Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three key studies are available.
- Bacterial reverse mutation assay (key): performed according to OECD Guideline 471 in S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 (Pharmakon Research International Inc., 1982). The substance was negative to induce reverse mutations in the presence and absence of an exogeneous metabolic activation system.
- Mammalian cell gene mutation test (Mouse lymphoma mutagenicity assay, key): performed according to a method equivalent to OECD Guideline 476 in Mouse lymphoma L5178Y cells (Seifried et al., 2006). The substance was demonstrated to be negative in the presence and absence of metabolic activation system.

- Rat hepatocyte primary culture/DNA repair test (key): performed according to OECD Guideline 482 in hepatocytes from male F344 rats (Pharmakon Research International Inc., 1988). The substance was demonstrated to be 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:
key study
Study period:
11 January 1982 - 21 January 1982
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Very abnormal lawn and pindat colonies observed at the highest test concentration (10000 µg/mL).
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): 4236-45-35, Order J-89
- Substance type: clear colorless liquid
- Physical state: liquid
- Analytical purity: responsibility of the Sponsor
- Lot/batch No.: # J-89
- Other: miscible in distilled water
- Stability: there was no apparent change in the physical state of the test or control articles during the assay.
Target gene:
Histidine gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Arcolor 1254-treated Sprague Dawley rat S9 liver homogenate
Test concentrations with justification for top dose:
10.000, 3333, 1000, 333 and 100 µg/plate
Vehicle / solvent:
solvent(s) used: distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation: 1 µg/plate for strains TA 1535 and TA100
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation: 150 µg/plate for TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation: 5 µg/plate for strains TA1538 and TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene: 5 µg/plate for all strains
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48-72h

NUMBER OF REPLICATIONS:
negative and positive controls: in triplicate
compound-treated plates: in duplicate

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition is tested at following concentration: 33.3, 10, 3.3 and 1 mg/mL with strains TA1538 and TA100 ( in duplicate)

Evaluation criteria:
- positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies.
- negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies
Statistics:
not applicable
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:
cytotoxicity
Remarks:
at 10000 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 10000 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: screening study showed cytotoxicity at 10000 ug/plate: abnormal lawn was observed with sparse growth.

COMPARISON WITH HISTORICAL CONTROL DATA: all solvent and positive controls used in the evaluation of the test article were within the acceptable range of mean historical data.

Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results:
negative

The substance showed no mutagenic effects in S. typhimurium tester strains both with and without metabolic activation.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Peer-reviewed study performed according to a method similar to OECD Guideline 476.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): 3-methoxypropylamine
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Cells were screened for the presence of mycoplasma after cryopreservation. New cultures were initiated at approximately 3 month intervals from cells stored in liquid N2.
Cells grown in Fischer's medium supplemented with 10% horse serum and 0.02% pluronic F-68
Periodically "cleansed" against high spontaneous background: yes, at approximately 3 month intervals
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced liver S9 from male Sprague-Dawley rats
Test concentrations with justification for top dose:
range of concentrations from 500 to 4000 µg/mL
Vehicle / solvent:
No data
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethylsulfonate at 4.7 x 1E-06 M (or methylmethanesulfonate at 10-20 µg/mL) for the test -S9, and a positive control of 3-methylcholanthrene at 1.86 x 1E-05 M (or dimethylbenz[a]anthracene at 0.5-4 µg/mL) for the test +S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- A total of 1.2 x 1E07 cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4h at 37 ± 1°C, washed twice with growth medium, and maintained at 37 ± 1°C for 48 h in log-phase growth to allow recovery and mutant expression. Cells in the cultures were adjusted to 3 x 1E05/mL at 24h intervals. They were then cloned (1 x 1E06 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium containing Fischer's medium, 20% horse medium, 2 mM sodium pyruvate, 0.02% pluronic F-68, and 0.23% granulated agar. Resistance to trifluorothymidine was determined by adding TFT to the cloning medium for mutant selection. The 100 x stock solution of TFT in saline was stored at -70°C and was thawed immediately before use. Plates were incubated at 37± 1°C in 5% CO2 in air for 10-12 days and then counted with an Artek automated colony counter or ProtoCol colony counter. Only colonies larger than ~ 0.2 mm in diameter were counted.

DETERMINATION OF CYTOTOXICITY
The toxicity was determined both with and without liver S9. Cells at a concentration of 6 x 1E05/mL (6 x 1E06 cells total) were exposed for 4 h to a range of concentrations from 0.0005 to 10000 µg/mL. The cells were then washed, resuspended in growth medium, and incubated at 37 ± 1 °C for 48 hours.
Mutant frequencies were expressed as mutants per 1E06 surviving cells. Results from this study were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Doubling of mutant frequency was previously reported as representing a positive effect. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.

Evaluation criteria:
Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not examined
Positive controls validity:
not specified
Additional information on results:
No data
Remarks on result:
other: all strains/cell types tested
Conclusions:
In this mouse lymphoma test, the substance was observed to be negative with and without metabolic activation in L5178Y TK+/- mouse lymphoma cells.
Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 March 1988 - 4 May 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
GLP study conducted according to a method similar to OECD Guideline 482 without significant deviations. The study is not a standard genetic toxicity endpoint study.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Deviations:
no
GLP compliance:
yes
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Specific details on test material used for the study:
- Name of test material (as cited in study report): 5601-98-1, Order #88-004
- Substance type: clear colorless liquid
- Physical state: liquid
- Storage condition of test material: at room temperature in the original glass bottle received from the sponsor
- Lot/batch No.: 88-004
- Purity and stability: responsibility of the sponsor
Species / strain / cell type:
hepatocytes: obtained from male F344 rats
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
0.167, 0.5, 1.67, 5.0, 16.7, 50, 167, 500, 1667 and 5000 ug/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Deionized water
- Justification for choice of solvent/vehicle: Solvent was chosen based on the suggestion of the sponsor.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionized water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-acetamidofluorene at 1E-05 M (1E-07 M in the treatment medium)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

CELL EXPOSURE:
1 x 1E05 viable hepatocytes were inoculated into 12 well cluster dishes containing 15 mm diameter Thermanox plastic coverslips in WME containing 10% calf serum. The hepatocytes were allowed to attach for approximately 2 hours in a 37°C CO2 incubator. The cultures were rinsed and serum-free medium containing test compound and 10 uC/mL of 3H-thymidine (with specific activity 50-80 CI/mL) were added to each culture. Eighteen to 20 hours after exposure, the cultures were washed three times with 3 mL volumes of phosphate buffered saline by aspiration.

CELL FIXATION: The cells on coverslips were swelled in 1 % sodium citrate for 10-15 minutes and fixed in three 10 minute changes of 100 % ethanol:glacial acetic acid (3:1). The coverslips were air dried and mounted cell surface up on glass slides with permount. Slides were dipped in NTB-2 photographic emulsion in the dark, allowed to dry overnight and stored 4°C in light proof slide boxes containing desiccant for one week.

STAINING:
After seven days of exposure time, autoradiographics were developed in D19 (Eastman Kodak) at approximately 15°C for 4 minutes, washed in deionized water with 5 mL glacial acetic acid for 30 seconds, immersed in Fixer (Eastman Kodak) for 10 minutes, washed in running tap water for 5 minutes, dried, stained in Harris Alum hematoxylin followed by a dip rinse in acid alcohol, rinsing in running tap water for 2-5 minutes and a dip rinse in ammonium water. The slides were then rinsed in running tap water for 2-5 minutes, dipped in 70% ethyl alcohol, followed by a 10-60 second dip in eosin solution. The slides were then rinsed in 3 separate baths of 95% ethyl alcohol for 2 minute intervals, followed by rinsing in 3 separate baths of 100% ethyl alcohol for 2 minute intervals. The slides were air dried, and coverslipped in permount. Excess emulsion was scrapped off.

NUMBER OF REPLICATIONS: 3 coverslips per group

NUMBER OF CELLS EVALUATED: A total of 150 cells/group were counted

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was reported, but no data was provided in study report on how it was measured.
Evaluation criteria:
The test substance was reported positive when the minimum net grain count of 3 per nuclei was consistently observed in triplicate wells. Where possible a dose response profile should have been observed. Due to the need to initially screen the substance over a wide range of doses, an adequate dose response may have not been attained, and the substance may have been classified as a "suspect" genotoxicant. To resolve the genotoxic potential of the substance, the sponsor may have chosen to initiate a second experiment with dose levels closely bracketing the positive response which resulted in classifying the substance as a "suspect" genotoxicant.
Key result
Species / strain:
hepatocytes: male Fischer 344 rat
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at doses higher than 50 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:
The solvent and positive control values of net nuclear grain counts were within the acceptable range of mean historical data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The highest dose level scored in the assay was 50 ug/ml due to excessive toxicity observed at the higher dose levels. In addition to the 50 ug/ml dose level, 1.67, 5.0 and 16.7 ug/ml dose levels were also scored.


Conclusions:
The results for the substance were negative in the Rat Hepatocyte Primary Culture/DNA Repair Test under the conditions of the assay. These findings were based upon the inability of the substance to produce a mean net nuclear grain count of at least three times the vehicle control at dose levels up to the highest non-toxic dose level of 50 ug/ml.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Peer-reviewed study performed according to a method similar to OECD TG 471.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): 3-methoxypropylamine
Target gene:
histidine locus
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:
Liver S9 homogenate prepared from male Sprague-Dawley rats and Syrian golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight
Test concentrations with justification for top dose:
Concentration range: 100-10000 µg/plate.
Vehicle / solvent:
No data
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
For testing in the absence of S9 mix, 100 µL of the tester strain and 50 µL of the solvent of test chemical were added to 2.5 mL of molten selective top agar at 45 ± 2°C. When S9 was used, 0.5 mL of S9 mix, 50 µL of tester strain, and 50 µL of solvent or test chemical were added to 2.0 mL of molten selective top agar at 45°C ± 2 °C. After it was vortexed, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. after the overlay had solidified, the plates were incubated for 48 h at 37 ± 2°C.

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
The doses that were tested in the mutagenicity assay were selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study using strain TA100. Ten dose levels of the chemical, one plate per dose, were tested in both the presence and the absence of induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used.
Evaluation criteria:
For a test article to be considered positive, it had to induce at least a doubling time (TA98, TA100, and TA1535) in the mean number of revertants per plate of at least one tester strain. This increase in the mean revertants per plate had to be accompanied by a dose response to increasing concentrations of the test chemical. If the study showed a dose response with a less than 3-fold increase on TA1537 or TA1538, the response had to be confirmed in a repeat experiment.
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:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not examined
Positive controls validity:
not specified
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:
not specified
Untreated negative controls validity:
not examined
Positive controls validity:
not specified
Additional information on results:
No data
Remarks on result:
other: all strains/cell types tested
Conclusions:
In this Ames test, the substance was observed to be negative in Salmonella strains TA98, TA100, TA1535, and TA 1538 with and without metabolic activation.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
No data
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): méthoxy-3-propylamine
- Batch: 8904A0596
- Purity: 99.89%
- Supplier: Atochem, Usine de La Chambre
Target gene:
Histidine operon
Species / strain / cell type:
other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix from Aroclor 1254-induced rat liver (10%)
Test concentrations with justification for top dose:
Concentrations (µg/plate):
Bacterial toxicity study:
. TA 98 : 1000, 5000, 10000
. TA 100: 500, 1000, 5000
Genotoxicity study:
. First study: 50, 100, 500, 1000 and 5000
. Second study: 50, 100, 500, 1000 and 2500
Vehicle / solvent:
Solvent: Distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
Without metabolic activation:  Na azide: 5 µg/plate (TA 100, TA 1535),  2-nitrofluorene: 5 µg/plate (TA98, TA1538), 9-aminoacridine: 100 µg/plate (TA 1537). With metabolic activation: 2-aminoanthracene: 5 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation
NUMBER OF REPLICATIONS: 2
No. plates/concentration/study: 3
Evaluation criteria:
Genotoxic character is assigned to the product if, in the concentration range studied, the number of revertant colonies observed is at least equal to twice the number of spontaneous revertants and whether this increase is directly related to the concentration, or if the number of revertants/nmole is greater than 0.01. Another criterion is the reproducibility of this positive response.
Statistics:
Nonetes
Key result
Species / strain:
other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxic up to and including 5000 µg/plate on TA98 and TA100
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
Methoxypropylamine was non mutagenic at 50, 100, 500, 1000, 2500 and 5000 µg/plate, with or without metabolic activation on the 5 tester strains used.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

One key study is available.

- In vivo Micronucleus test: performed according to a method similar to OECD Guideline 474 in mouse CD1 male/female mice (Pharmakon Research International Inc., 1988). The substance was demonstrated to be negative.

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:
26 March 1988 - 14 June 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
GLP study performed according to a method similar to OECD guideline 474 without significant deviations.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): 5601-98-1, Order #88-004
- Substance type: clear colorless liquid
- Physical state: liquid
- Analytical purity: responsibility of the Sponsor
- Lot/batch No.: Order #88-004, recieved on March 25, 1988
- Stability under test conditions: no apparent change in its physical state
- Storage condition of test material: room temperature
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, Massachusetts
- Age at study initiation: 7 weeks
- Weight at study initiation: males: 28-32g, females: 22-27g
- Assigned to test groups randomly: yes, randomized by bodyweight, assigned to groups by use of a random number table
- Fasting period before study: no
- Housing: 5 per cage (according to sex and dose group)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 70%
- Air changes (per hr): no info
- Photoperiod (hrs dark / hrs light): 12 h dark/ 12 h light
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: distilled water
Details on exposure:
one single intraperitoneal dose per mouse at a volume of 10 mL/kg body weight
Duration of treatment / exposure:
1 single dose
Frequency of treatment:
once
Post exposure period:
test substance: 30, 48 and 72h
positive control: 30h
negative control: 48h
Dose / conc.:
75 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- triethylenemelamine
- Route of administration: intraperitoneal
- Doses / concentrations: 0.5 mg/kg
Tissues and cell types examined:
bone marrow of femur
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on dose range finding study with following doses: 10, 50, 100, 200, 500, 1000 and 2000 mg/kg bw .
All mice died from 200 mg/kg bw up to 2000 mg/kg bw dose groups. Due to the mortality observed and the appearance of mild to severe pharmacotoxic signs in the 100 mg/kg bw dose group, 75 mg/kg bw was selected as an estimate of the maximum tolerated dose.

DETAILS OF SLIDE PREPARATION:
All mice were sacrified by cervical dislocation. Femora were opened carefully at the proximal end with a scissors until a small opening to the marrow canal became visible. A 1 ml tuberculin syringe filled with approx. 0.2 ml fetal bovine serum was inserted into the marrow cavity and the marrow was gently flushed (to assure maximum dispersion) into 1.0 ml of fetal bovine serum in a 3 ml conical centrifuge tube. The femora were flushed with fetal bovine serum until all the marrow was out and the bone appeared almost translucent. The suspension was centrifuged at 1000 rpm for 5 minutes. The supernatant was removed leaving a small amount of fetal bovine serum with the remaining cell button. The button was mixed with a pasteur pipette to assure a homogenous mixture. The marrow smears were made by placing immediately a small drop of the cell suspension near the frosted end of a glass slide pre-cleaned in absolute ethanol and smeared by pulling the cell suspension behind with another pre-cleaned slide at a 45° angle. The slides were quickly dried on a slide warmer set at 56°C, dipped in absolute methanol and air dried.
Stained with Giemsa.

METHOD OF ANALYSIS:
Slides were screened for good preparation, i.e. well spread, undamaged, perfectly stained.
1000 polychromatic erythrocytes (PCE)(immature) were counted for the presence of micronuclei per animal. Also the number of normochromatic erythrocytes (NCE) (mature) present in these 1000 PCE was recorded.
Data are expressed as the number of micronucleated PCE versus total normal PCE in 1000 PCE per animal.
A total of 1000 PCE and NCE was also counted per animal. These data were expressed as the ratio PCE/NCE.
OTHER: Slides were coded randomly by study number and number designation. The code was kept on a separate sheet until the slides were evaluated. Following evaluation, slides were decoded. Coding of the slides was carried out by an individual not involved in the actual scoring.
Evaluation criteria:
If the spontaneous rate of micronuclei in the PCE is less than 0.2% and the positive control is statistically greater (p<0.05) than the spontaneous rate and at least seven animals per group survived the treatment, the results are deemed acceptable.
Statistics:
- one-tailed t-tests were used to make pairwise comparisons between each treatment group and its concurrent vehicle control for statistically significant increases in the number of micronucleated PCE.
- the proportion of PCE per 1000 erythrocytes per animal were evaluated by pairwise two-tailed t-tests after an arcsin transformation was performed.
All comparisons were made for each sacrifice time separately comparing treated groups versus the vehicle control group.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
abnormal gait, decreased activity, piloerection, decreased body tone, no mortality observed
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The substance did not produce any statistically significant increases in the number of micronucleated PCEs. There was a significant increase in PCE/NCE ratios for the 72h treated group as compared to the vehicle control. However, biological significance of this positive result, if any, is unknown.

RESULTS OF RANGE-FINDING STUDY
- Dose range: 10 - 2000 mg/kg
- Clinical signs of toxicity in test animals:
all mice died from 200 mg/kg up to 2000 mg/kg dose groups.
no mice died at the three lower dose groups.
100 g/kg: writhing, abnormal gait, decreased activity (immediately after treatment), decreased body tone, piloerection, abnormal gait and decreased activity (all, at 24h, 48h, 72h), abnormal stance, tremors, ptosis, cyanosis and hypothermia (1 female, 72h after treatment)


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
total micronucleated PCE: 17/10000 (30h), 9/10000 (48h), 15/10000 (72h), 14/10000 (neg control), 462/10000 (pos control)
- Ratio of PCE/NCE (for Micronucleus assay):
1.031 (30h), 1.384 (48h), 1.711 (72h), 1.191 (neg control), 0.758 (pos control)
- Appropriateness of dose levels and route: no data
- Statistical evaluation:
results of positive control are statistically different from results of negative control (induction of micronuclei and PCE/NCE ratio)
number of induced micronuclei is not statistically different from negative control.
PCE/NCE ratio is statistically different from negative control, only at 72h sacrifice time.
Conclusions:
The substance, at 75 mg/kg bw, was considered negative in the micronucleus test at the time intervals evaluated under the experimental conditions of this assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro:

Bacterial reverse mutation assay:

Pharmakon Research International Inc., 1982 performed an Ames (plate incorporation) test with S typhimurium strains TA1535, TA1537, TA98, TA100 and TA1538 with and without metabolic activation (Pharmakon Research International, 1982).

Following test concentrations were applied: 10000, 3333, 1000, 333 and 100 µg/plate (in triplicate). Solvent control and positive controls were run in triplicate. There were no observed increases in mutation frequency with and without metabolic activation at the tested dose levels. All solvent and positive controls used induced mutant frequency values which were within the acceptable limits of mean historical data. Cytotoxicity was observed at 10000 µg/plate in the range-finding test: an abnormal lawn was observed with sparse growth. A K1 and a K2 Ames test performed by resp. Bichet (1989) and Seifried et al. (2006) as well demonstrated that the substance is not genotoxic towards bacteria.

Rat Hepatocyte Primary Culture/DNA Repair Test:

Pharmakon Research International Inc. (1988) studied the gene mutation potential in rat hepatocytes (adult male F344 rats). Following doses were evaluated in triplicate: 0.167, 0.5, 1.67, 5.0, 16.7, 50, 167, 500, 1667 and 5000 µg/ml. A vehicle control (deionized water) and a positive control (1E-05 molar 2 -acetamidofluorene) were scored as well. Under the conditions of this study, the test substance was not genotoxic in the hepatocyte primary culture/DNA repair test. These findings were based upon the inability of the substance to produce a mean net nuclear grain count of at least three times the vehicle control at dose levels up to the highest non-toxic dose level of 50 ug/ml.

Cytogenicity test:

An in vitro cytogenicity study in mammalian cells does not usually need to be conducted if adequate data from an in vivo cytogenicity test are available. Pharmakon Research International Inc. (1988) performed an in vivo micronucleus test on CD1 mice (IUCLID section 7.6.2). Therefore, it is not necessary to perform an in vitro cytogenicity test for the test substance.

Mouse Lymphoma Cell Mutation Assay:

A mouse lymphoma mutagenicity assay was performed according to a method equivalent to OECD Guideline 476 in mouse lymphoma L5178Y cells (Seifried et al., 2006). In this test, the substance was observed to be negative with and without metabolic activation.

Genetic toxicity in vivo:

In vivo micronucleus test:

Pharmakon Research International (1988) performed an in vivo micronucleus test in male and female CD1 -mice via single intraperitoneal administration of 75 mg/kg the test substance (nominal concentration). Concurrent vehicle (distilled water) was used as a control. Triethylenemelamine (0.5 mg/kg) was used as a positive control substance. 5 animals per sex and per dose were tested. Bone marrow of the femur was evaluated. The substance, at 75 mg/kg bw, was considered negative in the micronucleus test at the time intervals evaluated under the experimental conditions of this assay (30, 48 and 72 hours post exposure).

Justification for classification or non-classification

Based on the available data and according to the criteria of the CLP Regulation (EC) 1272/2008, the test substance should not be classified for mutagenicity.