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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-12-06 to 2017-04-06
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 30, 2008
GLP compliance:
yes (incl. certificate)
Remarks:
Behörde für Gesundheit und Verbraucherschutz
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Method

Target gene:
His operon
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:
microsomal preparation derived from Aroclor 1254-induced rat liver
Test concentrations with justification for top dose:
31.6, 100, 316, 1000, 3160 or 5000 μg dimelamine pyrophosphate per plate

Dimelamine pyrophosphate was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 μg/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 μg/plate. Hence, 5000 μg dimelamine pyrophosphate/plate was chosen as the top concentration for the main study in the plate incorporation test and in the preincubation test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Because of the small solubility of the test item in all solvents recommended, dimelamine pyrophosphate was suspended in highly purified water.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-amino-anthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation and preincubation method)

DURATION
- Preincubation period: 20 minutes for the preincubation method
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Cytotoxicity was defined as a reduction in the number of colonies by more than 50% compared with the vehicle control and/or a scarce background lawn.
Evaluation criteria:
A test item is considered to show a positive response if:

- the number of revertants was significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
- in addition, a significant (p ≤ 0.05) concentration (log value)-related effect (Spearman's rank correlation coefficient) was observed;
- positive results were reproducible and the histidine independence of the revertants was confirmed by streaking random samples on histidine-free agar plates.

A test item for which the results does not meet the above mentioned criteria was considered as non-mutagenic in the AMES test.
Statistics:
Statistical methods were not reported.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, 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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test item precipitation was noted at all concentrations in all test strains.

Applicant's summary and conclusion

Conclusions:
In conclusion, under the present test conditions, dimelamine pyrophosphate tested up to a concentration of 5000 μg/plate, that led to test item precipitation, caused no mutagenic effect in the Salmonella typhimurium strains T A98, TA 100, TA 102, TA 1535, and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.