Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study lnitiation Date - February 20, 2017 - Experiment Completion Date April 10, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Adopted on 21 July, 1997
Deviations:
no
Principles of method if other than guideline:
- Principle of test:
The Study was performed to evaluate the mutagenic potential of Delmopinolo HCl using Salmonella typhimurium Strains TA98, TA100, TA1537, TA1535 and TA102 in Trial I (5 % v/v S9 mix and without metabolic activation) and Trial II (10% v/v S9 mix) experiments with following concentrations including vehicle (Distilled water) and positive controls in triplicates.

- Method
Plate incorporation


- Parameters analysed / observed:
The growth inhibitory effect (cytotoxicity) and mean revertant colonies per plate.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
ldentification Delmopinolo HCI
Appearance White powder
Batch number 2510468
Chemical Name 2-[3-( 4-propylheptyl)-4-morpholinyl)ethanol hydrochloride (1 :1)
CAS No. 98092-92-3
Purity 99.8%
Manufactured date June 15, 2016
Expiry Date June 15, 2021


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Room temperature (20° - 30 °C)
- The test material is stable under test condition

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Not performed

Method

Target gene:
Genetic markers:
- rfa Wall Mutation
- pKM101 Plasmid
- pAQ1 Plasmid
- uvrB Mutation
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
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 homogenate and S9 mix
Test concentrations with justification for top dose:
Trial I: 4.8828125, 9.765625, 19.53125, 39.0625 and 78.125 µg/plate.
Trial II: 2, 5, 12.5, 31.25 and 78.125 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water (Based on the solubility test).
Controls
Untreated negative controls:
other: Historical negative controls
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
Details on test system and experimental conditions:




Preliminary Cytotoxicity Study
In the cytotoxicity study, the experiment was performed using tester strains TA98 and TA100 in the absence and presence of metabolic activation (5% v/v S9 mix) using plate incorporation method. Eight concentrations of test item were tested in triplicate plates along with vehicle and positive controls.

Main Assay
In Trial I, the assay was performed using the tester strains TA98, TA100, TA1535, TA1537 and TA102 in the absence and presence of the S9 mix (5% v/v S9 mix) using plate incorporation method. Since, there was reduction in number of revertant colonies and background lawn in the cytotoxicity assay from the tested concentration of 156.25 to 5000 µg/plate, the Trial I was performed using all the tester strains TA98, TA100, TA1535, TA1537 and TA102 along with the vehicle and positive controls in both presence and absence of metabolic activation.
In Trial II, the assay was performed to confirm the negative results. The tester strains TA1535, TA1537, TA98, TA100 and TA102 were tested only in the presence of S9 metabolic activation system (10% v/v S9 mix) using the plate incorporation method. Five concentrations of the test item were tested in triplicate plates along with the vehicle and positive controls.


Evaluation criteria:
Acceptance Criteria
The Bacterial reverse mutation assay is considered acceptable if it meets the following criteria:
- Regular background growth in the solvent control
- The positive control substances should produce a significant increase in revertant colony frequencies
- The spontaneous reversion rates in the solvent control is in the range of historical data.
Statistics:
No formal hypothesis testing will be performed to analyse the data. Once criteria for a valid assay have been met, responses observed in the assay are evaluated as follows:

- Criteria for a Positive Response

Tester Strains TA98, TA100 and TA102
For a test item to be considered positive, it must produce at least a 2–fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response (minimum of 2 to 3 concentrations) to increasing concentrations of the test item.

Tester Strains TA1537 and TA1535
For a test article to be considered positive, it must produce at least a 3–fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response (minimum of 2 to 3 concentrations) to increasing concentrations of the test item.

- Criteria for a Negative Response
A test item for which the results do not meet the above criteria is considered non mutagenic in this test.

Results and discussion

Test results
Key result
Species / strain:
other: TA98, TA100, TA1537, TA1535 and TA102
Remarks:
TA98, TA100, TA1537, TA1535 and TA102
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:
Trial I
Trial I was performed both in the presence (5 % v/v S9 mix) and absence of metabolic activation system along with the vehicle and the positive control with the test item concentration with a spacing factor of 2. There was no increase in the number of revertant colonies up to the tested concentration of 78.125 µg/plate in the presence (5% v/v S9 mix) and absence of metabolic activation, when compared to the vehicle control. No precipitation was observed from in tested concentration of 78.125 µg/plate both in the presence and absence of metabolic activation.

Trial II
Trial II was conducted to confirm the negative results observed in Trial I. For the negative confirmation, the test item concentration with a spacing factor of 2.5 in presence of metabolic activation and concentration of metabolic activation (S9 fraction) was altered to 10% v/v. Trial II was conducted with all the tester strains along with vehicle and positive control. The spontaneous revertant colonies were within the acceptable range and did not show any increase in revertant colonies with respect to dose concentration. No precipitation was observed in the tested concentration of 78.125 µg/plate in the presence of metabolic activation.



Remarks on result:
other: Delmopinolo HCl is non-mutagenic.

Any other information on results incl. tables

Revertant Colonies - Cytotoxicity Test

Test

Concentration

(µg/plate)

TA 98

TA 100

- S9

+ S9

- S9

+ S9

Mean

SD

Mean

SD

Mean

SD

Mean

SD

VC

18.67

2.08

18.33

1.15

119.00

11.53

119.33

6.03

39.0625

21.33

2.52

21.67

2.52

109.00

9.17

115.67

15.37

78.125

17.00

2.00

17.33

2.52

77.33

9.45

99.67

7.64

156.25

11.67

1.53

15.67

1.53

64.67

9.87

72.00

11.27

312.5

0.67

1.15

10.33

2.08

12.33

6.81

32.67

2.08

625

0.00

0.00

1.67

1.15

0.00

0.00

1.00

1.00

1250

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

2500

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

5000

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

PC

381.33

9.02

395.33

9.02

694.67

7.02

709.33

3.06

Revertant Colonies – Trial  I

 Plate Incorporation Method [Presence of metabolic activation (+S9 5% v/v S9 Mix)]

Test Concentration

(µg/plate)

TA 1537

TA 1535

TA 102

TA 98

TA 100

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

VC

6.00

0.00

11.67

1.53

310.00

12.49

20.67

2.89

124.33

13.87

4.8828125

10.33

1.53

13.33

0.58

326.67

13.61

20.00

2.65

134.33

6.11

9.765625

6.33

1.53

13.00

1.00

317.33

15.01

23.67

2.31

125.33

8.62

19.53125

8.67

2.08

9.67

2.52

297.33

8.08

23.33

0.58

148.67

18.04

39.0625

6.67

1.15

12.33

2.08

287.00

4.58

19.00

1.00

134.67

7.02

78.125

3.33

1.53

12.33

1.53

269.33

8.62

16.33

2.08

94.67

9.45

PC

235.00

6.08

400.00

9.17

1127.33

33.31

426.00

9.17

733.00

10.82

Plate Incorporation Method [Absence of metabolic activation (-S9)]

Test Concentration

(µg/plate)

TA 1537

TA 1535

TA 102

TA 98

TA 100

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

VC

9.00

1.00

11.33

2.52

297.67

9.02

20.67

1.53

122.33

7.02

4.8828125

9.33

2.52

10.33

2.52

344.67

20.60

22.00

1.73

139.00

5.00

9.765625

7.33

2.52

12.67

3.06

316.67

17.01

23.33

1.53

134.33

6.43

19.53125

6.00

1.73

10.33

1.53

296.67

11.68

23.00

0.00

145.33

6.66

39.0625

6.33

0.58

7.00

1.00

305.67

12.66

19.00

2.00

144.67

6.81

78.125

2.67

1.53

10.00

1.00

264.00

8.00

15.67

4.51

99.00

9.54

PC

212.67

13.05

400.67

11.37

1089.33

22.74

440.00

13.11

718.00

19.08

 Revertant Colonies -Trial II

Plate Incorporation Method [Presence of metabolic activation (+S9 10% v/v S9 Mix)]

Test Concentration

(µg/plate)

TA 1537

TA 1535

TA 102

TA 98

TA 100

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

VC

7.67

1.15

10.33

1.53

292.67

8.08

21.00

3.00

117.33

9.29

2

7.67

1.15

9.00

1.00

312.67

5.03

20.00

1.00

129.67

14.22

5

9.33

1.53

11.67

1.53

306.00

8.72

19.67

3.06

113.33

13.32

12.5

8.33

1.53

11.67

0.58

288.67

8.08

20.33

3.06

123.33

1.53

31.25

10.00

1.00

13.67

0.58

286.00

7.21

20.33

3.51

110.67

14.84

78.125

5.00

1.73

9.67

1.15

268.00

14.42

16.00

1.73

100.00

9.17

PC

221.33

14.50

426.67

9.02

1090.00

17.09

423.33

13.32

720.00

18.33

Key: SD = Standard Deviation, µg = Microgram, VC = Vehicle Control, PC = Positive Control, + S9 = Presence of S9, S9 = Rat liver Homogenate at 9000g.  

Applicant's summary and conclusion

Conclusions:
On the basis of the results of this study, it is concluded that Delmopinolo HCl is non-mutagenic as it did not induce (point) gene mutations at histidine locus by base pair changes or frame-shift in the presence and absence of metabolic activation system in all five tester strains of Salmonella typhimurium TA1537, TA1535, TA98, TA100 and TA102.
Executive summary:

Delmopinolo HCl  is non-mutagenic.