Prometryn

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames, +S9 negative, -S9 negative, S. typhimurium: TA1535, TA1537, TA98, TA100, OECD 471, Ogorek 1987

- in vitro mammalian cell gene mutation test (MLA), +S9 negative, -S9 negative, L5178/TK mouse lymphoma cells, equivalent to OECD 490, Beilstein 1985

- in vitro chromosome aberration study, +S9 negative, -S9 negative, human lymphocytes, equivalent to OECD 473, Strasser 1986

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:

17 Jul 1987 to 24 Aug 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no TA 102 or E.coli WP2 tested

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his-locus

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Aroclor 1254 induced rat liver
- concentration or volume of S9 mix and S9 in the final culture medium : 0.3 mL

Test concentrations with justification for top dose:

20, 78, 313, 1250 and 5000 µg/ 0.1 mL

Vehicle / solvent:

- Solvent used: acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

cyclophosphamide

other: daunorubicin-HCl (D-HCl), 9(5)-aminoacridine hydrochloride monohydrate (9-AHM), 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

PRELIMINARY TOXICITY TEST
A preliminary toxicity test is carried out with strain TA 100 without activation with the concentrations ranging from 0.08 to 5000 ug/0.1 mL. The protocol used is the same as in the main test.

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3 petri dishes
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 hours at 37 °C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition

Evaluation criteria:

CRITERIA FOR A POSITIVE RESPONSE
The test substance is considered to be positive in this test system if one or both of the following conditions are met:
- at least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration level for one or more of the following strains: TA 98, TA 1535 and TA 1537,
- a reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strain TA 100.
Generally a concentration-related effect should be demonstrable.

ASSAY ACCEPTANCE CRITERIA
A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TOXICITY TEST
Nine concentrations of the test material, ranging from 0.08 to 5000 µg/0.1 mL were tested to determine the highest concentration to be used in the mutagenicity assay. From the results obtained, the highest concentration suitable for the mutagenicity tests was found to be 5000 µg/0.1 mL.

TEST-SPECIFIC CONFOUNDING FACTORS
At the concentrations of 1250 and 5000 µg/0.1 mL the substance precipitated in soft agar.

Conclusions:

Under the conditions in the study (OECD 471, GLP), no evidence of the induction of point mutations by the test material, or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments.

Executive summary:

In a GLP compliant study performed according to OECD guideline 471 the test material was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The investigations were performed on strains TA 98, TA 100, TA 1535 and TA 1537 without and with microsomal activation at the following concentrations: 20, 78, 313, 1250 and 5000 µg/0.1 mL. In order to confirm the results, the experiments were repeated. 

The test material in solution was analysed to confirm the intended concentrations to be used in the mutagenicity tests. The measured concentrations were in agreement with the nominal test concentrations. In the experiments performed without and with microsomal activation, none of the tested concentrations of the test material led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. No signs of bacterial toxicity or precipitation were evident in the study.

No evidence of the induction of point mutations by the test material, or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments. The substance was found to be non-mutagenic in this study. 

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Jul 1985 to 17 Nov 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Principles of method if other than guideline:

The test material tested for mutagenic effects on L5178Y/TK mouse lymphoma cells in vitro. The investigations were performed with and without microsomal activation at concentrations of 10, 20, 40, 60, 80, 90 and 100 µg/mL. This test system permits the detection of forward point mutations in mammalian cells induced by chemical substances or by their metabolites. Mutagenic effects manifest themselves in the occurrence of mutants insensitive to 5-bromodeoxyuridine (BUdR). A mutagenic effect of a substance is demonstrable on comparison of the number of colonies in the treated and control cultures. To ensure that any mutagenic effects of metabolites of the test substance formed in mammals are also detected, experiments are performed in which the cultures are treated with the compound in the presence of an activation mixture (rat-liver microsomes and co-factors).

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
L5178Y/TK +/- cells with the selected concentrations at a cell density of 3 x 10^5 cells/mL in round-bottomed flasks.

MEDIA USED
F10P-medium (Fischer’s medium plus antibiotics and 10% hose serum)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Rat liver microsomal fraction S9 was prepared from Aroclor 1254 induced livers of RAI rats (Tif: RAIf(SPF).
- S9 mix contained: 0.50 mM NADP, 2.92 mM isocitrate, 2.33 mM tris(hydroxymethyl)-amino-methane, 0.17 mM MgCl2 and 8.33 µL/mL S9-fraction.

Test concentrations with justification for top dose:

10, 20, 40, 60, 80, 90 and 100 µg/mL

Vehicle / solvent:

- Solvent used: ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

CYTOTOXICITY TEST
A preliminary toxicity test was performed to determine the concentrations to be used in the mutagenicity assay. The concentration to be selected as the highest for the mutagenicity assay is that causing approximately 85% reduction in the relative suspension growth in comparison with the control after a 4-hour treatment followed by a 72-hour suspension growth. If in the toxicity test an 85% reduction in the relative suspension growth is not obtainable at any concentration, the highest concentration is also used as the highest in the mutagenicity experiment.
The L5178Y/TK +/- cells used in this preliminary test were taken from cultures in the exponential phase of growth. The substance was dissolved in Ethanol and applied in F10P-medium (Fischer’s medium plus antibiotics and 10% horse serum). The final concentration of the solvent (Ethanol) in the medium was 0.1%.The cells were exposed for four hours to seven concentrations ranging from 1.563 to 100 µg/mL of the test substance. After removal of the test substance, the cells were washed and incubated in the same growth medium (F10P) for three days. Cell counts were performed and registered daily and the cell number in each case was adjusted to the initial count (3 x 10^5 cells/mL).The percentages of the relative suspension growth in comparison with the control were evaluated .This preliminary toxicity test was performed with and without metabolic activation.
The concentration calculated to produce about 85% reduction in the relative suspension growth in comparison with the control is used as the highest in the mutagenicity experiment together with six further concentrations corresponding to factors of 0.9, 0.8, 0.6, 0.4, 0.2 and 0.1.

MUTAGENICITY TEST
The mutagenicity test was carried out by treating L5178Y/TK +/- cells with the selected concentrations at a cell density of 3 x 10^5 cells/mL in round-bottomed flasks. The procedure employed is based on that reported by CLIVE and SPECTOR (1975).
T/T +/- cells from growing stock cultures were cleansed of spontaneous TK -/- mutants by exposure for 24 hours to THMG (a combination of thymidine, hypoxanthine, methotrexate and glycine). This procedure ensures a low background of TK -/- mutant clones. After an additional three-days incubation period in THG (thymidine, hypoxanthine and glycine) medium the cells are ready to be used for the mutagenicity experiment.
The cells were treated for four hours, both in the presence and absence of rat liver S9-activation system, with the six preselected concentrations of the test substance, with the positive control, or with the solvent, or remained untreated as negative control. Ethylmethane sulfonate (EMS) 0.75 µL/mL, a mutagen not requiring S9 activation, and dimethylnitrosamine (DMN) 4.00 µL/mL which requires activation, were used as positive controls.
After treatment, the cells were washed once with 25 mL F10P to remove the test substance, resuspended in F10P medium and allowed grow for three days to express the induced forward TK -/- mutants. Cell counts were performed and registered daily and the cell number in each case was adjusted to the initial count (3 x 10^5 cells/mL).
At the end of the expression period, for mutant selection as well as for viability control cultures were set up in culture tubes containing 5 mL of a semi-solid agar cloning medium. For mutant selection, eight tubes were prepared at each concentration containing 4 x 10^5 cells per tube in cloning medium with BUdR at a final concentration of 50 µg/mL. For viability control four tubes for each concentration were set up containing 200 cells per tube in cloning medium without BUdR.
The incubation time was 16 days for mutant selection and 12 days for viability control in the experiments with and without metabolic activation. At the end of the incubation period, the numbers of colonies in the mutagenicity-test tubes and in viability control cultures were determined with the aid of a Colony Counter. The values obtained from the viability control served to normalize the results received from the mutagenicity, i.e. to calculate according to a 100% viability of the cells seeded in cultures of the mutagenicity test.
The results are expressed in termes of the number of induced TK -/- mutants/10^6 surviving cells.

Evaluation criteria:

- The test substance is generally considered to be mutagenic in this test system if the colony count exceeds that of the solvent control by a factor of more than 2.5 at any concentration.
- Positive control values are not used as reference points, but are also included to ensure the current cell population response to direct and pro-mutagens under appropriate treatment conditions.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
In a preliminary toxicity test, with concentrations ranging from 1563 to 100 µg/mL, in the experiments with and without microsomal activation an 85% reduction in the relative suspension growth was not obtained and therefore the concentrations of 10, 20, 40, 60, 80, 90 and 100 µg/mL were used or mutagenicity testing with and without metabolic activation.

MUTAGENICTY TEST
In the mutagenicity test without metabolic activation the mutant frequency of the solvent control was 51.3 x 10^-6. The mutant frequencies of the treated cultures were 35.7 x 10^6 at the highest concentration and 44.0 x 10^-6, 33.3 x 10^6, 26.8 x 10^-6, 39.0 x 10^-6, 34.1 x 10^-6 and 33.9 x 10^-6 down to the lowest. Thus, mutant factors of 0.70, 0.86, 0.65, 0.52, 0.76, 0.67 and 0.66 were calculated. By contrast, the positive control yielded a mutant-frequency value of 312.5 x 10^-6, which in comparing with the respective control value of 15.9 x 10^-6 gave a factor of 19.7.

In the study of effects of treatment with the test material in the presence of activation mixture, the mutant frequency value of the solvent control was 36.63 x 10^-6. The mutant frequencies of treated cultures were 59.5 x 10^-6 at the highest concentration and 43.3 x 10^-6, 44.9 x 10^-6, 69.2 x 10^-6, 51.3 x 10^-6, 35.9 x10^-6 and 32.5 x 10^-6 at the six concentrations down to the lowest. Comparison of the mutant frequencies of the treated cultures with that of the solvent control revealed a factor of 1.64 at the highest concentration and factors of 1.19, 1.24, 1.91, 1.42, 0.99 and 0.90 down to the lowest. In the concurrent positive-control study with metabolic activation using DMN (4.00 µg/mL),comparison with the negative control yielded a factor of 3.52.

Conclusions:

It is concluded that under the given experimental conditions no evidence of mutagenic effects of the test material was observed in this mammalian forward mutation system.

Executive summary:

The test material was tested for mutagenic effects on L5178Y/TK mouse lymphoma cells in vitro, in a non-GLP study equivalent to an OECD guideline 490 study. The investigations were performed with and without microsomal activation at concentrations of 10, 20, 40, 60, 80, 90 and 100 µg/mL. No signs of bacterial toxicity or precipitation were evident in the study. In the experiment performed without microsomal activation, the number of mutant cell colonies in all treatment groups was lower than in the solvent control. In the investigation in which the cultures were additionally treated with an activation mixture, comparison of the frequency of mutant colonies in the treated cultures with that of the solvent control gave factors of less than 2 at all test concentrations. Thus, in the investigations with and without microsomal activation a mutant factor greater than 2.5 was not detectable at any of the concentrations tested. The concurrent positive control with EMS (0.75 µL/mL) without activation gave a factor of 19.7 in comparison with the solvent control. In the experiment with metabolic activation, the positive control substance DMN (4.00 µL/mL) produced a 3.52-fold increase in the number of mutant cell colonies in comparison with the solvent control.

It is concluded that under the given experimental conditions no evidence of mutagenic effects of the test material was observed in this mammalian forward mutation system.  

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 May 1986 to 24 Sep 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human blood

Details on mammalian cell type (if applicable):

The human blood in this experiment was obtained from a normal donor by venipuncture. The white cells were separated from heparinized peripheral blood by spontaneous sedimentation of the erythrocytes and thereafter maintained in conventional blood culture medium.

Cytokinesis block (if used):

Colcemide (0.4 µg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Rat liver microsomal fraction S9 was prepared from Aroclor 1254 induced livers of male RAI rats. The co-factors used were NADP and Iso-citric acid.
- 1.0 mL S9 mix mixture contained: 0.15 mL S9 fraction and 0.2 mL of the solution with the co-factors and 0.65 mL medium. In the experiments in which the substance was metabolically activated, 1.0 mL of the activation mixture was added to 9.0 mL of cell suspension.

Test concentrations with justification for top dose:

- without S9: 7.5, 15.0, 30.0, 60.0 and 120.0 µg/mL
- with S9: 20.0, 40.0, 80.0 and 160.0 µg/mL

Vehicle / solvent:

- Solvent used: DMSO
- The final concentraiton of the solvent i the medium was 1%

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

CYTOXICITY TEST
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the mutagenicity assay. The concentration to be selected as the highest for the mutagenicity assay is that causing approximately 50% suppression of mitotic activity in comparison with the control after a 3-hour treatment followed by a 24-hour recovery phase. The human blood in this experiment was obtained from a normal donor by venipuncture. The white cells were separated from heparinized peripheral blood by spontaneous sedimentation of the erythrocytes and thereafter maintained in conventional blood culture medium. The preincubation time before treatment was 46 hours. The substance dissolved in DMSO was added (1:100) to the cell suspension in chromosome medium .The cells were exposed for three hours to fourteen concentrations ranging from 0.12 to 1000 µg/mL of the test substance. After removal of the test substance, the cells were washed and incubated in chromosome medium for 24 hours. The percentages of mitotic suppression in comparison with the controls were evaluated by counting at least 1000 cells per concentration. This preliminary toxicity test was performed with and without metabolic activation.
The concentration calculated to produce about a 50% suppression of mitotic activity in comparison with the control is used as the highest in the mutagenicity experiments together with four further concentrations corresponding to factors of 0.5, 0.25, 0.125, 0.0625.

MUTAGENICITY TEST
The mutagenicity tests were carried out by treating human lymphocytes with the selected concentrations (7.5, 15.0, 30.0, 60.0 and 120.0 µg/mL without microsomal activation and 10.0, 20.0, 40.0, 80.0 and 160.0 µg/mL with microsomal activation). The white blood cells were prepared in the same manner as in the toxicity test. About 46 hours before exposure to the test substance, a series of Falcon flasks was seeded with human lymphocytes. Subsequently, the cells were treated for three hours, both in the presence and in the absence of rat-liver S9 activation system, with the five preselected concentrations of the test substance, with the positive control, or with the vehicle as negative control. In the experiments in which the substance was metabolically activated, 1.0 mL of an activation mixture was added to 9.0 mL of cell suspension. 1.0 mL activation mixture contained: 0.15 mL S9 fraction of liver from rats induced with Aroclor 1254 and 2.0 mL of a solution of co-factors and 0.65 mL medium. Mitomycin C 0.8 µg /ml, a mutagen not requiring S9 activation, and cyclophosphamide 5.0 µg/mL, which requires activation, were used as positive controls. After treatment, the cells were washed twice with 10 mL Hanks BSS to remove the test substance, resuspended in chromosome medium and allowed to grow for 43.5 hours. Two and a half hours prior to harvesting, the cultures were treated with Colcemide 0.4 µg/mL. The experiment was terminated by hypotonic treatment (0.075 M KCl solution) of the cells, followed by fixation (methanol:acetic acid, 3:1). Drop preparations were made by the air-drying technique.

SCORING OF SLIDES
Prior analysis the slides were coded. 100 complete metaphase figures altogether from cultures of
following aberrations:
a) specific aberrations: breaks, exchanges, deletions, fragments and minutes,
b) unspecific aberrations: gaps, premature chromosome condensation and chromosome decay,
c) numerical aberrations (metaphases with >2n chromosomes).

Key result

Species / strain:

lymphocytes: human blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
The highest concentration selected for the mutagenicity assay without microsomal activation was 120.0 µg/mL and with microsomal activation was 160.0 µg/mL.

MUTAGENICITY TEST
One hundred metaphase plates from two culture flasks each from the cultures treated with the various concentrations of the test substance, with the vehicle alone or with the positive control substances were examined. In the experiment without microsomal activation, at the control cultures and at the cultures treated with the test material, with the concentrations of 7.5, 15.0, 30.0 and 60.0 µg/mL no specific aberrations were found. At the concentration of 120.0 µg/mL one metaphase with one minute and one metaphase with two minutes were detected. One unspecific aberration in the form of a chromatid gap was found at the concentration of 120.0 µg/mL. In the experiment with microsomal activation, the chromosome displays of the cultures treated with the vehicle alone and of the cultures treated with the concentrations of 40.0 and 80.0 µg/mL revealed no metaphases with specific chromosomal aberrations. At the concentrations of 10.0 and 20.0 µg/mL one metaphase each with a minute was found. The preparations of the cultures treated with the concentration of 160.0 µg/mL were not scoreable as a result of the cytotoxic property of the substance. Three unspecific aberrations in the form of chromatid gaps were found at the concentration of 20.0 µg/mL. Treatment of the cultures with mitomycin C, 0.8 µg/mL, and cyclophosphamide, 10.0 µg/mL, as positive controls caused a marked increase in most types of aberrations.

Conclusions:

It is concluded that, under the given experimental conditions (GLP), no evidence of mutagenic effect was obtained in human lymphocytes in vitro treated with the test material.

Executive summary:

The test material was tested for mutagenic effects on human lymphocytes in vitro in a GLP compliant study equivalent to an OECD guideline 473 test. The experiments were performed without microsomal activation at concentrations of 7.5, 15.0, 30.0, 60.0 and 120.0 µg/mL and with microsomal activation at concentrations of 10.0, 20.0, 40.0, 80.0 and 160.0 µg/mL. This test system permits the detection of structural chromosome aberrations in human lymphocytes in vitro induced by the test substance or by its metabolites. One hundred metaphase plates from the vehicle control, from the cultures treated with the various concentrations of the test material and from the positive controls were examined. Due to cytotoxicity observed in a preliminary cytotoxicity test, the highest concentrations tested were limited to 120 g/mL without and to 160 g/ml with microsomal activation. Precipitation was not observed in the study. In the experiment performed without microsomal activation, no metaphases with specific chromosomal aberrations were found in the cultures treated with the vehicle alone or with the concentrations of 7.5, 15.0, 30.0, 60.0 µg/mL of the test material. At the concentration of 120.0 µg/mL one metaphase with a specific chromosomal aberration in the form of a minute, i.e. a form of chromosomal intra-arm interchange, and one metaphase with two minutes were seen. In the experiment performed with microsomal activation, the cultures treated with the vehicle alone and the cultures treated with the concentrations of 40.0 and 80.0 µg/mL revealed no metaphases with specific chromosomal aberrations. One metaphase with a minute was registered each at the concentrations of 10.0 and 20.0 µg/mL. Owing to the cytotoxicity of the substance at the highest concentration of 160.0 µg/mL, a metaphase analysis was not feasible. In both experiments the incidence of specific chromosomal aberrations was within the frequency observed generally in control cultures and therefore can be considered spontaneous in origin. The treatment of the cultures with mitomycin-C, 0.8 µg/mL and cyclophosphamide, 10.0 µg/mL, respectively (positive controls), resulted in a high incidence of specific chromosomal aberrations (18% and 6%).

It is concluded that, under the given experimental conditions, no evidence of chromosomal aberrations was obtained in human lymphocytes treated in vitro with the test material. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- in vivo mouse erythrocyte micronucleus test, negative, OECD 474, Ceresa 1988

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

29 Sep 1987 to 6 Oct 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

26 May 1983

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

19 September 1984

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

June 19, 1987

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

Tif:MAGf

Sex:

male/female

Route of administration:

oral: gavage

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The results indicate that under the given experimental conditions (OECD 474, GLP) no evidence of mutagenic effects was obtained in mice treated with the test material.

Executive summary:

The test material was administered by oral gavage to groups of 8 female and 8 male mice each in the control, 480, 960 and 1920 mg/kg dose groups in a GLP compliant study performed according to OECD guideline 474. In the first part of this study the animals were treated once with the highest applicable dose of 1920 mg/kg and sacrificed 16, 24 and 48 hours thereafter. In the second part of this study the animals were treated once with doses of 480, 960 and 1920 mg/kg and sacrificed 24 hours thereafter. Smears were prepared from the bone marrow of all treated animals. The test material in suspension was analysed to confirm the intended concentration to be used in the mutagenicity tests. The measured concentrations were in agreement with the nominal concentrations. The experiments were performed to evaluate any mutagenic effect on polychromatic erythrocytes in bone marrow cells in vivo. In the first experiment the bone marrow smears for the animals treated with the dose of 1920 mg/kg of the test material showed no statistically significant increase (p>0.05) in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at the sampling times of 16, 24 and 48 hours. Therefore, the animals were sacrificed 24 hours after treatment in the second part of the experiment. Also in this second experiment the bone marrow smears from the animals treated with 480, 960 and 1920 mg/kg of test material showed no statistically significant increase (p>0.05) in the number of micronucleated polychromatic erythrocytes in comparison with the animals of the negative control group. The respective positive control experiments with cyclophosphamide (64 mg/kg) yielded an average of 2.22% (first part) and 2.25% (second part) polychromatic erythrocytes with micronuclei. This is significantly different from the controls (0.06% and 0.03% respectively) treated with the vehicle (CMC 0.5%) alone.

The results indicate that under the given experimental conditions no evidence of mutagenic effects was obtained in mice treated with the test material.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro OECD 471 (Ogorek 1987)  

In a GLP compliant study performed according to OECD guideline 471 the test material was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The investigations were performed on strains TA 98, TA 100, TA 1535 and TA 1537 without and with microsomal activation at the following concentrations: 20, 78, 313, 1250 and 5000 µg/0.1 mL. In order to confirm the results, the experiments were repeated. 

The test material in solution was analysed to confirm the intended concentrations to be used in the mutagenicity tests. The measured concentrations were in agreement with the nominal test concentrations. In the experiments performed without and with microsomal activation, none of the tested concentrations of the test material led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. No signs of bacterial toxicity or precipitation were evident in the study.

No evidence of the induction of point mutations by the test material, or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments. The substance was found to be non-mutagenic in this study. 

    

In vitro, equivalent to OECD 490 (Beilstein 1985)  

The test material was tested for mutagenic effects on L5178Y/TK mouse lymphoma cells in vitro, in a non-GLP study equivalent to an OECD guideline 490 study. The investigations were performed with and without microsomal activation at concentrations of 10, 20, 40, 60, 80, 90 and 100 µg/mL. No signs of bacterial toxicity or precipitation were evident in the study. In the experiment performed without microsomal activation, the number of mutant cell colonies in all treatment groups was lower than in the solvent control. In the investigation in which the cultures were additionally treated with an activation mixture, comparison of the frequency of mutant colonies in the treated cultures with that of the solvent control gave factors of less than 2 at all test concentrations. Thus, in the investigations with and without microsomal activation a mutant factor greater than 2.5 was not detectable at any of the concentrations tested. The concurrent positive control with EMS (0.75 µL/mL) without activation gave a factor of 19.7 in comparison with the solvent control. In the experiment with metabolic activation, the positive control substance DMN (4.00 µL/mL) produced a 3.52-fold increase in the number of mutant cell colonies in comparison with the solvent control.

It is concluded that under the given experimental conditions no evidence of mutagenic effects of the test material was observed in this mammalian forward mutation system.  

    

In vitro, equivalent to OECD 473 (Strasser 1986)  

The test material was tested for mutagenic effects on human lymphocytes in vitro in a GLP compliant study equivalent to an OECD guideline 473 test. The experiments were performed without microsomal activation at concentrations of 7.5, 15.0, 30.0, 60.0 and 120.0 µg/mL and with microsomal activation at concentrations of 10.0, 20.0, 40.0, 80.0 and 160.0 µg/mL. This test system permits the detection of structural chromosome aberrations in human lymphocytes in vitro induced by the test substance or by its metabolites. One hundred metaphase plates from the vehicle control, from the cultures treated with the various concentrations of the test material and from the positive controls were examined. Due to cytotoxicity observed in a preliminary cytotoxicity test, the highest concentrations tested were limited to 120 µg/mL without and to 160 µg/ml with microsomal activation. Precipitation was not observed in the study. In the experiment performed without microsomal activation, no metaphases with specific chromosomal aberrations were found in the cultures treated with the vehicle alone or with the concentrations of 7.5, 15.0, 30.0, 60.0 µg/mL of the test material. At the concentration of 120.0 µg/mL one metaphase with a specific chromosomal aberration in the form of a minute, i.e. a form of chromosomal intra-arm interchange, and one metaphase with two minutes were seen. In the experiment performed with microsomal activation, the cultures treated with the vehicle alone and the cultures treated with the concentrations of 40.0 and 80.0 µg/mL revealed no metaphases with specific chromosomal aberrations. One metaphase with a minute was registered each at the concentrations of 10.0 and 20.0 µg/mL. Owing to the cytotoxicity of the substance at the highest concentration of 160.0 µg/mL, a metaphase analysis was not feasible. In both experiments the incidence of specific chromosomal aberrations was within the frequency observed generally in control cultures and therefore can be considered spontaneous in origin. The treatment of the cultures with mitomycin-C, 0.8 µg/mL and cyclophosphamide, 10.0 µg/mL, respectively (positive controls), resulted in a high incidence of specific chromosomal aberrations (18% and 6%). It is concluded that, under the given experimental conditions, no evidence of chromosomal aberrations was obtained in human lymphocytes treated in vitro with the test material. 

    

In vivo OECD 474 (Ceresa 1988)  

The test material was administered by oral gavage to groups of 8 female and 8 male mice each in the control, 480, 960 and 1920 mg/kg dose groups in a GLP compliant study performed according to OECD guideline 474. In the first part of this study the animals were treated once with the highest applicable dose of 1920 mg/kg and sacrificed 16, 24 and 48 hours thereafter. In the second part of this study the animals were treated once with doses of 480, 960 and 1920 mg/kg and sacrificed 24 hours thereafter. Smears were prepared from the bone marrow of all treated animals. The test material in suspension was analysed to confirm the intended concentration to be used in the mutagenicity tests. The measured concentrations were in agreement with the nominal concentrations. The experiments were performed to evaluate any mutagenic effect on polychromatic erythrocytes in bone marrow cells in vivo. In the first experiment the bone marrow smears for the animals treated with the dose of 1920 mg/kg of the test material showed no statistically significant increase (p>0.05) in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at the sampling times of 16, 24 and 48 hours. Therefore, the animals were sacrificed 24 hours after treatment in the second part of the experiment. Also in this second experiment the bone marrow smears from the animals treated with 480, 960 and 1920 mg/kg of test material showed no statistically significant increase (p>0.05) in the number of micronucleated polychromatic erythrocytes in comparison with the animals of the negative control group. The respective positive control experiments with cyclophosphamide (64 mg/kg) yielded an average of 2.22% (first part) and 2.25% (second part) polychromatic erythrocytes with micronuclei. This is significantly different from the controls (0.06% and 0.03% respectively) treated with the vehicle (CMC 0.5%) alone. The results indicate that under the given experimental conditions no evidence of mutagenic effects was obtained in mice treated with the test material.

Justification for classification or non-classification

Based on the available data classification for genetic toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.