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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
other information
Study period:
October 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
June 2020
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator
- Solubility and stability of the test substance in the solvent/vehicle: According to ICH Draft Consensus Guideline M7, formulations were prepared freshly prior to start of treatment. Thus, no stability test in the solvent was performed.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING

- Final preparation: clear colorless 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, 16, 50, 160, 500, 1600, 5000 µg/plate (+/-S9 mix, all strains)








Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
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
Key result
Species / strain:
S. typhimurium, other: TA1535, TA100, TA1537, TA98 and TA102
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
test item precipitation was not observed.

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



solvent control


794716246

16


62202022252

50


02492320227

160


00000

500


00000

1600


00000

5000


00000

 Positive control


874132351879472

Dose ( µg per plate )



With metabolic activation (liver S9 mix)



 



TA 1535



 TA 100



 TA 1537



 TA 98



TA 102



solvent control


8104722345

16


81021421400

50


72701219395

160


00000

500


00000

1600


00000

5000


00000

 Positive control


123311521623451081


 


 


 


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:
Evidence of mutagenic activity.
Executive summary:

The test item Chloro nitro PP, dissolved in DMSO, was administered in doses of up to and including 5000 μg per plate without and with S9 mix on five Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA1535, TA100, TA1537, TA98 and TA102.
Doses up to and including 16 μg per plate did not cause any bacteriotoxic effects. At higher doses the test item induced a strain-specific bacteriotoxic effect. This range could also be used strain specific up to 160 μg per plate for assessment purposes. Test item precipitation did not occur.
Evidence of mutagenic activity of the test item was seen. In the preincubation modification, biologically relevant increase in the mutant count, in comparison to the solvent controls, was observed in two of the strains tested, without and with S9 mix, under the experimental conditions applied.
The employed positive controls induced a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding solvent controls.
Therefore, the test item is considered to be mutagenic in the preincubation modification of the Salmonella/microsome test.