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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: negative


HPRT: negative


Chromosomal aberration test: negative


UDS assay: negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1982-06-15 to 1982-07-02
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
purity of test item not stated; bacterial strains did not include TA102 or E.coli WP2 (to detect oxidizing mutagens or cross-linking agents)
GLP compliance:
no
Remarks:
At the time the study was performed, GLP was not compulsory.
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:

- source of S9: rat liver

- method of preparation of S9 mix: Male Emd: Wi-AF/Han (SPF) rats weighing 100 - 160 g (aged 7 - 8 weeks) are given a single i.p. injection of Aroclor 1254 (500 mg/kg body weight) diluted in peanut oil. The animals receive drinking water and Altromin standard diet ad libitum. On day 4, about 16 hours before sacrifice, the animals remain without food. On day 5 the animals are sacrificed. Their abdomens are thoroughly disinfected with 70% ethanol, are opened and the livers removed. The livers are collected in ice-cooled sterilized beakers containing 0.15 M KCl.
The beakers, previously tared, are then weighed and the liver transferred to a sterile glass potter homogenizer with Teflon pestle containing 3 mL of 0.15 M KCl per each gram of liver wet-weighed. The homogenate is transferred to sterilized steel centrifuge tubes and spun at 9000 xg for 15 minutes at 0 – 2°C. Finally, the supernatant fluid is decanted and transferred into sterilized and precooled plastic tubes. The S-9 is then frozen in a dry ice-acetone mixture and stored at -80°C. The protein content of the S-9 preparation is determined by the biuret method. To 2 mL S-9 (or albumin solution as standard) 2 mL cold trichloroacetic acid solution (20 %) are added. The precipitate is washed with cold water and then solved in 0.2 M NaOH by ultrasonic technique. After diluting in 0.2 M NaOH the biuret reagent is added and the mixture incubated at 37°C. Then extinction is read at 546 nm. The S-9 mix was prepared according to Ames et al (1975).

- concentration or volume of S9 mix and S9 in the final culture medium: 0.1 mL S-9 in S-9 mix; 4.8 mg S-9-protein per mL S-9 mix

- quality controls of S9: Every solution and S-9 mix used in an experiment is routinely tested by plating 0.1 mL (or 0.5 mL) each on a nutrient broth agar plate. In case of a positive result, the whole experiment would be considered invalid.
Test concentrations with justification for top dose:
125, 250, 500, 1250, 2500 and 5000 µg/plate
Test articles are investigated up to the limit of solubility or toxicity.
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: The test item is soluble in DMSO.
- Justification for percentage of solvent in the final culture medium: less than 1%
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-1,2-phenylene diamine
Remarks:
TA1538, without S-9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA98, without S-9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-Methyl-N-nitro-N-nitrosoguanidine
Remarks:
TA1535, without S-9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA98, TA100, TA1535, TA1537, TA1538, with S-9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537, without S-9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: daunomycin
Remarks:
TA98, without S-9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1-Ethyl-2-nitro-3-nitrosoguanidine
Remarks:
TA100, TA1535, without S-9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA100, without S-9 mix
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 4
- Number of independent experiments: 2

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: reduction in number of spontaneous revertants, a cleaning of the background lawn, degree of survival
Evaluation criteria:
Not specified.
Statistics:
Kruskal-Wallis-Test
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True 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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True 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:
cytotoxicity
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTOR
- Precipitation and time of the determination: Because of the low solubility of the test article in the test medium it precipitated from the top agar layer at 1250, 2500, and 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES
- In a preliminary experiment, toxicity is determined in a wide range of chemical concentrations (50 - 10 000 µg/plate). Toxicity becomes evident by a reduction in the number of spontaneous revertants, a clearing of the background lawn, or by the degree of survival of treated cultures: Test articles are investigated up to the limit of solubility or toxicity. Since the test item is non-toxic in the preliminary test it was tested up to 5000 µg/plate in the main test.

STUDY RESULTS
- Cytotoxicity results: The compound was tested over a series of concentrations. The choice of the concentration range was mainly influenced by the endeavor to achieve a toxic effect in the highest concentration. This effect was not observed in these experiments macroscopically or during the routinely conducted microscopical examination of the plates with the exception of one tester strain (TA100) where a toxic effect was observed at 5000 µg/plate.
- Concurrent vehicle negative and positive control data: The validity of the mutation assay can be assessed by the results obtained for the positive and negative controls. The negative control mutant frequencies were all in the normal range, and the positive control copounds yielded normal mutant frequencies that were greatly in excess of the background.
- Data of main test: The results of the experiments conducted on the compound in the absence and presence of a metabolic system were all negative. The repeat experiments were also negative. These results show that the test article has no mutagenic potential in this test system. Statistical evaluation of the data using the Kruskal-Wallis-Test proved that no significant increase in revertant colonies occurs when the test article is examined in this test system.

HISTORICAL CONTROL DATA: not reported
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine Operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: rat liver
- method of preparation of S9 mix: rats have been treated with Aroclor 1254 (500 mg/kg bw). On day 5 the animals are sacrificed and livers are removed. The livers are collected in ice-cooled sterilized beakers containing 0.15 M KCl and homogenized. The homogenate was centrifuged at 9000 xg for 15 minutes.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.1 mL
- quality controls of S9: not specified
Test concentrations with justification for top dose:
10, 50, 100, 500, 1000, 5000 µg/plate
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: The test item is soluble in DMSO.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
TA1535, without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)-acrylamide
Remarks:
TA 98, TA 100, E.coli, without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537, without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA1538, without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
TA100, TA98, TA1538, TA1535, TA1537, E.coli, with S9-mix
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)
Evaluation criteria:
Not specified.
Statistics:
none
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True 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:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True 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:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True 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:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True 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:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1985-11-27 to 1985-11-29
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Version / remarks:
1986
Deviations:
yes
Remarks:
no statistics reported
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: unscheduled DNA synthesis (UDS) test
Species / strain / cell type:
hepatocytes:
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: The hepatocyte suspension was prepared from the liver of 8- 12 weeks old male WISTAR CF HB rats (weight 150 - 200 g). After narcotizing the rats with sodium pentobarbital the liver was perfused at a rate of 15 mL/min for 15 minutes through the vena portae with modified Seglen's medium adjusted to pH 7.4 and maintained at 37° C. Then the liver was perfused for 20 min with the perfusion medium supplemented with collagenase (0.05 % w/v, type IV). The perfusion was terminated after 25 - 35 minutes when the liver was swollen. The hepatocytes were removed from the liver and washed twice with the perfusion solution without collagenase. The crude cell suspension was filtered through a 40 µm stainless steel mesh to yield a single cell suspension. For estimating the viability the cells were stained with trypan blue. The viability obtained by this preparation method was 86 %.
- Suitability of cells: Rat hepatocytes are non-dividing cells and are predominantly not in S-phase. Rat hepatocytes are non-transformed cells. They also retain normal detoxification pathways and may, therefore, be more realistic cellular test units than cells from most continuous cell line.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:
The isolated hepatocytes were washed, centrifugated and transferred into L-15 culture medium supplemented with HEPES (15 mM), penicillin (100 µg/mL), (100 µg/mL), glucose (1.5 mg/mL), insulin (0.5 µg/mL) and NaHCO3 (2.2 mg/mL). The medium was adjusted to pH 7.4. The cells have been incubated at 37°C.
Metabolic activation:
not applicable
Metabolic activation system:
Primary rat hepatocytes retain normal detoxification pathways and therefore no external metabolic activation system was needed.
Test concentrations with justification for top dose:
1, 3.3, 10, 33, 100 µg/mL
The toxicity of the test substance was determined in a pre-experiment by measuring the dose-related incorporation of radioactive thymidine up to the limit of solubility of the test substance. According to these data the dose range was chosen.
Vehicle / solvent:
- Solvent used: acetone
- Justification for choice of solvent: Substance is not soluble in medium or water.
- Justification for percentage of solvent in the final culture medium: Final concentration in medium 1% is nontoxic to cells.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
25.64 µg/mL medium in DMSO
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 6
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 3.5 -4 x10E6 in 4 mL medium supplemented with hydroxyurea (15 mM)
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
Aliquots containing 3.5 - 4 x 10E6 hepatocytes were incubated in 4 mL of culture medium supplemented with hydroxyurea (15mM) in flasks for one hour in an orbital water bath set at 100 oscillations per minute. Then the dissolved test substance and (3H)-TdR (0.7 µCi/mL) was added and the incubation was continued for another 3 hours. If a non-aqueous solvent is used, its final concentration does not exceed 1 %. The incubation temperature was 37° C.
The 4 hour incubation period was terminated by placing the flasks into an ice bath. The cells were washed twice with 2 mL of ice-cold phosphate-buffered saline and thymidine (0.5 mg/mL) was added to each sample. The nuclei of the cells were isolated by lysing the cells with 2 mL of buffer (10 mM Tris-HCl; 15 mM 1.5 mM MgC12; Nonidet P40 0.5%, pH 8.5). The lysis was accomplished after 10 minutes and the nuclei were pelleted.
Isolation of DNA:
The nuclei were lysed in 2.5 mL "lysis solution" (2.5 mM EDTA; 2 % SDS; 0.1 M glycine; 1 mg/mL proteinase K, pH 10) for 30 minutes. The DNA was precipitated by adding 2.5 L trichloro-acetic acid (TCA), 10 %. This preparation was kept at 4°C overnight. The DNA was spun down the next day and the pellet was redissolved in 1 mL of TCA 5 % (20 minutes at 90°C).
LSC determination:
0.2 mL aliquots were taken for liquid scintillation counting in Aqualuma (BAKER, Deventer, The Netherlands) using a liquid counter. (Packard TRI-CARB 460 CD Liquid Scintillation System)
Determination of the DNA content of the samples:
0.2 mL aliquots were taken for the determination of the DNA content by colorimetric estimation according to the method described by BURTON (1956).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: The toxicity of the test substance was determined by measuring the dose-related incorporation of radioactive thymidine up to the limit of solubility of the test substance.
Evaluation criteria:
Positive responses are these in which a clear increase in the (3H)-TdR incorporation above the level of the negative controls can be demonstrated for at least one concentration of the test substance. By a case by case decision biometrical analysis is applied to confirm the increase. When inconclusive results are obtained a second independent experiment is recommended. A test substance is regarded as negative, when the (3H)-TdR incorporation is indistinguishable from the background level up to cytotoxic concentrations or the limit of solubility, respectively.
Statistics:
Not performed.
Key result
Species / strain:
hepatocytes:
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Concentrations higher than 100 µg/mL precipitated in the medium.

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test substance was determined in a pre-experiment by measuring the dose-related incorporation of radioactive thymidine up to the limit of solubility of the test substance. According to these data the dose range was chosen. Five concentrations over two decades were tested.
Concentration in µg/mL % Survival after 3 hours treatment
absolute relative
0 73 100
0.4 55 75
1.0 60 82
4.0 64 88
10.0 50 68
40.0 41 56
100.0 59 81

STUDY RESULTS
- The (3H)-TdR incorporation given as disintegrations per minute (dpm) per µg DNA was not enhanced in the experimental groups treated for three hours with concentrations of 1.0; 3.3; 10.0; 33.0; 100.0 µg/mL of the test substance. The values for dpm/µg DNA are comparable to that of the negative control. In addition no indication of a concentration dependency was observed. The positive control substance DMBA induced a 2,1 fold increase of the dpm/µg DNA as compared to the negative control thus demonstrating the sensitivity of the test system.

Table 1: Measured radioactivity in testing solutions






































Dose Group



dpm / µg DNA (mean± SD)



Negative control (solvent)



138.3 ±82.7



1.0 µg/mL



153.1 ±77.3



3.3 µg/mL



85.4±27.4



10.0 µg/mL



123.8±22.5



33.0 µg/mL



90.7±49.9



100.0 µg/mL



69.3±19.1



Positive control



283.7±38.1



 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1985-11-11 to 1985-12-13
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Version / remarks:
1983
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
The gene for HGPRT located on the X-chromosome of the V79 cells.
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: cellbank of test laboratory
- Suitability of cells: The V79 cell line has been used for many years in experiments with success.
- Absence of Mycoplasma contamination: not specified
- Methods for maintenance in cell culture: subcultured twice a week
- Cell cycle length, doubling time or proliferation index : doubling time 12-16 h
- Modal number of chromosomes: 22

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: MEM medium supplemented with 10% fetal calf serum (FCS). Cells have been cultured at 37°C, 5% CO2
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: rat liver
- method of preparation of S9 mix: The S9 liver microsomal fraction is obtained from the liver of 8 - 12 weeks old male Wistar rats, strain CFHB (weight ca. 150-200 g) which had been given a single i. p. injection of 500 mg/kg bw Aroclor 1254 in olive oil 5 days previously. After cervical dislocation the livers of 5 animals are removed, washed in 150 mM KCl and homogenized. The homogenate, diluted 1:3 in KCl centrifuged cold at 9000 g for 10 minutes. The supernatant which contains microsomes is frozen in ampoules of 2 or 5 mL and stored in liquid nitrogen. For standardization a protein estimation is made.

- concentration or volume of S9 mix and S9 in the final culture medium: Before the experiment an appropriate quantity of S9 supernatant is thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.3 mg/mL in the cultures. The concentration in the S9 preparation is between 30 and 45 mg/mL. The composition of the cofactor solution is concentrated to yield the following concentrations in the S9 mix: 8 mM MgCl2; 33 mM KCl; 5 mM glucose-6-phosphate; 5 mM NADP; 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment the S9 mix is stored in an ice bath.

- quality controls of S9: not specified
Test concentrations with justification for top dose:
5, 10, 25 and 50 µg/mL
The test substance could be solubilized up to 50 µg/mL, higher concentrations precipitated in the medium.
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: The solvent was chosen because it proved to be relatively nontoxic in the concentrations applied.
- Justification for percentage of solvent in the final culture medium: final concentration in the medium 1 % is non-toxic.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
Remarks:
with S9 mix, 15.4 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 mix, 1 mg/mL
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1x 10E6 cells per flask in 30 mL medium for mutagenicity test
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hours at 37°C

Treatment scheme:
Day 1: Subculturing of a log-phase culture
a) About 400 cells in 5 mL medium/25 cm2-plastic-flask for plating efficiency; in duplicate per experimental point
b) 1 x 10E6 cells in 30 mL medium/175 cm2-plastic-flask for the mutagenicity test, 1 flask per experimental point
Day 2: Treatment of a) and b)
Day 5: Subculturing of b) in 175 cm2-plastic flasks.
Day 8: Fixation and staining of colonies in a)-flasks = plating efficiency and dose relationship
Day 9: Subculturing of b) in five 80 cm2-plastic-flasks containing selective medium: mutant selection (about 6 x 10E5 cells/flask): subculturing of b) in two 25 cm2-flasks for plating efficiency (about 500 cells/flask). For the selection of mutants the medium was supplemented with 11 µg/mL thioguanine.
Day 16: Fixation and staining of colonies in b) - derived flasks seeded on day 9.
Staining was done with 10% methylene blue in 0.01 n-KOH solution. The stained colonies with more than 50 cells were counted with a preparation.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: plating efficiency
Evaluation criteria:
The test substance is classified as mutagenic if it induces with at least one of the test substance concentrations reproducibly a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment.

The test substance is classified as mutagenic if there is a reproducible concentration - related increase in the mutation frequency. Such evaluation may be considered independently of the enhancement factor for the induced mutants.

However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate as compared to the range normally found (10-30 mutants per 10E6 cells) a seemingly concentration-related increase of the mutations within this range will be regarded as to be irrelevant.
Statistics:
Not necessary to perform as there is no reproducible increase of the mutation rates in the test groups treated with the test substance .
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: no cytotoxicity, but tested up to maximum solubilisable concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: The test substance could be solubilized up to 50 µg/mL. Higher concentrations precipitated in the medium.

RANGE-FINDING/SCREENING STUDIES
- The toxicity of the test substance was determined in a pre-experiment by establishing the concentration related plating efficiency. According to these data the concentration range of 5, 10, 25 and 50 µg/mL was chosen in the main test with and without S9 mix.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: The sensitivity of the test system was demonstrated by the enhanced mutation rates found in the groups treated with the positive control substances compared to the negative control.

- Results from cytotoxicity measurements: No cytotoxicity was detected, plating efficiency was 84.3 - 106.7% in test substance-treated cultures across the two experiments performed.

- Genotoxicity results: With no concentration of the test substance a reproducible enhancement of the mutation rate over the range of the negative controls was found, neither without nor with metabolic activation by S9 mix.

HISTORICAL CONTROL DATA: "spontaneous mutation rate [...] normally found" was given as 10-30 mutants per 10E6 cells.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1985-08-19 to 1985-09-20
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
Version / remarks:
1983
Deviations:
yes
Remarks:
only short exposure treatment protocol was used, historical control data was not reported
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: cellbank of test laboratory
- Suitability of cells: The V79 cell line has been used for many years in experiments with success.
- Absence of Mycoplasma contamination: not specified
- Methods for maintenance in cell culture: subcultured twice a week
- Cell cycle length, doubling time or proliferation index : doubling time 12-16 h
- Modal number of chromosomes: 22

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: MEM medium supplemented with 10% fetal calf serum (FCS). Cells have been cultured at 37°C, 5% CO2
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: rat liver
- method of preparation of S9 mix: The S9 liver microsomal fraction is obtained from the liver of 8-12 weeks old male Wistar rats (weight ca. 200-250 g) which had been given a single i.p. injection of 500 mg/kg bw Aroclor 1254 in olive oil 5 days previously. After cervical dislocation the livers of 5 animals are removed, washed in 150 mM KCl and homogenized. The homogenate, diluted 1:3 in KCl is centrifuged cold at 9000 g for 10 minutes. The supernatant which contains microsomes is frozen in ampoules of 2 or 5 mL and stored in liquid nitrogen.
- concentration or volume of S9 mix and S9 in the final culture medium: Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.3 mg/mL. The protein concentration in the S9 preparation is between 30 and 45 mg/mL. This gave the S9 mix. The composition of the cofactor solution was as follows: 8 mM MgCl2; 33 mM KCl; 5 mM glucose-S-phosphate; 5 mM NADP; 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment the S9 mix was stored in ice bath.
- quality controls of S9: not specified
Test concentrations with justification for top dose:
4, 25 and 50 µg/mL with and without S9 mix
Higher concentrations of test substance could not be achieved due to lack of solubility.
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: The solvent was chosen because it proved to be relatively nontoxic in the concentrations applied.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9-mix, 2.79 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9-mix, 1.25 mg/mL
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 4
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 3 x 10E4 - 2 x 10E5 cells per Quadriperm dish
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Test substance was added in medium with 2% FCS and with or without S9 mix for 4 hours.
- 5, 15.5 or 25.5 h after start of treatment, colcemid was added and cells were prepared 2-2.5 hours later for analysis
- total time from start of treatment to fixation: 7, 18 or 28 hours.

CHROMOSOME ABERRATION:
- Spindle inhibitor: 5, 15.5 and 25.5 h after the start of the treatment colcemid was added (0.2 µg/mL culture medium)
- Methods of slide preparation and staining technique used including the stain used: Cells were treated on the slides with a hypotonic solution (0.4 % KCl) for 20 min at 37°C. After incubation in the hypotonic solution the cells were fixed with glacial acetic acid/methanol 1:3. The next day the cells were stained with aceto-orcain.
- Number of cells spread and analysed per concentration: The selection of metaphases was performed on coded slides using ZEISS and LEITZ photomicroscopes with attached television equipment. The number of chromosomes per metaphase was determined on the television monitor. Only metaphases with 22 ± 1 chromosomes were included in the analysis. 400 well spread metaphases were scored per experimental group (= 100 metaphases/slide). Gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural aberrations. In the positive control groups with EMS and CPA only 200 metaphases were scored.
- Determination of polyploidy: no
- Determination of endoreplication: no

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
1.The test substance is classified as mutagenic if it induces with one of the concentrations tested a significantly increased aberration rate as compared with the negative controls. The significance is obvious either by an enhancement of the rate clearly over the control range or it is proven by adequate biometry.
2.The test substance is classified as mutagenic if there is a concentration related increase in the aberration rates as compared with the solvent controls.
3. The test substance is considered negative when item 1. and 2. do not apply.
Statistics:
Not necessary to perform as the chromosomal aberration rates found in the groups treated with the test substance are in the range of the negative (solvent) control.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 50 µg/mL with S9 mix at 7 and 18 h preparation time
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Not observed for the used concentrations in the main study.

RANGE-FINDING/SCREENING STUDIES: In preliminary testing, the concentration of 50 µg/mL without S9 did not reduce the mitotic activity whereas it did in the presence of S9 at sampling intervals of 7 and 18 hours. No concentration above 50 µg/mL could be achieved because of the limited solubility of the test material.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: The sensitivity of the test system was demonstrated by the clearly enhanced mutation rates found in the groups treated with the positive control substances compared to the negative control.
- Results from cytotoxicity measurements: see table
- Genotoxicity results: With no concentration of the test substance an enhancement of the chromosome aberration rates over the range of the negative controls was found, neither without nor with metabolic activation by S9 mix.

HISTORICAL CONTROL DATA: Not provided.

Mitotic index






















































































































































































Substance



test substance concentration [µg/ml]



preparation
time (hours)



S9 mix



                               mitotic index*


                 absolute(%)       relative (%)



Negative control



-



7



-



3.0



100.0



Test substance



50



7



-



3.0



100.0



Solvent control



0



7



+



2.7



100.0



Test substance



50



7



+



1.7



64.5



Negative control



-



18



-



3.4



100.0



Solvent control



0



18



-



3.7



100.0



Positive-
control EMS



-



18



-



3.6



98. 6



Test substance



4



18



-



2.7



71.6



Test substance



25



18



-



4.1



110.8



Test substance



50



18



-



4.5



120.3



Negative control



-



18



+



10.6



100.0



Solvent control



0



18



+



12.0



100.0



Positive-
control CPA



-



18



+



5.4



44.6



Test substance



4



18



+



11.1



92.3



Test substance



25



18



+



7.6



63.3



Test substance



50



18



+



8.8



73.3



Solvent control



0



28



-



7. 3



100. 0



Test substance



50 µg



28



-



10.3



140.8



Solvent control



0 µg



28



+



3.3



100.0



Test substance



50 µg



28



+



3.7



111.3


       

*mean value of four flasks


 


 


Aberrant cells following treatment with the test substance:









































































































































































































































Test group



Test substance concentration in medium [µg/ml]



Scoring time point [h]



S9 mix



Number of cells analysed



Aberrant cells (#)



Aberrant cells (%)



incl. gaps



excl. gaps



incl. gaps



excl. gaps



exchanges



Negative control (solvent)



0


 



7



-



400



8



1



1.00



0.25



0.00



+



400



5



1



1.25



0.25



0.00



Test group



50



7



-



400



6



5



1.50



1.25



0.25



+



400



6



2



1.50



0.50



0.00



Negative control (untreated)



-



18



-



400



11



4



2.75



1.00



0.00



+



400



8



3



2.00



0.75



0.00



Negative control (solvent)



0



18



-



400



9



5



2.25



1.25



0.25



+



400



6



2



1.50



0.50



0.00



Test group



4



18



-



400



11



7



2.75



1.75



0.50



+



400



17



5



4.25



1.25



0.25



25



18



-



400



6



3



1.50



0.75



0.00



+



400



7



5



1.75



1.25



0.25



50



18



-



400



20



8



5.00



2.00



0.00



+



400



7



2



1.75



0.50



0.00



Positive control (EMS)



-



18



-



200



46



43



23.00



21.50



17.00



Positive control (CPA)



-



18



+



200



47



43



23.50



21.50



11.0



Negative control (solvent)



0



28



-



400



10



1



2.50



0.25



0.00



+



400



5



2



1.25



0.50



0.00



Test group



50



28



-



400



14



4



3.50



1.00



0.00



+



400



12



6



3.00



1.50



0.00



 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information




Genotoxicity in vitro:


The test substance was tested in vitro in two independent Ames tests, an HPRT test, a chromosomal aberration test and an UDS assay.


 


In an Ames test similar to OECD guideline 471, the test substance was tested in the Salmonella strains TA 1537, TA 1538, TA 98, TA 100 and TA 1535 at doses up to 5000 µg/plate with and without metabolic activation. Two independent experiments were performed with 4 plates per experimental condition. No cytotoxicity was observed except for TA 100 at concentrations of 5000 µg/plate, however precipitation was observed in all bacterial strains starting at 1250 µg/plate. No increased reverse mutation rate was observed in any strain at any of the doses tested. However, several methodological weaknesses were identified: the purity of the test substance was not stated and the bacterial strains did not include a strain to detect oxidizing or cross-linking mutagens. Further, only a plate incorporation test was performed.


To evaluate the mutagenicity in the Ames test, another study report can be taken into consideration. In a protocol according to the Japan MAFF guidelines and under GLP, the test substance was tested in Salmonella strains TA 100, TA 1535, TA 98, TA 1537, TA 1538 and E.coli WP2uvrA at doses up to 5000 µg/plate. While only one experiment was conducted, it included two plates per dose. According to the report, no genotoxicity was observed, however no details on cytotoxicity or precipitation on the test substance were available.


Taking the information from both studies together, it can be concluded that the test substance was not mutagenic under the conditions of the Ames test as described.


 


In order to clarify mutagenicity in mammalian cells, an HPRT test was conducted according to OECD guidelines and under GLP. V79 cells were incubated with test substance at concentrations up to 50 µg/ml (which was the highest achievable concentration without precipitation) with and without metabolic activation. Two independent experiments were performed, no reproducibly increased mutation rate was observed. Therefore, the test substance was considered not mutagenic under the conditions of this assay.


 


In vitro clastogenicity was assessed with a chromosomal aberration test according to OECD and EPA guidelines and under GLP. V79 cells were incubated with up to 50 µg/ml test substance (highest achievable concentration without precipitation) with and without metabolic activation for 4 hours. Analysis time points were 7, 18 and 28 hours after start of treatment. Four cultures were used for each test condition, a repetition was considered not necessary due to the clearly negative results observed. No increases in aberrant cells were observed in treated cultures as compared to the concurrent controls at any time point analyzed. The positive and negative / solvent controls were considered valid. Therefore, the test substance was not clastogenic under the conditions of this assay.


 


Finally, a UDS assay was conducted similar to OECD guideline 482 and under GLP. Primary rat hepatocytes were isolated and incubated with the test substance up to 100 µg/ml (highest achievable concentration without precipitation) and 3H-labeled thymidine for 3 hours. Addition of a metabolic system was considered not necessary since freshly isolated primary hepatocytes have sufficient metabolic capacity. After incubation, the nuclei were isolated, lysed and the DNA extracted. The respective radioactivity was measured in each sample (6 per experimental condition). No increased radioactive signal was detected in treated samples as compared to the concurrent controls while a clearly elevated radioactive signal was provided by the positive control. Therefore, the substance was considered not genotoxic in this UDS assay.


 




Justification for classification or non-classification

Based on the data available and taking into consideration the criteria laid down in regulation (EC) 1272/2008 (CLP), no classification for genotoxicity is warranted.