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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
February-March 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
method of treatment is restricted to preincubation
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
method of treatment is restricted to preincubation
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
(2Z)-2-fluoro-3-(morpholin-4-yl)acrylaldehyde
EC Number:
813-789-9
Cas Number:
152873-67-1
Molecular formula:
C7H10FNO2
IUPAC Name:
(2Z)-2-fluoro-3-(morpholin-4-yl)acrylaldehyde
Test material form:
solid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator, approx. 4°C
- Solubility and stability of the test substance in the solvent/vehicle: The test item is an impurity of a drug substance. Thus, according to ICH Draft Consensus Guideline M7, no stability test in the solvent was performed.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING

- Final preparation of a solid: solution (only used at the same day) - The purity of the test item was not taken into account for the calculation of the dosages.

Method

Target gene:
Histidine gene locus
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced male Sprague Dawley rat liver S9 mix
Test concentrations with justification for top dose:
0, 100, 250, 500, 1000, 2500, 5000 µg/plate (+/-S9 mix, all strains)








Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Remarks:
No solvent control was used since sufficient evidence was available in the literature and from testing laboratory experience, indicating that the solvents used had no influence on the spontaneous mutant counts of the used strains.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (TA 1535, TA 100), 4-nitro-1,2-phenylene diamine (TA 1537), 2-Nitrofluoren (TA 98), Cumene hydroperoxide (TA 102), 2-aminoanthracene (all strains).
Details on test system and experimental conditions:
METHOD: each concentration including the controls was tested in triplicate.
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA1535, TA100, TA1537 and TA98 this increase should be about twofold as compared to the solvent controls. For TA102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA1535, TA100, TA1537, TA98 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Summary of results from the Salmonella mutagenicity assay with FMA (mean values of revertants per plate)

Dose (µg per plate)

Without metabolic activation

 

TA 1535

 TA 100

 TA 1537

 TA 98

 TA 102

0

9

165

6

17

213

100

7

160

7

15

208

250

8

166

8

17

216

500

8

167

7

19

212

1000

9

155

8

17

206

2500

9

160

7

18

234

5000

12 

 170

8

15 

219

 Positive control

685

1274

55

1405

505 

Dose ( µg per plate )

With metabolic activation (liver S9 mix)

 

TA 1535

 TA 100

 TA 1537

 TA 98

TA 102

0

9

210

8

27

294

100

9

212

8

26

240

250

8

224 

8

28

277

500

11

251

8

25

279

1000

8

193

7

22

289

2500

10

177

10

25

296

5000

11 177 9 26 277

 Positive control

153

3021 

241 

2270 

853 

Historical negative control data demonstrated that no deleterious or mutagenic effects were induced by the chosen solvent. The positive controls sodium azide, 4-nitro-1,2 -phenylene diamine, 2-nitrofluoren, cumene hydroperoxide and 2-aminoanthracene increased mutant counts to well over those of the solvent controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

None of the five strains used showed a dose-related and biologically relevant increase inmutant counts over those of the solvent controls in the preincubation test. This applied both to the tests with and without S9 mix.

 

 

Applicant's summary and conclusion

Conclusions:
negative
Executive summary:

The mutagenic potential of the test material was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471. Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. Test item precipitation did not occur. Historical negative control data demonstrated that no deleterious or mutagenic effects were induced by the chosen solvent. The positive controls increased mutant counts to well over those of the solvent controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix. None of the five strains used showed a dose-related and biologically relevant increase in mutant counts over those of the solvent controls in the preincubation test. This applied both to the tests with and without S9 mix. Due to these results the test item has to be regarded as non-mutagenic.