide in the absence or presence of 4% S9 liver microsomal fraction. N/A, no colonies present in the samples selected with TG, i.e. no mutation frequency could be determined.

 

 

With S9

Without S9

Neg. control

N/A

N/A

Pos. control

N/A

4.18x10-4

0.5 mM

N/A

N/A

1.0 mM

N/A

N/A

2.5 mM

N/A

N/A

5.0 mM

N/A

N/A

Conclusions:
Based on the results of the current study, it can be concluded that the given test chemical does not give rise to gene mutations when exposed at ≤ 5.0 mM for 3 hrs or more, and it does not give rise to cytotoxicity in CHO cells at concentrations of ≤ 5.0 mM.
Executive summary:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the given test chemical as per OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test) when administered to Chinese Hamster Ovary (CHO) cells.

A preliminary dose-finding study was conducted prior to the main study. A range of different test chemical concentrations were tested in 96-well plates and analyzed by two commonly used assays, i.e. the colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bicinchoninic acid (BCA) assay to assess cell viability and protein concentration, respectively.

From the basis of the results from the MTT and BCA assays, test concentrations of the test chemical was chosen to be included in the gene toxicity test.

In the genotoxicity test, the given test chemical was administered to CHO cells for 3 hrs at the dose levels of 0, 0.5, 1.0, 2.5 or 5.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test.

The results showed indication of gene mutations occurring only in the positive control ENU while no other treatment gave rise to gene toxicity. No cytotoxic effects was observed in CHO cells when exposed towards test chemical for 3 hrs in the absence or presence of S9 liver microsomal fraction.

Based on the results of the current study, it can be concluded that the given test chemical does not give rise to gene mutations when exposed at ≤ 5.0 mM for 3 hrs or more, and it does not give rise to cytotoxicity in CHO cells at concentrations of ≤ 5.0 mM.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in vitro:

Available experimental data from study report and literature was evaluated to determine the mutagenic nature of the chemical. The studies are summarized below.

Ames assay:

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 above data 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.

 


Another study A bacterial reverse mutation test was performed to determine the mutagenic nature of the test substance. The study was performed using Salmonella typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without S9 metabolic activation system. The test was performed as a preincubation assay at six different concentrations with 10 mg/plate being the maximum concentration. Cells were pre-incubated with both the test chemical and S9-mix for 20 min. The number of revertant (His+) colonies was scored after 48 hours of incubation. The result was considered positive if the number of revertant colonies was found twice the number of the controls (exposed to the appropriate solvent or untreated). The test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 in the presence and absence of S9 metabolic activation system. Hence, it is considered as Non-mutagenic (negative) in vitro in the test system used under the certain experimental conditions.

 

Another study on bacterial reverse mutation assay was performed for the test chemical using Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 with and without rat and hamster liver S9 mix. The test chemical was dissolved in ET95 (lab 1) and DMSO (lab 2). The study was performed using the preincubation protocol at five dose levels of 0, 100, 333, 1000, 3333 or 10000 µg/plate (lab 1 and 2) with incubation period of 48 hrs in the presence and absence of S9 mix. The final dose level selection was based on the results of a preliminary range-finding study conducted with TA100 in the presence and absence of S-9. No mutagenic response was noted for the test compound in the preliminary dose range finding study and the main study performed. The given test chemical did not induce mutation in the Salmonella typhimurium strain TA100, TA1535, TA98, TA1537 with and without rat and hamster liver S9 mix in both lab 1 and lab 2 studies and hence is negative for gene mutation in vitro.

 

 

In vitro mammalian chromosome aberration study:

The in vitro mammalian chromosomal aberration study was conducted to determine the chromosomal aberration induction potential of test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on29thJuly 2016.

The experiment was conducted using human peripheral blood lymphocytes. Blood was drawn from a healthy volunteer, by venous puncture using heparinised syringe. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 hours mitogenic stimulation. The cells were treated with metaphase arresting substance (colcemid) 3 hours prior to harvesting and stained. The slides were analysed with the aid of microscope and a minimum number of 1000 of cells per culture were counted and number of metaphases were recorded in different fields to determine the mitotic index. In total, 300 well-spread metaphases per concentration and control were scored for chromosomal aberrations. Based on the solubility and precipitation test, Dimethyl sulfoxide (DMSO) was selected as the vehicle for treatment. The pH of test item in culture medium was assessed at 0 hour and 4 hours after incubation at 37°C. No significant change in pH was observed at 0 hour and 4 hours when compared with negative control. Hence, 2.0 mg/mL was selected as the highest concentration for cytotoxicity test. Before conducting the chromosomal aberration study, the test chemical was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.0 (NC), 0.0 (VC), 0.5 (T1),1.0 (T2) and 2.0 (T3) mg/mL of culture media. Cytotoxicity was observed in the treated concentration of 2.0(T3) mg/mL both in the absence and in the presence of metabolic activation (1%). In the absence of S9 mix, the mean mitotic index observed was 10.05 (NC), 9.93 (VC), 5.43 (T1), 4.19 (T2), 3.58 (T3) and 8.20 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.19 (NC), 9.92 (VC), 5.49 (T1), 4.47 (T2), 3.03 (T3) and 8.49 (PC). The test concentration 2.0(T3) mg/ mL showed more than 60% reduction on the mitotic index in treated culture both in the presence or absence of metabolic activation when it was compared to the respective vehicle control, which confirms extensive cytotoxicity effect. Thereby, this concentration was not selected for the main study. Hence, 1.0 mg/mL of culture media was selected as the highest concentration for main study both in the presence and in the absence of metabolic activation.

The main study was performed in two independent phases:

In Phase I experiment, the cultures were exposed to test chemical for a short period of time (4 hours) both in the absence and in the presence of metabolic activation system (1%).The mean percentage of aberrant cells was 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.667 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.333 (PC)in the presence of metabolic activation at the concentration of 0.0 (NC), 0.0 (VC) 0.250 (T1), 0.5 (T2) and 1.0 (T3) mg/mL and positive controls, respectively. Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamide monohydrate at the concentration of30 µg/mL in the presence of metabolic activation (1%) caused significant increases in percent aberrant cells (≥10.33% aberrant cells).Even though the analysis did not reveal any statistical significance, the increases were biologically significant. During thetreatment with the test item both in the absence and presence of S9 mix, there were slight to moderater eductions of mitotic index observed at low (0.250 mg/mL) and middle (0.5 mg/mL) doses. The observed mean mitotic indexin the absence of metabolic activation were 10.03, 9.79,6.64,5.11,4.30and 8.73andin the presence of metabolic activation were 10.18, 9.89,6.80,5.59,4.10and 8.69 for 0.0 (NC), 0.0 (VC) 0.250 (T1), 0.5 (T2) and 1.0 (T3) mg/mL and 30 µg/mL (+S9), 600 ug/ml (-S9) of PC concentrations.

The Phase II experiment was performed to confirm the negative results obtained in the absence and in the presence of metabolic activation in Phase I. In the absence of metabolic activation, test items concentrations used were 0.0 (NC), 0.0 (VC) 0.250 (T1), 0.5 (T2) and 1.0 (T3) mg/mL and 600 µg/mL (PC; ethylmethanesulfonate). In the presence of metabolic activation (2%), test items concentrations used were 0.0 (NC), 0.0 (VC) 0.250 (T1), 0.5 (T2) and 1.0 (T3) mg/mLand30 µg/mL(PC; cyclophosphamide monohydrate).The duration of exposure to the test item in presence of metabolic activation system was 4 hours and in absence of metabolic activation was 24 hours. The mean percent aberrant cells were 0.333 (NC), 0.333 (VC) 0.333 (T1), 0.667 (T2), 0.667 (T3) and 10.333 (PC) in the absence of metabolic activation and 0.333 (NC),0.667 (VC),0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.000 (PC) in the presence of metabolic activation at the concentration of 0.0 (NC), 0.0 (VC) 0.250 (T1), 0.5 (T2) and 1.0 (T3) mg/mL of culture and positive control. Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamide monohydrate at the concentration of 30 µg/mL in the presence of metabolic activation (2%) caused significant increases in percent aberrant cells (≥10.00% aberrant cells) .Though the analysis did not reveal any statistical significance, the increases were biologically significant. The increased frequency of aberrations observed in the concurrent positive control groups (Phase I and II)validated the sensitivity of the test system, suitability of the methods and conditions employed in the experiment. Treatment with test item both in the absence and presence of S9 mix induced slight to moderate reductions of mitotic index at low (0.250 mg/mL) and middle (0.5 mg/mL) doses. The observed mean mitotic index in the absence of metabolic activation were 10.13, 9.90,6.40,5.05,4.20and 8.29 andin the presence of metabolic activation were 10.03, 9.90,6.14,5.35,4.37and 8.68 for 0.0 (NC), 0.0 (VC) 0.250 (T1), 0.5 (T2) and 1.0 (T3) and 30 µg/mL(+S9), 600 ug/ml(-S9) of PC concentrations, respectively.

Hence from the above data it can be concluded that, the test chemical was found to be not mutagenic for chromosomal aberration in human peripheral blood lymphocyte both in presence (1% and 2%) and in the absence of metabolic activation under the specified conditions. Hence it is not likely to be classified as a gene mutant as per the criteria mentioned in CLP regulation.

Another study of in vitro mammalian chromosomal aberration was performed to determine the mutagenic nature of test chemical. The cells were exposed to the test material at three different doses with 2 mg/ml being the maximum concentration for 48 hr. Colcemid (0.2 µg/ml in final concentration) was added to the culture 2 hrs before cell harvesting. The slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation (liver microsome solution, S9 mix) systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others were recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9 %, equivocal if it was between 5.0 and 9.9 %, and positive if it was more than 10.0 %. The results showed that the given test chemical did not give a rise of the number of cells with chromosome aberrations and/or induce polyploid cells when CHL cells were exposed to 2 mg/ml of test chemical in the absence of S9-mix. Thus, it is considered as Non-mutagenic (negative) when CHL cells are exposed to the test chemical without metabolic activation under the presented experimental conditions.

 

In another study, chromosomal aberration study was performed to determine the mutagenic nature of the test chemical. The cells were exposed to the test material at three different doses with 0.125 mg/mL being the maximum concentration for 48hr. Colcemid (final concn 0.2 µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. The test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line CHL and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian cell gene mutation assay:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the given test chemical as per OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test) when administered to Chinese Hamster Ovary (CHO) cells.

A preliminary dose-finding study was conducted prior to the main study. A range of different test chemical concentrations were tested in 96-well plates and analyzed by two commonly used assays, i.e. the colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bicinchoninic acid (BCA) assay to assess cell viability and protein concentration, respectively.

From the basis of the results from the MTT and BCA assays, test concentrations of the test chemical was chosen to be included in the gene toxicity test.

In the genotoxicity test, the given test chemical was administered to CHO cells for 3 hrs at the dose levels of 0, 0.5, 1.0, 2.5 or 5.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test.

The results showed indication of gene mutations occurring only in the positive control ENU while no other treatment gave rise to gene toxicity. No cytotoxic effects was observed in CHO cells when exposed towards test chemical for 3 hrs in the absence or presence of S9 liver microsomal fraction.

Based on the results of the current study, it can be concluded that the given test chemical does not give rise to gene mutations when exposed at ≤ 5.0 mM for 3 hrs or more, and it does not give rise to cytotoxicity in CHO cells at concentrations of ≤ 5.0 mM.

Based on the available data on experimental study available for the target chemical, the given chemical does not exhibit gene mutation in vitro by Ames assay, In vitro mammalian chromosome aberration study and In vitro mammalian cell gene mutation assay. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation. 

Justification for classification or non-classification

Based on the available data on experimental study available for the target chemical, the given chemical does not exhibit gene mutation in vitro by Ames assay, In vitro mammalian chromosome aberration study and In vitro mammalian cell gene mutation assay. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.