6-amino-1,3-dimethyluracil

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic Toxicity:

- in vitro: Gene mutation (Bacterial reverse mutation assay / Ames test): negative in all strains ± S9 (S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, E. coli WP2 uvr A, ±S9, OECD 471/472, GLP)

- in vitro: Gene mutation (Bacterial reverse mutation assay / Ames test): negative in all strains ± S9 (S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, ±S9, OECD 471)

- in vitro: Chromosome aberrations: positive -S9 (48h treatment, 48h fixation); negative –S9 (24h treatment, 24h fixation); negative +S9 (3h treatment, 24h fixation); negative –S9 (24h treatment, 24h fixation); negative +S9 (3h treatment, 24h fixation); (human lymphocytes, ± S9, OECD 473, GLP)

- in vitro: Cell cycle distribution (cytotoxicity): the proportion of cells in the G1/0-phase was increased, simultaneously the proportion of cells in the S-phase was reduced dose-dependently (CHO cells, 48h treatment)

- in vitro: Gene mutation in mammalian cells: negative ± S9 (mouse lymphoma L5178Y cells, ± S9, OECD 476, GLP)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-06-05 - 1997-06-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was performed acc. OECD TGs 471 and 472 of 1983, which are combined equal to the recent version of OECD TG 471.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Guideline no. 471: "Genetic Toxicology: Salmonella typhimurium Reverse Mutation Assay". (adopted May 26, 1983)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Version / remarks:

Guideline no. 472: "Genetic Toxicology: Escherichia coli Reverse Mutation Assay", with strain WP2uvrA only (adopted May 26, 1983)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

European Economic Community (EEC), Directive 92/69/EEC. Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity;
B.13: "Mutagenicity: Escherichia coli - Reverse Mutation Assay", with strain WP2uvrA only
B.14: "Mutagenicity: Salmonella typhimurium - Reverse Mutation Assay". EEC Publication no. L383 (adopted December, 1992)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark, stable under storage conditions

Target gene:

his (S. typhimurium); trp (E. coli)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:
Dr, Bruce N. Ames, University of California at Berkeley, U.S.A. Salmonella typhimurium strains)
TA100 received 18-02-1993, used batch: TA100.250297
TA98 received 21-02-1991, used batch: TA98.250297
TA1535 received 14-12-1994, used batch: TA1535.250297
TA1537 received 14-12-1994, used batch: TA1537.250297
- Suitability of cells: Recommended test system in international guidelines (e.g. EPA, OECD, EEC).

MEDIA USED
- Periodically 'cleansed' against high spontaneous background: yes
The Salmonella typhimurium strains were regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Prof. Dr. B.A. Bridges, University of Sussex, Brighton, U.K., Escherichia coli strain received 23-10-1987 used batch: EC.290197
- Suitability of cells: Recommended test system in international guidelines (e.g. EPA, OECD, EEC).

MEDIA USED
- Periodically 'cleansed' against high spontaneous background: yes
The strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male rat liver S9

Test concentrations with justification for top dose:

Top dose: 5 mg/plate
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.
Selection of an adequate range of doses was based on a dose range finding test with strain TA100 and the WP2uvrA strain, both with and without S9-mix. Eight concentrations were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay. The highest concentration of test article used in the subsequent mutagenesis assay was 5 mg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

methylmethanesulfonate

other: daunomycin (DM), TA98, 4 µg/plate in saline, -S9; 2-Aminoanthracene (2-AA), 5 µg/plate (WP2uvrA), 2.5 µg/plate (TA1537), 1 µg/plate (TA1535, TA98, TA100) in DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 10exp8 / plate

DURATION
- Exposure duration: 48h

SELECTION AGENT (mutation assays): his / trp minimal agar

NUMBER OF REPLICATIONS: The test substance were tested in triplicate in each strain and condition, in two independent experiments.

DETERMINATION OF CYTOTOXICITY
To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

Rationale for test conditions:

Recommended test system in international guidelines (e.g. EPA, OECD, EEC).

Evaluation criteria:

ACCEPTABILITY OF ASSAY

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:

a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain.

Strain S9 mix Minimum value Maximum value
TA1535 +S9 4 21
-S9 4 24
TA1537 +S9 3 22
-S9 3 28
TA98 +S9 12 50
-S9 14 58
TA100 +S9 55 223
-S9 63 247
WP2uvrA +S9 5 26
-S9 5 21

b) The positive control chemicals should produce responses in all tester strains which are within the laboratory historical range documented for each positive control substance.
Furthermore, the mean plate count should be at least two times the concurrent vehicle control group mean.

c) The selected dose range should include a clearly toxic concentration as demonstrated by the preliminary range-finding test or should extend to 5 mg/plate.

DATA EVALUATION AND STATISTICAL PROCEDURES
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.

Key result

Species / strain:

S. typhimurium TA 1535

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 applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

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 applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

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 applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium 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 applicable

Positive controls validity:

valid

Key result

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 applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none stated
- Effects of osmolality: none stated
- Precipitation: none observed

RANGE-FINDING/SCREENING STUDIES:
6-amino-1,3-dimethyluracil was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.
Precipitate
The test substance did not precipitate in the top agar.
Precipitation of 6-amino-1,3-dimethyluracil on the plates was not observed at the start or at the end of the incubation period in tester strain TA100 and WP2uvrA.
Toxicity
To determine the toxicity of 6-amino-1,3-dimethyluracil, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.
No reduction of the bacterial background lawn and no decrease in the number of revertants was observed.
Based on these data, 6-amino-1,3-dimethyluracil was tested in the mutation assay up to concentrations of 5000 µg/plate in the absence and presence of S9-mix.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
see acceptance criteria

Remarks on result:

other: consistent in both independent experiments

Conclusions:

The study was performed acc. OECD TGs 471 and 472 of 1983 being hence equal to the recent OECD TG 471. Positive and negative controls gave the appropriate results. Hence, the results can be considered to be sufficiently reliable to assess the potential of 6-amino-1,3-dimethyluracil to induce gene mutations in bacteria.
In strain TA100, in the presence of S9-mix, 6-amino-1,3-dimethyluracil showed 1.6-fold increases in the number of revertant (His+) colonies in both experiments. Since these increases were within the historical control data range and not two-fold, this test result is not considered to be a positive response. Strain TA100, in the absence of S9-mix, and all other 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.
Based on the results of this study it is concluded that 6-amino-1,3-dimethyluracil is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

6-amino-1,3-dimethyluracil 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 acc. OECD TGs 471 and 472 of 1983.

6-amino-1,3-dimethyluracil was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix.

In strain TA100, in the presence of S9-mix, 6-amino-1,3-dimethyluracil showed 1.6-fold increases in the number of revertant (His+) colonies in both experiments. Since these increases were within the historical control data range and not two-fold, this test result is not considered to be a positive response.

Strain TA100, in the absence of S9-mix, and all other 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.

The negative and strain-specific positive control values were within the laboratory background historical ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that 6-amino-1,3-dimethyluracil is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only 4 strains tested

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

20 - 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: [DMSO]
- Justification for choice of solvent/vehicle:

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 10 µg 2-aminoanthracene (dissolved in DMSO) for TA 100, TA 98, TA 1537 and TA 1535

Remarks:

with S-9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 5 µg N-methyl-N -nitro-N-nitrosoguanidine (MNNG) (dissolved in DMSO) for the strains TA 100 and TA 1535

Remarks:

without S-9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 10 µg 4-nitro-o-phenylendiamine (dissolved in DMSO) for the strain TA 98

Remarks:

without S-9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 100 µg 9-aminoacridine chloride monohydrate (dissolved in DMSO) for the strain TA 1537

Remarks:

without S-9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation;


DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results

Key result

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Preincubation test

Dose µg/plate	metabolic activation	TA98			TA100			TA1535			TA1537
		Trial 1	Trial 2	Trial 3	Trial 1	Trial 2	Trial 3	Trial 1	Trial 2	Trial 3	Trial 1	Trial 2	Trial 3
0	+	33	34	30	115	95	91	11	12	12	10	11	8
20	+	29	31	29	95	90	94	13	11	18	9	9	10
100	+	35	34	34	91	97	91	15	10	11	11	9	10
500	+	37	32	37	97	94	95	15	9	16	10	12	10
2500	+	31	33	31	92	90	106	15	12	15	9	9	8
5000	+	34	31	32	90	89	98	17	12	13	8	9	8
10 µg 2-Aminoanthracene	+	507	518	466	588	705	710	125	123	123	123	107	109
0	-	22	23	29	123	115	107	19	13	17	7	10	13
20	-	22	23	22	107	107	131	16	13	11	12	9	11
100	-	29	25	20	145	148	127	14	16	18	10	8	9
500	-	18	24	23	112	103	115	18	11	9	10	12	8
2500	-	21	21	18	121	121	111	15	13	16	11	12	5
5000	-	24	18	25	123	103	102	17	13	14	9	7	7
5 µg MNNG	-				893	898	836	1122	1028	1023
10 µg 4-Nitro-o-phenylendiamin	-	507	518	466
100 µg 9-amninoacridine chloride monohydrate	-										633	536	557

Standard Plate test

Dose µg/plate	metabolic activation	TA98			TA100			TA1535			TA1537
		Trial 1	Trial 2	Trial 3	Trial 1	Trial 2	Trial 3	Trial 1	Trial 2	Trial 3	Trial 1	Trial 2	Trial 3
0	+	31	32	32	123	114	116	13	14	15	13	9	8
20	+	32	32	34	104	106	102	17	14	10	8	11	7
100	+	35	28	31	105	108	100	12	13	13	8	11	8
500	+	36	33	33	106	101	103	14	22	11	9	10	11
2500	+	31	27	40	108	92	103	18	9	7	12	10	7
5000	+	29	26	35	88	107	105	10	7	7	11	14	8
10 µg 2-Aminoanthracene	+	1040	1130	1040	2050	2050	2150	241	255	193	144	145	114
0	-	24	24	21	113	118	108	-	16	18	8	10	9
20	-	21	22	22	98	95	104	13	13	14	14	8	11
100	-	21	17	18	118	121	115	17	18	17	8	10	7
500	-	20	30	23	125	111	123	-	12	17	8	7	12
2500	-	23	20	21	141	129	134	18	16	21	10	8	7
5000	-	20	18	20	122	128	135	17	15	-	8	9	5
5 µg MNNG	-				1670	1850	1820	2150	-	1690
10 µg 4-Nitro-o-phenylendiamin	-	765	818	804
100 µg 9-amninoacridine	-										473	513	434

Conclusions:

Interpretation of results (migrated information):
negative

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-06-11 - 1997-08-20 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Remarks:

in vivo micronucleus study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 473: "Genetic Toxicology: In Vitro Mammalian Cytogenetic Test", (adopted May 26, 1983).

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

European Economic Community (EEC), Directive 92/69/EEC. Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity; B.10: "Mutagenicity: In Vitro Mammalian Cytogenetic Test". EEC Publication no. L383 (adopted December, 1992).

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro mammalian cell chromosome aberration test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark, stable under storage conditions

Target gene:

n/a

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:
Healthy adult male volunteers
dose range finding study: age 30, AGT = 14,0 h (Dec. 1996)
experiment 1: age 33, AGT = 14.2 h (Dec. 1997)
experiment 2: age 30, AGT = 14.0 h (Dec. 1997)
(AGT- Average Generation Time of the celIs)
Blood samples were taken from healthy adult male volunteers by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. The blood samples were stored at a temperature between 4 and 25°C. Within 4 h after blood collection, lymphocyte cultures were started.
- Suitability of cells: Recommended test system in international guidelines (e.g. EPA, OECD, EEC).
- Sex, age and number of blood donors if applicable: see above
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Methods for maintenance in cell culture if applicable:
Cell culture conditions: Whole blood was cultured in F10 complete culture medium with Phytohaemagglutinin (Murex). Per culture (in the absence of S9-mix 5 ml F10 complete culture medium and in the presence of S9-mix 4.8 ml F10 complete culture medium and 0.4 ml whole blood) 0,1 ml (9 mg/ml) Phytohaemagglutinin was added.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
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-glutamlne (2 mM), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively), sodium bicarbonate (1.2 g/l) and 30 U/ml heparin.
All Incubations were carried out in a humid atmosphere (80-95%) containing 5% CO2 in air in the dark at 37°C. The temperature, humidity and CO2-percentage were monitored during the experiment.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

Range-finding study: 0, 100, 333, 1000, 3330, 5455 µg/ml
Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assay considering the highest dose level is 5000 µg/ml or has an inhibition of the mitotic index of 50% or greater whereas the mitotic index of the lowest dose level is approximately the same as the mitotic index of the solvent control.
Experiment 1
Without S9-Mix 100, 333, 560, 1000, 1778 and 2140 µg/ml culture medium (24 h treatment time, 24 h fixation time)
333, 560, 1000, 1778 and 2140 µg/ml culture medium (48 h treatment time, 48 h fixation time)
With S9-mix 1000, 3330 and 5000 µg/ml culture medium (3 h treatment time, 24 h fixation time)
5000 µg/ml culture medium (3 h treatment time, 48 h fixation time)
Experiment 2
Without S9-mix 100, 333 560, 1000, 1333, 1778 and 2140 µg/ml culture medium (24 h treatment time, 24 h fixation time)
With S9-mix 1000, 3330 and 5000 µg/ml culture medium (3 h treatment time, 24 h fixation time)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48h
- Exposure duration: 24 h and 48 h in the absence of S9-mix or 3 h in the presence of S9-mix.
- Expression time (cells in growth medium): After 3 h treatment, the celIs exposed to the test substance in the presence of S9-mix were rinsed once with 5 ml of HBSS and incubated in 5 ml culture medium for another 20-22 h (24 h fixation time) or for 44-46 h (48 h fixation time).
The cells which were treated for 24 h and 48 h in the absence of S9-mix were not rinsed after treatment but were worked up immediately after 24 h and 48 h (24 h and 48 h fixation time).
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h or 48 h

SPINDLE INHIBITOR (cytogenetic assays):
During the last 3 h of the culture period, cell division was arrested by addition of the spindle inhibitor colchicine (0.5 µg/ml medium).

STAIN (for cytogenetic assays): stained for 10-30 min with 5% (v/v) Giemsa solution in tap water

NUMBER OF REPLICATIONS: duplicates, and two slides / per culture

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
During the last 3 h of the culture period, cell division was arrested by addition of the spindle inhibitor colchicine (0.5 µg/ml medium). Thereafter the cell cultures were centrifuged for 5 min at 1300 rpm (150 g) and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol : acetic acid fixative (3:1 v/v).
Based on the mitotic index of the dose range finding test and the first cytogenetic assay, appropriate dose levels were selected for the second cytogenetic assay. For the independent repeat the 24 h fixation time is needed only.
Preparation of slides: Fixed cells were dropped onto cleaned slides which were immersed for 24 hours in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the study identification number and group number. Two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10-30 min with 5% (v/v) Giemsa solution in tap water. Thereafter slides were rinsed in tap-water and allowed to dry. The dry slides were cleared by dipping them in xylene before they were embedded in MicroHount and mounted with a coverslip.

NUMBER OF CELLS EVALUATED: The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): At least 100 metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was > 25 in 50 metaphases no more metaphases were examined. Only metaphases containing 46 chromosomes were analysed. The number of cells with aberrations and the number of aberrations were calculated.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Rationale for test conditions:

Recommended test system in international guidelines (e.g. EPA, OECD, EEC).

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 0.9, standard deviation=1.0) aberrant cells per 100 metaphases in the absence of S9-mix; gaps excluded and min=0, max=5 (mean=0.7, standard deviation=0.9) aberrant celIs per 100 metaphases in the presence of S9-mix; gaps excluded).
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
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:
a) 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 were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

STATISTICAL PROCEDURES
The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each treatment group was compared to that of the solvent control using Chi-square statistics:

Chi² = {(N-1) x (ad-bc)²} / {(a+b) (c+d) (a+c) (b+d)}

Where:
b = the total number of aberrant cells in the control cultures
d =the total number of nonaberrant celIs in the control cultures
n0 = the total number of cells scored in the control cultures
a = the total number of aberrant cells in treated cultures to be compared with the control.
c = the total number of nonaberrant cells in treated cultures to be compared with the control.
n1 = the total number of cells scored in the treated cultures
N = sum of n0 and n1

If P [Chi² > {(N-1) x (ad-bc)²} / {(a+b) (c+d) (a+c) (b+d)}] (two-tailed)
is small (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.

Key result

Species / strain:

lymphocytes: human

Remarks:

Experiment 1

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

Without S9-mix induced statistically and biologically significant increases in the number of cells with chromosome aberrations, all concentrations tested, with a dose-response relationship, at the 48 hours treatment time with a 48 hours fixation time.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human

Remarks:

Experiment 1

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

The test item did not induce statistically or biologically significant increase in the number of cells with chromosome aberrations at the 24 hours treatment time with a 24 hours fixation time.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human

Remarks:

Experiment 1

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

3 h treatment time, 24 h fixation time

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human

Remarks:

Experiment 2

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

24h treatment, 24h fixation, -S9; 3h treatment, 24h fixation, + S9

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
none stated

RANGE-FINDING/SCREENING STUDIES:
The pH and osmolarity of a concentration of 5000 µg /ml were 7.13 and 446 mOsm/kg respectively (compared to 7.18 and 475 mOsm/kg in the solvent control).
In the dose range finding, the highest concentration tested with the test item should be 5000 µg/ml. However, due to a dilution error the highest concentration tested in the dose range finding test was 5455 µg/ml. This had no further effect on the integrity of the results.

Conclusions:

Testing was performed scientifically reasonable according to OECD TG 473 under GLP. The number of cells with chromosome aberrations found in the solvent control cultures were within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. This test should be considered valid and the test item is clastogenic under the experimental conditions of this test in the absence of metabolic activation at the 48 hours treatment time with a 48 hours fixation time. The test item did not induce statistically or biologically significant increase in the number of cells with chromosome aberrations at the 24 hours treatment time with a 24 hours fixation time without S9, or in the presence of S9 after 3 h incubation and 24 h fixation time.
According to the recent OECD guideline (1997, Culture harvest time), In the first experiment, cells should be exposed to the test substance both with and without metabolic activation for 3-6 hours, and sampled at a time equivalent to about 1.5 normal cell cycle length after the beginning of treatment. If this protocol gives negative results both with and without activation, an additional experiment without activation should be done with continuous treatment until sampling at a time equivalent to about 1.5 normal cell cycle lengths. Certain chemicals may be more readily detected by treatment/sampling times longer than 1.5 cycle lengths. Auf der Maur P (Hum Genet. 1979 Jun 19;49(2):209-15) described the cell cycle length of human lymphocytes cultures in FCS as 14.7 hours, and that following PHA stimulation 90% of the lymphocytes divide once, about 65% divide for a second and about 40% divide for a third time.
So it can be concluded that the incubation time of 48 h was way longer than stipulated by the recent guideline, and the positive result could not be reliable and has to be treated with care. Further, after 48h incubation, a dose-dependent increase was noted above background, but not reaching the level of the positive control MMC-C. Per 200 cells, there were 1, 9, 12, and 17 cells with aberration and gaps for concentrations of 0, 560, 1000, and 1778 µg/ml, the latter having a mitotic index of 60%, compared to 72 aberrant cells of 0.1 µg/ml MMC-C. Hence it can be concluded that the substance is, if at all, not a very potent substance, and the assessment of the substance’ genotoxic properties should focus on the 3h and 24h incubation, which gave negative results.

Executive summary:

This report describes the effect of CAS 6642-31-5 on the induction of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix) in a OECD 473 study under GLP.

In the absence of S9-mix the test item was tested up to 1778 µg/ml for a 24 h treatment time with a 24 h fixation time and for a 48 h treatment time with a 48 h fixation time in the first experiment. In the second experiment it was tested up to 560 µg/ml for a 24 h treatment time with a 24 h fixation time.

 

In the presence of 1.8% (v/v) S9-fraction the test item was tested up to 5000 µg/ml for 3 h treatment time with a 24 h fixation time and for a 3 h treatment time with a 48 h fixation time in the first experiment. In the second experiment it was tested up to 5000 µg/ml for a 3 h treatment time with a 24 h fixation time.

 

Experiment 1

In the absence of S9-mix the test item induced statistically and biologically significant increases in the number of cells with chromosome aberrations, at all concentrations tested in the presence of a dose-response relationship, at the 48 hours treatment time with a 48 hours fixation time. the test item did not induce statistically or biologically significant increase in the number of cells with chromosome aberrations at the 24 hours treatment time with a 24 hours fixation time.

 

Experiment 2

Both in the absence and presence of S9-mix the test item did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations.

 

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.

 

Finally, it is concluded that this test should be considered valid and that the test item is clastogenic under the experimental conditions of this test in the absence of metabolic activation at. the 48 hours treatment time with a 48 hours fixation time. The test item did not induce statistically or biologically significant increase in the number of cells with chromosome aberrations at the 24 hours treatment time with a 24 hours fixation time without S9, or in the presence of S9 after 3 h incubation and 24 h fixation time.

Reference 4

Endpoint:

genetic toxicity in vitro, other

Remarks:

cell cycle distribution

Type of information:

experimental study

Adequacy of study:

other information

Study period:

1998-02-25 - 1998-02-27 (experimental phase)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

does not actually cover genotoxic endpoints as stipulated under REACH

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Analysis of any cell-cycle activity of Chinese hamster cells in vitro by flow cytometry

GLP compliance:

yes

Type of assay:

other: Analysis of Cell-Cycle Distribution

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
Cell line: ATCC (American Type Culture Collection) CCL 61 (ovary, Chinese hamster, CHO Kl).
- Source of cells: The CHO cell line ATCC CCL 61 (Chinese hamster ovary cells, CHO) was obtained from the AMERICAN TYPE CULTURE COLLECTION, Rockville, MD, USA.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
- Properly maintained: Yes. The cells were maintained in culture medium consisting of Nutrient Mixture F-12 supplemented with 10% fetal calf serum + Penicillin/Streptomycin 100 units/ml/100 µg/ml (Gibco AG, Basel, Switzerland) in 75 cm² tissue-culture (plastic) flasks. The cultures were incubated at 37°C in a humidified atmosphere of 85% at minimum containing 5% CO2. The cells were passaged twice weekly.
- Periodically checked for Mycoplasma contamination: Yes. The cell cultures were periodically checked for mycoplasma contamination.
- Periodically checked for karyotype stability: Genome stability of the cell line: The cell line CHO CCL 61 has been used for cytogenetic studies for several years. The stability of the genome of these cells is assessed regularly on the basis of the cytogenetic analysis of control cultures in the course of the cytogenetic studies. It is judged to be adequate for the particular purpose of cytogenetic studies.

Metabolic activation:

without

Test concentrations with justification for top dose:

100, 333, 560, 1000 and 1778 µg/ml culture medium
The purpose of the study was to get information on the cell cycle distribution of cultered cells exposed to similar conditions as the human lymphocytes in the previously performed chromosome aberration study.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

1% DMSO

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 8x10exp4 cells per flask of 75 cm² and 18 ml culture medium with 2 ml fetal calf serum

DURATION
- Preincubation period: 48h
- Exposure duration: 48 h

STAIN (for cytogenetic assays): DAPI-solution (5 µMol 4',6-diamidino-2-phenylindole in Tris-Puffer at pH 7.4 (Serva Heidelberg)

NUMBER OF REPLICATIONS: Each concentration including a solvent control was run in duplicate.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Rationale for test conditions:

The purpose of the study was to get information on the cell cycle distribution of cultered cells exposed to similar conditions as the human lymphocytes in the previously performed chromosome aberration study.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At time of harvest, no precipitates of the test substance were observed in the cultures.
- Other confounding effects: There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the test data.

Remarks on result:

other: After treatment of the culture with the test item at the concentration of 1778 µg/ml culture medium the proportion of cells in the G1/0-phase was increased by 53.3%. Simultaneously the proportion of cells in the S-phase was reduced by 53.4%.

Conclusions:

The study was conducted scientifically reasonable and is sufficiently documented to assess the influence of the test item on the cell cycle distribution. After treatment of the culture with the test item at the concentration of 1778 µg/ml culture medium the proportion of cells in the G1/0-phase was increased by 53.3%. Simultaneously the proportion of cells in the S-phase was reduced by 53.4%. This effect was also observed at the lower concentrations, however it was less pronounced. No effects were visible on the cells in the G2+M-phase.

Executive summary:

6-amino-1,3-dimethyluracil, identified as a light yellow powder, was investigated for cytotoxic activity on Chinese hamster ovary cells in vitro in the absence of a metabolic activation system by monitoring the cell cycle of the treated cells using flow cytometry.

The purpose of the study was to get information on the cell cycle distribution of cultured cells exposed to similar conditions as the human lymphocytes in the previously performed chromosome aberration study.

The test compound was suspended in DMSO and tested at the concentrations of 100, 333, 560, 1000 and 1778 µg/ml culture medium supplemented with fetal calf serum. DMSO was used as a solvent control at a final concentration of 10 µl/ml culture medium. The cells were treated for 48 hours without microsomal activation. Prior to cell harvest, the culture flasks were examined under a microscope for their cell growth.

Comparison with the solvent control revealed that at the concentration of 1778 µg/ml the proportion of cells in the G1/0-phase was increased by about 53%. The proportion of cells in the S-phase decreased by about 53%. The proportion of cells in G2+M-phase was similar to the control. At lower concentrations smaller changes in the proportions of the G1/0- and S-phase were also noted by comparison with the solvent control.

The number of cells present in the cultures at the end of the treatment was clearly reduced. This is confirmed by the result of microscopical inspection of the culture flask, which showed a reduced growth of cells in the treated cultures and by the increased proportion of control beads recorded in the treated cultures (used as internal standard).

 

The observations are consistent with two possible effects:

1) The relative increase of the cells in G1/0-phase indicates that these cells were hindered or delayed to enter the S-phase. This effect is also confirmed by the reduction of the relative amount of cells in the S-phase. The unchanged proportion of cells in G2+M-phase could indicate, that the number of cells leaving the S-phase was nearly constant. As a result of the treatment for 48 hours with the test item, the cells are assumed to be partially blocked in the G0/1-phase, whereas the cells in S-phase or G2+M-phase could pass these cell cycle phases normally. A calculation shows that at the highest concentration the delay or partial block in G1/0 caused an increase of the mean G1 phase by a factor of about 1.5.

2) Regarding the reduced cell growth in the cultures, a retardation of the cell cycle will have occurred during treatment with the test item. Additional, the cells are partially blocked to enter the S-phase just as to pass the G2+M-pliase. This can be explained by the facts of the relative increase of cells in G0/1-phase and the unchanged proportion of cells in the G2+M-phase, compared with the negative control. The proportion of cells passing the S-phase is reduced due to the cycle block, which took effect in the G1/S-phase.

 

CONCLUSIONS

It is concluded that under the given experimental conditions a remarkable concentration-dependent effect on the cell cycle was observed in Chinese hamster ovary cells treated in vitro with the test item.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-06-10 - 1997-07-14 (experimental phase)

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)

Version / remarks:

Organisation for Economic Co-operation and Development (OECD), OECO Guidelines for Testing of Chemicals, Guideline no. 476: "Genetic Toxicology: In Vitro Mammalian Cell Gene Mutation Tests", (adopted April 4, 1984).

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

European Economic Community (EEC), Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity; "Mutagenicity: In Vitro Mammalian Cell Gene Mutation Test". Directive 87/302/EEC, EEC Publication no. L133 (adopted May 30, 1988) and Directive 96/54/EEC, EEC Publication no. L248 (adopted September 30 1996).

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro mammalian cell forward gene mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark, stable under storage conditions

Target gene:

thymidine-kinase locus (TK-locus)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. A.G.A.C. Knaap, Department of Radiation Genetics and Chemical Mutagenesis of the State University of Leiden, The Netherlands (1981). This mouse lymphoma cell line was originally derived from the Fischer L5178Y line, isolated by Clive (1975).
- Suitability of cells: Recommended test system in international guidelines (e.g. EPA, OECD, EEC).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
F10 complete culture medium: F10 complete culture medium consisted of Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 10% (v/v) horse serum, L-glutamine (2 mM) and penicillin/streptomycin (50 U/ml and 50 µg/ml respectively).
Cell culture conditions: L5178Y mouse lymphoma cells were cultured in FIO complete culture medium. Cell density was preferably kept below 7 x 10exp5 cells/ml.
Exposition medium: Cells were exposed to the test item for 3 hours in F10 culture medium, buffered with 20 mM HEPES or for 24 hours in F10 complete culture medium.
Selective medium: Selective medium consisted of FIO complete culture medium, supplemented with 10% (v/v) horse serum and 5 µg/ml TFT (Sigma).
Non-selective medium: Non-selective medium consisted of F10 complete culture medium, supplemented with 10% horse serum.
Environmental conditions: All incubations were carried out in a humid atmosphere (80-95%) containing 5% CO2 in air in the dark at 37°C. The temperature and CO2-percentage were monitored during the experiment.
- Properly maintained: Stock cultures of these cells were stored in liquid nitrogen (-196°C).
- Periodically checked for Mycoplasma contamination: The cultures were checked for mycoplasma contamination.
- Periodically 'cleansed' against high spontaneous background: Prior to mutagenicity testing, the mouse lymphoma cells were grown for 1 day in F10 complete culture medium containing 10exp(-4) M hypoxanthine, 2 x 10exp(-7) M aminopterin and 1.6 x 10exp(-5) M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on medium containing hypoxanthine and thymidine only. After this period cells were returned to normal medium at least for 1 day before starting the experiment.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 33 to 5000 µg/ml in the absence and in the presence of S9-mix.
Both in the absence and presence of S9-mix no reduction was observed in the cell count of the remaining cells directly after treatment. In the cloning efficiency of the remaining cells no clear reduction in the number of colonies was observed compared to the solvent control.
Based on the results of the dose range finding test and the solubility test the following dose range was selected for mutagenicity testing:
Without S9-mix: 333, 1000, 3330 and 5000 µg/ml exposition medium
With S9-mix: 333, 1000, 3330 and 5000 µg/ml exposition medium
Based on the results of the first experiment, the following dose levels were selected for mutagenicity testing in experiment 2.
Without S9-mix: 333, 1000, 1778, 2344, 3330 and 5000 µg/ml exposition medium
With S9-mix: 333, 1000, 3330 and 5000 µg/ml exposition medium

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylsulphoxide of spectroscopic quality (Merck)

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): Per culture 8 x 10exp6 cells (10exp6/ml) were used

DURATION
- Preincubation period: none
- Exposure duration: Cell cultures in the absence of S9-mix were exposed for 24 hours to the test substance in exposition medium (F10 complete culture medium) and the cell cultures in the presence of S9-mix were exposed for 3 hours to the test substance in exposition medium
- Expression time (cells in growth medium): For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the 24 hours exposure time for the cell cultures without S9-mix. The cells were cultured for 3 days after the 3 hours exposure time for the cell cultures with S9-mix. During this culture period at least 4 x 10exp6 cells (if possible) were subcultured.
- Selection time (if incubation with a selection agent): For determination of the MF a total number of 5.76 x 10exp5 cells/ concentration were plated in three 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection). The microtiter plates for CE3 and MF were incubated for 11 days at 37°C in humified air with 5% CO2.
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: three 96-well microtiter plates (MF) per concentration, two independent experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After this incubation period the plates for the TFT-selection were stained for 2 h, by adding 0.5 µg/ml MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma) to each well. The plates for the CE3 and MF were scored with the naked eye or with the microscope.

NUMBER OF CELLS EVALUATED: each well contained 2000 cells in selective medium

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Rationale for test conditions:

Recommended test system in international guidelines (e.g. EPA, OECD, EEC).

Evaluation criteria:

ACCEPTABILITY OF ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls was > 50%.
b) At least at three of the four doses of the test substance, an acceptable number of surviving cells (10exp6) could be analysed for expression of the TK mutation.
c) The spontaneous mutant frequency in the untreated or solvent control was < 10 per 10exp5 clonable cells.
d) The positive controls (ethylmethanesulfonate and dimethylnitrosamine) induced significant (at least three-fold) increases in the mutant frequencies.
e) In the absence of S9-mix the selected dose range had to include a clearly toxic concentration or had to be treated for a 24 h exposure period

DATA EVALUATION AND STATISTICAL PROCEOURES
No formal hypothesis testing was done.
A test substance was considered positive (mutagenic) in the mutation assay if:
a) It induced at least a 3-fold increase in the mutant frequency compared to the solvent control in a dose-dependent manner; and
b) The results were reproducible in an independently repeated test.
A test substance was considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations showed a mutant frequency of at least three-fold compared to the solvent control.
b) The results were confirmed in an independently repeated test.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

see above

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

Experiment 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

With S9, after 24 h the cell count of the test substance concentration of 5000 µg/ml was reduced by 67% compared to the cell count of the solvent control. The cloning efficiency of the remaining eel Is direcUy after treatment showed a reduction of 95%.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

Experiment 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

-S9, 5000 µg/ml was too toxic for evaluation, after 24 h cell count of 3330 µg/ml was reduced by 63% compared to the cell count of the solvent control. The cloning efficiency of the remaining cells directly after treatment showed a reduction of 68%

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH / Effects of osmolality: The pH and osmolarity of a concentration of 5000 µg/ml were 7.13 and 446 mOsm/kg respectively (compared to 7.18 and 475 mOsm/kg in the solvent control).
- Precipitation: none stated

RANGE-FINDING/SCREENING STUDIES:
Dose range finding study
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 33 to 5000 µg/ml in the absence and in the presence of S9-mix.
Both in the absence and presence of S9-mix no reduction was observed in the cell count of the remaining cells directly after treatment. In the cloning efficiency of the remaining cells no clear reduction in the number of colonies was observed compared to the solvent control.
In the absence of S9-mix, the selected dose range did not include a clearly toxic concentration. Therefore, cell cultures in the mutagenicity test, in the absence of S9-fnix, were exposed for 24 hours to the test substance in exposition medium in the presence of serum.
Based on the results of the dose range finding test and the solubility test the following dose range was selected for mutagenicity testing:
Without S9-mix: 333, 1000, 3330 and 5000 µg/ml exposition medium
With S9-mix: 333, 1000, 3330 and 5000 µg/ml exposition medium

Remarks on result:

other:

Remarks:

In the absence of S9 metabolic activation, in the first experiment the test item induced a 8-fold significant increase in the mutant frequency at the TK-locus. However, this increase was observed only at one very toxic concentration. The relative cloning efficiency of the remaining cells was 5% compared to the solvent control, whereas only 33% of the cells initially survived the treatment. This means that the actual survival of the cells after treatment was only 1.6% (5% of 32%). Furthermore no dose relationship was observed.

Conclusions:

The study was conducted according to OECD 476 under GLP. Positive and negative controls gave the appropriate results. In conclusion, the test item is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

This report describes the effects of 6-amino-1,3-dimethyluracil on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells in the presence and absence of a metabolic system (S9-mix) according to OECD 476 under GLP.

In the first experiment, 6-amino-1,3-dimethyluracil was tested from 333 to 5000 µg/ml in the absence and presence of S9-mix. In the second experiment, 6-amino-1,3-dimethyluracil was tested from 333 to 3330 µg/ml in the absence of S9-mix and 6-amino-1,3-dimethyluracil was tested from 333 to 5000 µg/ml in the presence of S9-mix.

In the absence of S9 metabolic activation, in the first experiment, 6-amino-1,3-dimethyluracil induced a 8-fold significant increase in the mutant frequency at the TK-locus. However, this increase was observed only at one very toxic concentration. The relative cloning efficiency of the remaining cells was 5% compared to the solvent control, whereas only 33% of the cells initially survived the treatment. This means that the actual survival of the cells after treatment was only 1.6% (5% of 32%). Furthermore no dose relationship was observed.

In the absence of S9 metabolic activation, in the second experiment, 6-amino-1,3-dimethyluracil induced a 3-fold significant increase in the mutant frequency at the TK-locus at the test substance concentrations of 1778 and 3330 µg/ml. However, these increases were observed at toxic concentrations. The actual survival of the cells after treatment of 1778 µg/ml was only 9% (18% of 52%) and the actual survival of the cells after treatment of 3330 µg/ml was only 12% (32% of 37%). Furthermore, these increases were within our historical control data range.

Since, the increase in the first experiment was observed only at a very toxic concentration without a dose response relationship and the increases in the second experiment were observed only at toxic concentrations and were within our historical control data range, these increases were considered not to be biologically relevant and 6-amino-1,3-dimethyluracil is considered not to be mutagenic in the absence of S9-niix.

In the presence of S9 metabolic activation, in the first experiment, 6-amino-1,3-dimethyluracil induced a 3-fold significant increase in the mutant frequency at the TK-locus, However, no dose relationship was observed and this increase was observed only in one experiment. Therefore, this increase was considered not to be biologically relevant and 6-amino-1,3-dimethyluracil is considered not to be mutagenic in the presence of S9-mix.

Mutant frequencies induced by positive control chemicals were increased by 26-and 30-fold for EMS, in the first and second experiment respectively, and by 9- and 5-fold for DMN, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

It is concluded that 6-amino-1,3-dimethyluracil is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Genetic Toxicity:

- in vivo: Chromosome aberrations (Micronucleus test): negative (femur bone marrow erythrocytes, Swiss CD-1 mouse, m/f, 2000, 1000, 500 mg/kg, oral, single gavage, 24h or 48h, OECD 474, GLP)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-10-06 - 1997-11-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

European Economic Community (EEC), Directive 92/69/EEC. Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity; B.12: "Other Effects-Mutagenicity: In Vivo Micronucleus Test'. EEC Publication no. L383 (adopted December, 1992).

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for the Testing of Chemicals, Proposal for updating guideline 474: Mammalian Erythrocyte Micronucleus Test (Revised draft document, March 1996).

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vivo mammalian erythrocyte (somatic cell) micronucleus test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark, stable under storage conditions

Species:

mouse

Strain:

Swiss

Remarks:

CD-1(outbred-SPF-Quality)

Details on species / strain selection:

Recommended test system in international guidelines (e.g. EPA, FDA, OECD, EEC).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: Approximately 7 weeks
- Weight at study initiation: see attachments, mean weights of the respective treatment groups are given, varying from 33.0±1.6 - 35.8±3.1 g (males) and 25.6±2.2 - 28.8±2.5 g (females)
- Assigned to test groups randomly: Allocated to treatment groups as they came to hand from delivery boxes.
- Fasting period before study: yes, 3-4h
- Housing: The animals were housed in an air-conditioned room. Five animals per sex per group were housed in label led polycarbonate cages containing purified sawdust as bedding material (Woody SPF, supplied by B.M.I., Helmond, The Netherlands). Animals were identified by unique number on the tail.
- Diet (e.g. ad libitum): The animals were provided with standard pelleted laboratory animal diet (Carfil Quality BVBV, Oud-Turnhout, Belgium)
- Water (e.g. ad libitum): tap water
- Acclimation period: The acclimatisation period was at least 5 days before start of treatment under laboratory conditions. On arrival and prior to final assignment to study, all animals were clinically examined to ensure selected animals were in a good state of health.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-70%
- Air changes (per hr): 15/h
Fluctuations from these optimal conditions were noted, but were considered not to have affected integrity.
- Photoperiod (hrs dark / hrs light): The room is illuminated with 12 hours artificial fluorescent light and 12 hours dark per day.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 1% Aqueous carboxymethyl cellulose
- Amount of vehicle (if gavage or dermal): The dosing volume was 10 ml/kg body weight.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was suspended in 1% (w/v) carboxymethylcellulose (van 8oom B.V., Meppel, The Netherlands). The solution was treated with ultra-sonication to obtain a homogeneous suspension. Test substance concentrations were prepared directly prior to use.

Duration of treatment / exposure:

24h or 48h after single application

Frequency of treatment:

single application

Post exposure period:

24h or 48h

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

maximum tolerated (high) dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

intermediate dose

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

low dose

No. of animals per sex per dose:

5 / sex / dose
exception: females treated with 2000 mg/kg over 24 h (4 animals) and 48 h (6 animals)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
The positive control used in the micronucleus test was cyclophosphamide (CP; CAS no. 50-18-0; Endoxan, Asta-Werke, F.R.G.) dissolved in 0.9% (w/v) NaCl (Merck) in Milli-R0 water dosed at a single oral intubation of 50 mg/kg body weight.
- Route of administration: The route of administration and the volume administered of the positive control was the same as those of the test article.

Tissues and cell types examined:

femur bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The mice received a single oral intubation of a maximum tolerated (high), an intermediate and a low dose of the test item.
Selection of an adequate dose range for the Micronucleus test was based on a dose range finding study. Two dose groups, each comprising 3 males and 3 females, received a single dose of the test substance. The study duration was three days. During this period mortality and physical condition were recorded daily.
Based on the results of this dose range finding study dose levels of 2000, 1000 and 500 mg/kg body weight were selected as appropriate doses for the Micronucleus Test. After two hours, 2000 mg/bw animals were lethargic, then, no abnormalities were noted.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Single treatment, sampling was done after 24 or 48 h.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed by cervical dislocation at 24 and 48 h according to the above schedule after dosing of the vehicle, the test item and the positive control.
Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a smal1 opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of foetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min.
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide which was previously cleaned (24 h immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue) and marked with the study identification number and the animal number). The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were then air-dried and thereafter fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal.
Staining of the bone marrow smears: The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.

METHOD OF ANALYSIS:
Analysis of the bone marrow smears for micronuclei: All slides were randomly coded before examination. An adhesive label with study identification number and code was stuck over the marked slide At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

Evaluation criteria:

ACCEPTABILITY OF ASSAY
A micronucleus test is considered acceptable if it meets the following criteria:
a) The positive control substance induced a statistically significant (WiIcoxon Rank Sum Test, two-sided test at P (0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
b) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range.

DATA EVALUATION AND STATISTICAL PROCEDURES
A test substance is considered positive in the micronucleus test if: It induced a biologically as well as a statistically significant (WiIcoxon Rank Sum Test; two-sided test at P (0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.
A test substance is considered negative in the micronucleus test if: None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the combined data for both sexes nor in the data for male or female groups alone.
The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Statistics:

see above

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Animals treated with 2000 and 1000 mg/kg were lethargic on day 1

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: In a dose range finding study 12 animals (3 males and 3 females per group) were dosed orally with 2000 and 1000 mg/kg body weight (groups A and B respectively). 2h after dosinf, animals treated with 2000 mg/kg were lethargic. Based on the results of this dose range finding study dose levels of 2000, 1000 and 500 mg/kg body weight were selected as appropriate doses for the Micronucleus Test.
- Clinical signs of toxicity in test animals: Lethargy 2h after dosing, 2 females and 3 males dosed with 2000 mg/kg died.
- Evidence of cytotoxicity in tissue analyzed: Not observed in the range-finding study.
- Rationale for exposure: The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and use.

RESULTS OF DEFINITIVE STUDY
Mortality and systemic toxic signs: The mortality and systemic toxic signs of the animals treated with 2000 and 1000 mg/kg body weight are presented below. The animals treated with 500 mg/kg body weight showed no abnormalities. Negative and positive control groups showed no abnormalities also. Since two female animals died in group D, 2000 mg/kg body weight with a 48 h sampling time, animal no. 60 of group C, 2000 mg/kg body weight with a 24 h sampling time, was allocated to group D.
The mean number of micronucleated polychromatic erythrocytes scored in the test substance treated groups were compared with the corresponding control groups. Five male and five female animals were used in each treatment group.
- Induction of micronuclei (for Micronucleus assay):
Micronucleated polychromatic erythrocytes: No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of test substance treated animals.
The incidence of micronucleated polychromatic erythrocytes in the control animals were between or equal to the minimum and maximum value of the historical control data range {male: 0 and 3 (mean 0.7)) and {female: 0 and 3 (mean 0.5)}; indicated are means for n=126 and n=105 respectively.
Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes.
- Ratio of PCE/NCE (for Micronucleus assay):
Ratio polychromatic to normochromatic erythrocytes: The groups that were treated with the test item showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes
- Appropriateness of dose levels and route: The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and use.

Conclusions:

The study was conducted according to OECD 474 under GLP. Positive and negative controls gave the appropriate results. It is concluded that this test is valid and that the test item is not mutagenic in the micronucleus test under the experimental conditions described in this report.

Executive summary:

6-amino-1,3-dimethyluracil was tested in the Micronucleus Test in mice according to OECD 474 under GLP, to evaluate its genotoxic effect on erythrocytes in bone marrow.

Six groups each comprising 5 males and 5 females, received a single oral intubation.

Two groups were dosed with 500 mg/kg body weight, two groups were dosed with 1000 mg/kg body weight and two groups were dosed with 2000 mg/kg body weight.

Bone marrow from corresponding vehicle treated groups (A and B) served as negative controls. Bone marrow from a group (I) treated with a single oral intubation of cyclophosphamide (CP) at 50 mg/kg body weight served as positive control.

Bone marrow was sampled at 24 and 48 hours after dosing. Bone marrow from the positive control group (I), was harvested at 48 hours after dosing only.

No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with 6-amino-1,3-dimethyluracil.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes.

The groups that were treated with 6-amino-1,3-dimethyluracil showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls.

It is concluded that this test is valid and that 6-amino-1,3-dimethyluracil is not mutagenic in the micronucleus test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Problem formulation

The present MoAA aims to outline that there is no genotoxic potential of 1,3-Dimethyl-4-aminouracil.

 

Hypothesised Mode of action Statement

It is hypothesized that 1,3-Dimethyl-4-aminouracil does not exhibit a genotoxic mode of action, which is based on negative results in all Ames test, mouse lymphoma assay, in vivo micronucleus test and mostly in the available chromosome aberration assay. The only positive response is neither relevant in vivo nor directly related to a direct genotoxic mode of action.

 

Summary of data for use in Mode of Action Analysis

 

Species	Details / Dose	Results	References
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, E. coli WP2 uvr A	±S9, top dose: 5 mg/plate	negative	a) Ames test, OECD 471/472, GLP
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100	±S9, doses: 20 - 5000 µg/plate	negative	b)Ames test,OECD 471
human lymphocytes	Experiment 1: -S9, 48h treatment, 48h fixation,333, 560, 1000, 1778 and 2140 µg/ml	positive, dose-response	c) CA,OECD 473, GLP
human lymphocytes	Experiment 1:-S9,24h treatment, 24h fixation100, 333, 560, 1000, 1778 and 2140 µg/ml	negative	c) CA,OECD 473, GLP
human lymphocytes	Experiment 1: +S9,1000, 3330 and 5000 µg/ml (3h treatment, 24h fixation) / 5000 µg/ml (3h treatment, 48h fixation)	negative	c) CA,OECD 473, GLP
human lymphocytes	Experiment 2:-S9,24h treatment, 24h fixation100, 333 560, 1000, 1333, 1778 and 2140 µg/ml	negative	c) CA,OECD 473, GLP
human lymphocytes	Experiment 2: +S9,1000, 3330 and 5000 µg/ml (3h treatment, 24h fixation)	negative	c) CA,OECD 473, GLP
mouse lymphoma L5178Y cells	±S9, top dose: 5000 µg/ml	negative	d) MLA,OECD 476, GLP
Swiss CD-1 mouse, m/f, femur bone marrow erythrocytes	Doses: 2000, 1000, 500 mg/kg, oral, single gavage, 24h or 48h	negative	e) in vivo MNT, OECD 474, GLP
CHO cells	Doses: 100, 333, 560, 1000 and 1778 µg/ml, -S9	proportion of cells in the G1/0-phase was increased, simultaneously the proportion of cells in the S-phase was reduced dose-dependently	f) Cell cycle distribution (cytotoxicity)

 

 

Listing of key events identified for a specific Mode of Action

 

Key Event 1	No indication of genotoxic mode of action in most systems
Key Event 2	Dose-related increase of chromosome aberrations in human lymphocytes without S9 after48h treatment, 48h fixation
Key Event 3	Dose-related increase ofproportion of CHO cells in the G1/0-phase and reduction of proportion of cells in the S-phase

 

 

Bradford Hill Considerations for Weight of Evidence Analysis of available data/information for Mode of Action Analysis in experimental species

In the absence of S9-mix the test item induced statistically and biologically significant increases in the number of cells with chromosome aberrations, at all concentrations tested in the presence of a dose-response relationship, at the 48 hours treatment time with a 48 hours fixation time. The test item did not induce statistically or biologically significant increase in the number of cells with chromosome aberrations at the 24 hours treatment time with a 24 hours fixation time.

Both in the absence and presence of S9-mix the test item did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations.

 

According to the recent OECD guideline (1997, Culture harvest time), in the first experiment, cells should be exposed to the test substance both with and without metabolic activation for 3-6 hours, and sampled at a time equivalent to about 1.5 normal cell cycle length after the beginning of treatment. If this protocol gives negative results both with and without activation, an additional experiment without activation should be done with continuous treatment until sampling at a time equivalent to about 1.5 normal cell cycle lengths. Certain chemicals may be more readily detected by treatment/sampling times longer than 1.5 cycle lengths. Auf der Maur P (Hum Genet. 1979 Jun 19;49(2):209-15) described the cell cycle length of human lymphocytes cultures in FCS as 14.7 hours, and that following PHA stimulation 90% of the lymphocytes divide once, about 65% divide for a second and about 40% divide for a third time.

So it can be concluded that the incubation time of 48 h was way longer than stipulated by the recent guideline, and the positive result could not be reliable and has to be treated with care. Further, after 48h incubation, a dose-dependent increase was noted above background, but not reaching the level of the positive control MMC-C. Per 200 cells, there were 1, 9, 12, and 17 cells with aberration and gaps for concentrations of 0, 560, 1000, and 1778 µg/ml, the latter having a mitotic index of 60%, compared to 72 aberrant cells of 0.1 µg/ml MMC-C. Hence it can be concluded that the substance is, if at all, not a very potent substance, and the assessment of the substance’ genotoxic properties should focus on the 3h and 24h incubation, which gave negative results.

After this results, the possibility of modifications of cell cycle distributions was evaluated. The purpose of the cell cycle distribution analysisstudy was to get information on the cell cycle distribution of cultered cells exposed to similar conditions as the human lymphocytes in the previously performed chromosome aberration study. Any influence of a test compound on the DNA synthesis is detected by shifting the proportion of the G1/0-S- and G2+M-phases.

The flow histogram from the data obtained in the solvent control shows two typical peaks: a small peak with a maximum in channel 40 represents the internal standard beads, a second peak with its maximum in channel 100 represents the ceils in the G1/0- phase. During the S-phase, while the DNA content increases, the G1/0-peak moves to higher channel numbers with the maximum peak in channel 190, representing the G2+M-phase).

A comparison of the proportions of the G1/0-S- and the G2+M--phase of the treated cultures with those of the corresponding control culture indicates the influence of the test compound on the cell cycle activity.

After treatment of the culture with the test item at the concentration of 1778 µg/ml culture medium the proportion of cells in the G1/0-phase was increased by 53.3%. simultaneously the proportion of cells in the S-phase was reduced by 53.4%. This effect was also observed at the lower concentrations, however it was less pronounced. No effects were visible on the cells in the G2+M-phase.

The observations are consistent with two possible effects:

1) The relative increase of the cells in G1/0-phase indicates that these cells were hindered or delayed to enter the S-phase. This effect is also confirmed by the reduction of the relative amount of cells in the S-phase. The unchanged proportion of cells in G2+M-phase could indicate, that the number of cells leaving the S-phase was nearly constant. As a result of the treatment for 48 hours with the test item, the cells are assumed to be partially blocked in the G0/1-phase, whereas the cells in S-phase or G2+M-phase could pass these cell cycle phases normally. A calculation shows that at the highest concentration the delay or partial block in G1/0 caused an increase of the mean G1 phase by a factor of about 1.5.

2) Regarding the reduced cell growth in the cultures, a retardation of the cell cycle will have occurred during treatment with the test item. Additional, the cells are partially blocked to enter the S-phase just as to pass the G2+M-phase. This can be explained by the facts of the relative increase of cells in G0/1-phase and the unchanged proportion of cells in the G2+M-phase, compared with the negative control. The proportion of cells passing the S-phase is reduced due to the cycle block, which took effect in the G1/S-phase.

The retardation is commonly associated with toxic effects of the test item and may not directly be associated with a genotoxic event.

Further, the available in vivo micronucleus test (oral, gavage) revealed consistently negative results, in which the maximum dose possible was applied, and the sampling times were according to the guideline and hence appropriate.

No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with 6-amino-1,3-dimethyluracil. The groups that were treated with 6-amino-1,3-dimethyluracil showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis.

Hence, it can be clearly deducted that the observed induction of chromosome aberrations has no relevance for in vivo systems.

 

Uncertainties/Inconsistencies and Identification of Data Gaps

There are no uncertainties remaining. The given database is solid and provides even more information than stipulated under REACH for the present tonnage band. The database is of very high quality, and all possible inconsistencies were discussed above and shown to be not relevant.

 

Conclusions in relation to problem formulation

It was shown that that there is no genotoxic potential of 1,3-Dimethyl-4-aminouracil relevant for humans.

Justification for classification or non-classification

All available in vitro studies on 1,3-Dimethyl-4-aminouracil covering the endpoints gene mutation in bacteria and mammalian cells and micronucleus induction in human lymphocytes and mouse lymphoma cells and even in mice in vivo (micronucleus assay) gave negative results or were shown to pose no relevant potential in humans. All these stated above cover the main causes for genetic damage sufficiently and mimic due to the addition of S9 in vivo metabolism to a wide extent, and are partially even in vivo data. Based on the available data, there is no indication given that 1,3-Dimethyl-4-aminouracil should be classified as mutagen.