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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Relatively well reported study according to guideline/standard; however, it was noted that OECD guidelines 471 and 472 were used (study performed in June 1998), whereas the revised OECD guideline 471 was already in place (21 July 1997). In addition, due to infections only the results of strain TA100 without S9 mix were considered reliable and these results were incorporated directly into the main study (I) without further repeat.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Principles of method if other than guideline:
It was noted that OECD guidelines 471 and 472 were used (study performed in June 1998), whereas the revised OECD guideline
471 was already in place (21 July 1997).
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name of test compound: PDTN
Appearance: white crystalline solid
Chemical name: Acetonitrile,2,2',2'',2'''-(1,3-propanediyldinitrilo)tetrakis-
Batch no: JNN98038
Composition: PDTN 99.2±1.0% m/m; water 0.5±0.1% m/m
Storage: at room temperature in the dark
Expiry date: 1 June 2000

Method

Target gene:
Point mutations at GC base pairs (Salmonella strains) and at AT base pairs (E coli strain)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Periodically "cleansed" against high spontaneous background: yes
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Periodically "cleansed" against high spontaneous background: yes/no
Metabolic activation:
with and without
Metabolic activation system:
rat liver Aroclor 1254 induced
Test concentrations with justification for top dose:
Preliminary test: TA100 with and without S9 mix and WP2uvrA with and without S9 mix, at concentrations of 0, 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate. However, due to infections only the results of strain TA 100 without S9 mix were reliable and these
results were incorporated directly into the main study (I) without further repeat.
Main test (I and II): 0, 100, 333, 1000, 3330 and 5000 µg/plate, with and without S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not provided
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-aminoacridine, daunomycine, methylmethanesulfonate, 4-nitroquinoline N-oxide; see table below
Remarks:
- S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene; see table below
Remarks:
+ S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: until cultures reached ca. 10E9 cells/mL
- Exposure duration: 48 h


NUMBER OF REPLICATIONS: in triplo (each test)


DETERMINATION OF CYTOTOXICITY
- Method: reduction in background lawn, increase in size of microcolonies, reduction of revertant colonies

OTHER: no
Evaluation criteria:
No formal hypothesis testing was done. A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value,
with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent
control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is
considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
Not used.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
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:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: apparently not present

RANGE-FINDING/SCREENING STUDIES: largely failed due to an infection

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: no cytotoxicity present
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

There was not a 2-fold (or more) increase in the number of revertants. Therefore, PDTN was not mutagenic in the Ames test.
Executive summary:

PDTN was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA in two independent experiments. PDTN was tested up to concentrations of 5000 µg/plate both in the absence and presence of S9 -mix. PDTN did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9 -metabolic activation. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that PDTN is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.