Pyrimido[5,4-g]pteridine-2,4,6,8-tetramine, 4-methylbenzenesulfonate, base-hydrolyzed

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are reliable in vitro studies available to assess the potential of the test substance for gene mutations in bacteria and mammalian cells and cytogenicity in mammalian cells. All of them show that the substance is not genotoxic in vitro.

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

His: Salmonella
Trp: E. Coli

Species / strain / cell type:

other: Salmonella typhimurium strains: TA100, TA98, TA1535, TA1537; E. coli : WP2uvrA

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

first mutation assay (range finding test): 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
second mutation assay: 10, 33, 100, 333 and 1000 µg/plate

Vehicle / solvent:

Dimethyl sulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see: "Details on test system and conditions"

Details on test system and experimental conditions:

POSITIVE CONTROLS:
Without metabolic activation (-S9-mix):
TA1535: sodium azide (SA), 5 µg
TA1537: 9-aminoacridine (9AC), 60 µg
TA98: daunomycine (DM), 4 µg
TA100: methylmethanesulfonate (MMS), 650 µg
WP2uvrA: 4-nitroquinoline N-oxide (4-NQO), 10 µg

With metabolic activation (+S9-mix):
TA1537: 2-aminoanthracene (2AA), 2.5 µg
TA1535,TA98 and TA100: 2-aminoanthracene (2AA), 1 µg
WP2uvrA: 2-aminoanthracene (2AA), 5 µg; In the presence of 10% (v/v) S9-fraction, the concentration of 2AA was 10 µg/plate

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Incubation period: 48 hours. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli) were counted.
- COLONY COUNTING:
- Exposure duration: The revertant colonies (histidine independent/ tryptophan independent) were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

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.

Species / strain:

other: Salmonella typhimurium strains: TA100, TA98, TA1535, TA1537; E. coli : WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRECIPITATE: The test substance precipitated in the top agar at concentrations of 100 µg/plate and upwards. Precipitation of the test substance on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 µg/plate and upwards in all tester strains.

TOXICITY: No reduction of the bacterial background lawn and no biologically significant decrease in the number of revertants were observed.

MUTAGENICITY: No increase in the number of revertants was observed upon treatment with the test substance under all conditions tested (table 1). All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.

Table 1: Number of revertants in the control or after treatment with the test substance

First experiment (10 - 5000 µg/plate)
Strain	Metabolic activation system	mean revertants in Controls	maximum revertant factor	dose dependency	Assessment
TA 1535	no	11	1.27	no	negative
	yes	14	1.28	no	negative
TA 1537	no	12	0.67	no	negative
	yes	8	1.13	no	negative
TA 98	no	15	1.20	no	negative
	yes	22	1.27	no	negative
TA 100	no	85	1.24	no	negative
	yes	98	1.05	no	negative
E. coli WP2 uvrA	no	12	1.08	no	negative
	yes	10	1.40	no	negative
Second experiment (10 - 1000 µg/plate)
Strain	Metabolic activation system	mean revertants in Controls	maximum revertant factor	dose dependency	Assessment
TA 1535	no	18	1.11	no	negative
	yes	14	1.57	no	negative
TA 1537	no	3	1.67	no	negative
	yes	3	1.67	no	negative
TA 98	no	19	1.05	no	negative
	yes	19	1.11	no	negative
TA 100	no	68	1.16	no	negative
	yes	65	1.15	no	negative
E. coli WP2 uvrA	no	12	1.16	no	negative
	yes	13	1.38	no	negative

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Peripheral human lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9-mix

Test concentrations with justification for top dose:

0.3, 1 and 3 µg/ml

Vehicle / solvent:

Dimethyl sulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9- mix activation: Mitomycin - 0.5 µg/ml for a 3 h exposure period, 0.2 µg/ml for a 24 h exposure period and 0.1 µg/ml for a 48 h exposure period. + S9-mix activation: Cyclophosphamide - 15 µg/ml for a 3 h exposure period (24 h fixation time).

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 3 hours in the first cytogenetic assay; 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix in the second cytogenetic assay.
- Expression time (cells in growth medium): 20-22 hours in the first cytogenetic assay; 44-46 hours in the second cytogenetic assay.
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hours

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 u.g/ml medium)
STAIN (for cytogenetic assays): 10-30 min with 5% (v/v) Giemsa solution in tap water

NUMBER OF REPLICATIONS: in duplicate

NUMBER OF CELLS EVALUATED: At least 100 metaphase chromosomes per culture

DETERMINATION OF CYTOTOXICITY
- Method: The mitotic index as determined by counting the number of metaphases per 1000 cells. At least three analysable concentrations were used. Chromosomes of metaphase spreads were analysed of those cultures with an inhibition of the mitotic index of about 50 % or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations.

Type and identity of media: F10 complete culture medium consisted of Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 20 % (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Gibco), L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 (microg/ml respectively), sodium bicarbonate (1.2 g/l) and 30 U/ml heparin.

Evaluation criteria:

ACCEPTABILITY OF ASSAY:
A chromosome aberration test was considered acceptable if it met the following criteria:
a) The numbers of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range {min=0, max=5 (mean=0.8, standard deviation=1.0) aberrant cells per 100 metaphases in the absence of S9-mix; gaps excluded and min=0, max=5 (mean=0.8, standard deviation=0.9) aberrant cells per 100 metaphases in the presence of S9-mix; gaps excluded; for n=785 and 680 respectively}.
b) The positive control substances should produce a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
c) A homogeneous response between the replicate cultures is observed.

DATA EVALUATION AND STATISTICAL PROCEDURES:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) it induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
b) a statistically significant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each exposure group was compared to that of the solvent control using Chi-square statistics.
If P< 0.05 the hypothesis that the incidence of cells with chromosome aberrations is the same for both the treated and the solvent control group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95 % confidence level.

Species / strain:

other: Peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- PRECIPITATION: At a concentration of 3 µg/ml the test substance precipitated in the culture medium. Therefore, a concentration of 3 µg/ml was used as the highest concentration of the test substance.
- MUTAGENICITY: Under the conditions tested, the test substance is not clastogenic as it does not induce chromosome aberrations in cultured human peripheral blood lymphocytes.

Table 1: Chromosome aberrations in donor cultures treated with test substance

1st cytogenetic assay with and without metabolic activation (3h exposure time, 24 h fixation time)
Concentration (µg/mL)	Metabolic activation	Metaphases	Mitotic Index (%)	No. of cells with aberrations (+ gaps)	No. of cells with aberrations (-gaps)	Chromatid gaps	Chromosome gaps	Chromatid break	Chromosome break
Vehicle Ctrl.	without	200	100	3	0	3	0	0	0
	with	200	100	2	1	1	0	1	0
0.3	without	200	86	1	1	0	0	0	1
	with	200	88	1	0	1	0	0	0
1.0	without	200	75	1	1	0	0	1	0
	with	200	101	2	2	0	0	1	1
3.0	without	200	81	2	1	1	0	1	0
	with	200	94	2	1	1	0	1	0
Positive Ctrl.	without	200	79	44***	44***	0	0	21	18
	with	200	55	32***	32***	1	0	19	11
2nd cytogenetic assay without metabolic activation (24h exposure time, 24 h fixation time)
Concentration (µg/mL)	Metabolic activation	Metaphases	Mitotic Index (%)	No. of cells with aberrations (+ gaps)	No. of cells with aberrations (-gaps)	Chromatid gaps	Chromosome gaps	Chromatid break	Chromosome break
Vehicle Ctrl.	without	200	100	2	1	0	1	1	0
0.3	without	200	82	4	3	1	0	1	3
1.0	without	200	73	1	0	1	0	0	0
3.0	without	200	62	3	2	1	0	2	0
Positive Ctrl.	without	200	36	36***	35***	1	0	23	10
2nd cytogenetic assay without metabolic (48h exposure time, 48 h fixation time)
Concentration (µg/mL)	Metabolic activation	Metaphases	Mitotic Index (%)	No. of cells with aberrations (+ gaps)	No. of cells with aberrations (-gaps)	Chromatid gaps	Chromosome gaps	Chromatid break	Chromosome break
Vehicle Ctrl.	without	200	100	0	0	0	0	0	0
0.3	without	200	87	2	2	0	0	2	0
1.0	without	200	83	2	1	1	0	1	0
3.0	without	200	61	0	0	1	0	2	0
Positive Ctrl.	without	200	41	38***	37***	1	0	19	10
2nd cytogenetic assay with metabolic activation (3h exposure time, 48 h fixation time)
Concentration (µg/mL)	Metabolic activation	Metaphases	Mitotic Index (%)	No. of cells with aberrations (+ gaps)	No. of cells with aberrations (-gaps)	Chromatid gaps	Chromosome gaps	Chromatid break	Chromosome break
Vehicle Ctrl.	with	200	100	2	2	0	0	2	0
0.3	with	200	78	2	1	1	0	1	0
1.0	with	200	71	0	0	0	0	0	0
3.0	with	200	51	1	0	1	0	0	0
Positive Ctrl.	with	200	b)	39***	39***	0	0	29	12
*: p< 0.05
**: p < 0.01
***: p< 0.001
b) CP was fixed after 24 hours. Therefore, the mitotic index could not be calculated as percentage of control.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hgprt

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F12 medium, plus antibiotics and 10% FCS
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from induced rats

Test concentrations with justification for top dose:

1st Experiment
without S9 mix (4-hour exposure period)
0; 18.8; 37.5; 75; 150; 300; 600 μg/mL
with S9 mix (4-hour exposure period)
0; 37.5; 75; 150; 300; 600; 1 200 μg/mL
2nd Experiment
without S9 mix (24-hour exposure period)
0; 5; 10; 20; 40; 250; 500; 1 000; 2 000 μg/mL
with S9 mix (4-hour exposure period)
0; 5; 10; 20; 40; 250; 500; 1 000; 2 000 μg/mL
3rd Experiment
with S9 mix (4-hour exposure period)
0; 15; 30; 60; 300; 600; 1 000 μg/mL

Vehicle / solvent:

culture medium (Ham's F12)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation Migrated to IUCLID6: 300 μg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with metabolic activation Migrated to IUCLID6: 20 μg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 20 - 24h
- Exposure duration: 4h or 24h
- Expression time (cells in growth medium): 5 - 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 16 days

SELECTION AGENT (mutation assays): 6-thioguanine


NUMBER OF REPLICATIONS: Population doubling time 12 - 16h

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than
50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should fall within our historical negative control data range of 0 – 15.95 mutants per 10exp6 clonable cells
• The positive controls both with and without S9 mix must induce distinctly increased mutant frequencies (historical positive control data given in Appendix to study report)
• At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions should be tested. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.


A finding is assessed as positive if the following criteria are met:
• Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
• Evidence of reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a dos-response relationship.

Statistics:

Due to the negative findings, a statistical evaluation was not carried out.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

In all experiments in the absence and the presence of metabolic activation at least the highest concentrations tested for gene mutations were clearly cytotoxic. The 1st Experiment in the presence of metabolic activation missed the requirements of the

current OECD Guideline 476 and was discontinued, due to the observation of test substance precipitation in all dose groups. Thus, a repeat experiment was performed. On the basis from the results of the present study, the test substance did not cause any

biologically relevant increase in the mutant frequencies both without S9 mix and after adding a metabolizing system in three experiments performed independently of each other.

Conclusions:

The substance is not mutagenic in cultivated mammalian cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in bacteria:

In a GLP conform study according to OECD guideline 471, the potential of the test substance to induce gene mutations using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA was investigated in two independent experiments (Notox B.V., 2001).

In the first mutation assay, the test substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix. The test substance precipitated on the plates at dose levels of 1000 µg/plate and upwards. The bacterial background lawn was not reduced at all concentrations tested and no biologically significant decrease in the number of revertants was observed.

In the second mutation assay, the test substance was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix. The test item precipitated on the plates at the dose level of 1000 µg/plate. The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed.

The test substance did not induce a dose-related, two-fold, 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 the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

Gene mutation in mammalian cells:

In a GLP conform study according to OECD guideline 476, the substance was assessed for its potential to induce gene mutations in CHO cells in vitro (BASF 2011a)

After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations.

In all experiments in the absence and the presence of metabolic activation at least the highest concentrations tested for gene mutations were clearly cytotoxic. The 1st Experiment in the presence of metabolic activation missed the requirements of the current OECD Guideline 476 and was discontinued, due to the observation of test substance precipitation in all dose groups. Thus, a repeat experiment was performed. On the basis from the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies both without S9 mix and after adding a metabolizing system in three experiments performed independently of each other.

 

Cytogenicity in mammalian cells:

In a GLP conform study according to OECD guideline 473, the substance was assessed for its potential to induce structural chromosome aberrations in cultured peripheral human lymphocytes in vitro (Notox B.V., 2001). The possible clastogenicity of the test substance was tested in two independent experiments.

In the first cytogenetic assay, the test substance was tested up to 3 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix. The test substance precipitated in the culture medium at this dose level.

In the second cytogenetic assay, the test substance was tested up to 3 µg/ml for a 24 h or 48 h continuous exposure time with a 24 h or 48 h fixation time in the absence of S9-mix. In the presence of 1.8 % (v/v) S9-fraction the test substance was tested up to 3 µg/ml for a 3 h exposure time with a 48 h fixation time.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

The test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix, in two independently repeated experiments.

Under the conditions tested, the test substance is not clastogenic as it does not induce chromosome aberrations in cultured human peripheral blood lymphocytes.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008:

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008.