Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): S. typhimurium TA 98, TA 100, TA 102, TA 1535, TA 1537: negative with and without metabolic activation (OECD 471)

Mammalian mutagenicity (mouse lymphoma assay): negative with and without metabolic activation (OECD 490)

Mammalian micronucleus (micronucleus test): negative with and without metabolic activation (OECD 487)

Link to relevant study records

Referenceopen allclose all

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
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
Target gene:
His operon
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.
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.
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.
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.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-01-17 to 2017-04-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
July 29, 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Behörde für Gesundheit und Verbraucherschutz
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Sex, age and number of blood donors: Human peripheral blood was obtained by venipuncture from young, healthy, non-smoking male or female individuals with no known recent exposures to genotoxic chemicals or radiation, and collected in heparinised vessels.
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Normal (negative control) cell cycle time: 1.5 cell cycles

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Freshly prepared blood lymphocytes were seeded with 5 mL of Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin. After 48 hours, the cultures were centrifuged (10 minutes at 800 – 900 rpm) and the medium was replaced by 4.5 mL of fresh Ham’s F10 medium with fetal calf serum (FCS). Cultures were then treated with the test material, positive, or negative control. After exposure, the medium was removed and the
cultures were washed twice with Ham’s F10 medium with FCS. After addition of
5 mL Chromosome complete medium containing 5 μg/mL Cytochalasin B the cultures
were incubated for further 20 hours at 37°C.
Cytokinesis block (if used):
5 μg/mL CytoB
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rats
Test concentrations with justification for top dose:
250, 500, 1000 and 2000 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The test item was completely dissolved in highly purified water for concentrations of 10 and 3.16 μg/mL medium in the preliminary test.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: 0.01 μg colchicine/mL, -S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 h
- Exposure duration: 4 or 24 h
- Expression time: 4 or 24 h
- Selection time: 20 h
- Fixation time: 24 h

SPINDLE INHIBITOR (cytogenetic assays): 5 μg/mL of CytoB

STAIN (for cytogenetic assays): 10% Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

NUMBER OF CELLS EVALUATED: 2000

DETERMINATION OF CYTOTOXICITY
- Method: The evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI).
Evaluation criteria:
The assay was considered acceptable if the the following criteria was met:

- The concurrent negative control was considered acceptable for addition to the laboratory historical negative control database (Poisson-based 95% control
limits). If the concurrent negative control data fell outside the 95% control limits, inclusion of historical control data was acceptable as long these data were not extreme outliers.
- Concurrent positive controls induced responses that were compatible with those generated in the laboratory’s historical positive control data base and produced a statistically significant increase compared with the concurrent negative control.
- Adequate number of cells, cell proliferation criteria and concentrations were analysable.

A test chemical was considered to be clearly positive if, in any of the experimental conditions examined:

- at least one of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control;
- the increase was dose-related in at least one experimental condition when evaluated with an appropriate trend test;
- any of the results were outside the distribution of the historical negative control data (Poisson-based 95% control limits)

A test chemical was considered clearly negative if, in all experimental conditions examined:

- none of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control;
- there was no concentration-related increase when evaluated with an appropriate trend test;
- all results were inside the distribution of the historical negative control data (Poisson-based 95% control limits).
Statistics:
Not reported
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
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
- Effects of pH: For the 4 hr exposure, +S9, the concentrations of 1000 and 2000 μg/mL revealed decreases in pH of 6.59 or 6.15 compared to the control with a pH of 7.68.
- Precipitation: Test item precipitation was noted at all tested concentrations in the experiments without and with S9 mix.

RANGE-FINDING/SCREENING STUDIES: In a preliminary experiment without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 μg/mL were employed. Test item precipitation was noted starting at a concentration of 316 μg/mL medium in the experiments without and with S9 mix (24- or 4-hour exposure). No signs of cytotoxicity were noted up to the top concentration of 2000 μg/mL medium. Hence, 2000 μg/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation.

HISTORICAL CONTROL DATA
- Positive historical control data: In the positive control cultures the micronucleus frequencies were increased to 22.0 or 27.5 micronucleated cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, -S9, respectively. In the positive control culture the micronucleus frequency was increased to 22.0 micronucleated cells per 1000 binucleate cells for the 4-hour exposure, +S9.
- Negative (solvent/vehicle) historical control data: In the negative control cultures the micronucleus frequencies were 4.5 or 5.5 micronucleated cells per 1000 binucleated cells for the 4-hour and 24-hour exposure, -S9, respectively, were observed. In the negative control cultures the mean micronucleus frequencies were 6.5 micronucleated cells per 1000 binucleated cells for the 4-hour, +S9.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: The evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI).

Test with metabolic activation (4-hour exposure)

The micronucleus frequencies of cultures treated with the concentrations of 250, 500, 1000 and 2000 μg dimelamine pyrophosphate/mL medium (4-h exposure) in the presence of metabolic activation ranged from 3.0 to 8.0 micronucleated cells per 1000 binucleated cells. The slightly dose-related increase in micronuclei up to the top concentration of 2000 μg/mL medium is considered to be incidental and of no biological relevance, as the frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls. Nevertheless it is known that changes in pH cause artifactual positive results. The concentrations of 1000 and 2000 μg dimelamine pyrophosphate/mL medium revealed decreases in pH of 6.59 or 6.15 compared to the control with a pH of 7.68.

Conclusions:
Under the present test conditions (OECD 487 and GLP compliant), dimelamine pyrophosphate tested up to a concentration of 2000 μg/mL medium in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-01-02 to 2017-03-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
May 30, 2008
GLP compliance:
yes (incl. certificate)
Remarks:
Behörde für Gesundheit und Verbraucherschutz
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
Thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection, 0801 University Blvd., Manassas, VA 20110-2209, USA

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cells were maintained in growth medium RPMI 16401 with glutamax medium supplemented with Pluronic F682, gentamycin, amphotericin B1 and horse serum (10% by volume). Treatment medium is growth medium with a reduced horse serum content (5% by volume). Plating medium is growth medium with increased horse serum content (approx. 20% by volume) but without Pluronic® F68. Selection medium is plating medium that contains 3 μg/mL of TFT.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9-mix
Test concentrations with justification for top dose:
10.0, 31.62, 100, 316.2, 1000 and 2000 μg dimelamine pyrophosphate/mL

Based on the results of a preliminary assay, the top concentration of 2000 μg/mL medium was selected.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The test item was completely dissolved in highly purified water for a concentration of 10 μg/mL medium in the preliminary test.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 2 x 10E5 cells/mL

DURATION
- Exposure duration: 3 h, ±S9 or 24 h, -S9
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10 to 12 days
SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2 replications, +S9

NUMBER OF CELLS EVALUATED: four 96-well microtiter plates (average 2 x 10E3 cells/well)

DETERMINATION OF CYTOTOXICITY
- Method: suspension growth (SG; the number of times the cell number increased from the starting cell density); relative total growth (RTG; the product of the relative suspension growth [RTG] and the relative plating efficiency [RPE])
Evaluation criteria:
An assay was considered acceptable for evaluation if the following criteria were met:

- Adequate number of cells and concentrations were analysable;
- The criteria for the selection of highest concentration were consistent with protocol;
- Data of the untreated/solvent control met acceptance criteria;
- The positive control demonstrated an absolute increase in total mutant frequency (MF) (i.e., an induced MF of at least 300 x 10-6). At least 40% of the induced MF were reflected in the small colony MF, or the positive control increased in the small colony MF of at least 150 x 10-6 above that seen in the concurrent untreated/solvent control (a small colony induced MF of 150 x 10-6); and
- Mutation Frequencies of both, negative and positive controls fell within the normal range (historical data).

The test material was considered to be clearly positive if the increase in MF above the concurrent background exceeded the GEF (Global Evaluation
Factor, 126 x 10-6) and the increase was concentration related. The test material was considered to be clearly negative if there was no concentration related response or there was an increase in MF that did not exceed the GEF. In cases when the response was neither clearly negative nor clearly positive expert judgement and/or further investigations were used.
Statistics:
Statistical methods were not reported.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: A slight decrease in pH compared to the control was noted at concentrations of 1000 and 2000 μg/mL medium. However, no relevant changes in pH of the test item formulations at the lower concentrations compared to the negative control were noted.
- Effects of osmolality: No changes in osmolality of the test item formulations compared to the negative control were noted up to the top concentration of 2000 μg/mL medium.
- Precipitation: Test material precipitation was noted starting at a concentration of 250 μg dimelamine pyrophosphate/mL medium in all experiments.

RANGE-FINDING/SCREENING STUDIES: In a preliminary study, cytotoxicity was noted at the top concentration of 2000 μg/mL in the absence and presence of metabolic activation (24-hour or 3-hour exposure, respectively). Test item precipitation was noted starting at a concentration of 316.2 μg/mL medium. A slight decrease in pH compared to the control was noted at concentrations of 1000 and 2000 μg/mL medium. However, no relevant changes in pH of the test item formulations at the lower concentrations compared to the negative control were noted. No changes in osmolality of the test item formulations compared to the negative control were noted up to the top concentration of 2000 μg/mL medium.

HISTORICAL CONTROL DATA
- Positive historical control data: The positive controls Methylmethanesulfonate (MMS) and 3-Methylcholanthrene (3-MC) caused pronounced increases in the mutation frequency of 582.70 and 466.65 mutant colonies per 10E6 cells in the case of MMS and of 395.02 and 416.10 mutant colonies per 10E6 cells in the case of 3-MC. As the increase in the small colony mutation frequency was at least 150 x 10-6 above the concurrent negative control, an absolute increase in total mutation frequency was at least 300 x 10E-6 for the positive controls and the mean relative total growth (RTG) greater than or equal to 10%, the acceptance criteria for the positive controls were met.
- Negative (solvent/vehicle) historical control data: The negative controls had mutation frequencies of 96.61 or 119.75 mutant colonies per 10E6 cells in the experiments without metabolic activation (3- or 24-hour exposure, respectively) and 50.42 or 105.96
mutant colonies per 10E6 cells in the experiment with metabolic activation; therefore, all mutation frequencies were within the historical data-range. The mean cloning efficiencies of the negative controls from the Mutation Experiments were between the range 65% to 120%. Hence, the acceptance criteria were met.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytotoxicity was noted starting at a concentration of 500 μg dimelamine pyrophosphate/mL medium in the 24-hour exposure experiment without metabolic activation and at the top concentration of 2000 μg/mL in the presence of metabolic activation.

In addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.14 to 1.17 for dimelamine pyrophosphate-treated cells and ratios of 0.34 and 0.92 for the negative controls.

Conclusions:
Dimelamine pyrophosphate did not exhibit any clastogenic potential at the concentration-range investigated. According to the evaluation criteria for this assay, these findings indicate that dimelamine pyrophosphate, tested up to cytotoxic concentrations, neither induced mutations nor had any chromosomal aberration potential.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

not applicable

Additional information

Bacterial Mutagenicity

In an Ames assay conducted in accordance with OECD 471 and GLP compliant, dimelamine pyrophosphate (CAS 13518-93-9) was examined in the 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535, and TA1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver) (LPT, 2017). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.

 

No signs of cytotoxicity were noted up to the top concentration of 5000 μg/plate in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, in any test strain. Test item precipitation was noted at all concentrations in all test strains. No increase in revertant colony numbers as compared with control counts was observed for dimelamine pyrophosphate, tested up to a concentration of 5000 μg/plate, that led to test item precipitation, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

 

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.

Mammalian Mutagenicity

In a gene mutation assay conducted in accordance with OECD 490 and GLP compliant, cultured mammalian cells (L5178Y TK +/-) were treated with dimelamine pyrophosphate (CAS 13518-93-9) both in the presence and absence of metabolic activation by a liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced rats (LPT, 2017). The test was carried out employing two exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours, the experiment with S9 mix was carried out in two independent assays.

Cytotoxicity was noted starting at a concentration of 500 μg dimelamine pyrophosphate/mL medium in the 24-hour exposure experiment without metabolic activation and at the top concentration of 2000 μg/mL in the presence of metabolic activation. Test item precipitation was noted starting at a concentration of 250 μg dimelamine pyrophosphate/mL medium in all experiments.

The mutation frequencies of the cultures treated with dimelamine pyrophosphate ranged from 51.14 to 76.51 mutant colonies per 10E6 cells (3-hour exposure), from 59.22 to 98.14 mutant colonies per 10E6 cells (24-hour exposure) in the experiments without metabolic activation. The highest concentrations of 1000 and 2000 μg/mL in the 24-h exposure assay were almost completely cytotoxic and, hence not used for evaluation for mutant colonies. In the experiments with metabolic activation, mutation frequencies ranged from 55.12 to 84.56 mutant colonies per 10E6 cells (3-hour exposure, first assay) and from 87.76 to 144.22 mutant colonies per 10E6 cells (3-hour exposure, second assay). The results were within the range of the negative control values and the normal range of 50 to 170 mutants per 10E6 viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation. In addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.14 to 1.17 for dimelamine pyrophosphate-treated cells and ratios of 0.34 to 0.92 for the negative controls.

Under the present test conditions, dimelamine pyrophosphate, tested up to the cytotoxic concentration of 2000 μg/mL medium, in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK + /mammalian cell mutagenicity test.

Mammalian Cytogenicity

In an in vitro micronucleus assay that was conducted in accordance with OECD 487 and GLP compliant, human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals were treated with dimelamine pyrophosphate (CAS 13518-93-9). The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. The harvesting time was 20 hours after the end of exposure. The cytokinesis-block technique was applied.

In the main study no signs of cytotoxicity were noted up to the top concentration of 2000 μg dimelamine pyrophosphate/mL medium in the experiments without and with metabolic activation. Test item precipitation was noted at all tested concentrations in the experiments without and with S9 mix.

The micronucleus frequencies of cultures treated with the concentrations of 250, 500, 1000 and 2000 μg dimelamine pyrophosphate/mL medium in the absence of metabolic activation (4- and 24-hour exposure, respectively) ranged from 3.5 to 7.0 micronucleated cells per 1000 binucleated cells. There was no dose-related increase in micronuclei up to the top concentration of 2000 μg/mL medium. The frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls.

The micronucleus frequencies of cultures treated with the concentrations of 250, 500, 1000 and 2000 μg dimelamine pyrophosphate/mL medium (4-h exposure) in the presence of metabolic activation ranged from 3.0 to 8.0 micronucleated cells per 1000 binucleated cells. The slightly dose-related increase in micronuclei up to the top concentration of 2000 μg/mL medium is considered to be incidental and of no biological relevance, as the frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls. Nevertheless it is known that changes in pH cause artifactual positive results. The concentrations of 1000 and 2000 μg dimelamine pyrophosphate/mL medium revealed decreases in pH of 6.59 or 6.15 compared to the control with a pH of 7.68.

Under the present test conditions, dimelamine pyrophosphate tested up to a concentration of 2000 μg/mL medium in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test.

Justification for classification or non-classification

The available data on genetic toxicity of dimelamine pyrophosphate do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.