Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Four reliable studies were available for this endpoint (Klimisch 1 studies): 2 studies performed according to OECD guideline 471 (one key and one supporting study), 1 study performed according to OECD guideline 476, and 1 performed according to OECD guideline 482. None of these studies observed any genotoxic effect.

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:
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 (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: J-98

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: no data
- Stability under test conditions: no apparent change in the physical state of the test or control articles during the assay

OTHER SPECIFICS:
- Name of test material (as cited in study report): 4236-44-30
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S-9 fraction of rat liver homogenate obtained from Arcolor 1254-treated Sprague Dawley rats
Test concentrations with justification for top dose:
12, 40, 120, 400 and 1200 µg/plate
Vehicle / solvent:
-solvent(s) used: distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
with and without metabolic activation
True negative controls:
no
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
sodium azide
Remarks:
TA1535 and TA100=10 µg/ml
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
with and without metabolic activation
True negative controls:
no
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
2-nitrofluorene
Remarks:
TA1538 and TA98=50 µg/ml
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
with and without metabolic activation
True negative controls:
no
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
9-aminoacridine
Remarks:
TA1537=1500 µg/ml
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
with and without metabolic activation
True negative controls:
no
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-anthramine
Remarks:
in all strains=50 µg/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation);


DURATION
- Preincubation period: 48h
- Exposure duration: 48-72h



SELECTION AGENT (mutation assays): histidine and biotin



NUMBER OF REPLICATIONS:
positive and negative controls: in triplicate
treated groups: in duplicate
tester strains: treated with five levels of the test compound




DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition is tetsted at the following concentrations: 10,000, 3333, 1000, 333 and 100 µg/plate with strains TA1538 and TA100 ( in duplicate).

Evaluation criteria:
revertant colonies are counted (Artek Counter Model 880)
- 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



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 determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: dose levels for the Preliminary Toxicity Screen were 10,000, 3333, 1000, 333 and 100 µg/plate.
Tester Strains TA1538 and TA100 showed complete inhibition at 10,000 ug/plate, excessive inhibition at 3333 µg/plate and moderate inhibition a t 1000 µg/plate.


COMPARISON WITH HISTORICAL CONTROL DATA: all positive and negative controls were within acceptable range of mean historical data


Remarks on result:
other: all strains/cell types tested
Conclusions:
The results for the test article 4236-44-30, Order# 5-98, were negative in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at 1200, 400, 120, 40 and 12 µg/plate.
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)
Principles of method if other than guideline:
The substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.
GLP compliance:
yes (incl. certificate)
Remarks:
Experimental Toxicology and Ecology
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Abl. Nr. 85-4113
- Date of production: November 16, 2000

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: was guaranteed by the sponsor

OTHER SPECIFICS:
- Name of test material (as cited in study report): Polyetheramin T 403; Substance No.: 01/0618-1
- Analytical purity: 93.9 %
- Homogeneity: Homogeneous by visual inspection
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9-mix prepared from Sprague Dawley rat livers after Aroclor 1254 activation
Test concentrations with justification for top dose:
0, 22, 110, 550, 2750, 5500 µg/plate (SPT)
0, 4, 20, 100, 500, 1500 µg/plate (SPT)
0, 10 ,30 ,100, 300, 1000 µg/plate (PIT)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see details below
Details on test system and experimental conditions:
Standard plate test
Test tubes containing 2 ml portions of soft agar kept in a water bath at 45°C, and the remaining components are added in the following order:
0.1 ml test solution or vehicle
0.1 ml bacterial suspension
0.5 ml S-9 mix (in tests with metabolic activation) or
0.5 ml phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates. After incubation at 37°C for 48 -72 hours in the dark, the bacterial colonies (his+ revertants) are counted.


Preincubation test
0.1 ml test solution or vehicle, 0.1 ml bacterial suspension and 0.5 ml S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx.30 seconds.


Both Tests
In each experiment 3 Test plates per dose per control used. After incubation at 37°C for 48 -72 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Positive Controls:
with S-9 mix: 2.5 µg 2-aminoanthracene (2-AA)(dissolved in DMSO) for the strains TA 100, TA 98, TA 1537and TA 1535
60 µg 2-aminoanthracene (2-AA)(dissolved in DMSO) for the strain E. coli WP2 uvrA

without S-9 mix: 5 µg N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) (dissolved in DMSO) for the strains TA 100 and TA 1535
10 µg 4-nitro-o-phenylendiamine (NOPD) (dissolved in DMSO) for the strain TA 98
100 µg 9-aminoacridine (AAC) chloride monohydrate (dissolved in DMSO) for the strain TA 1537
5 µg 4-nitroquinoline-N-oxide (4-NQO) (dissolved in DMSO) for the strain E. coli WP2 uvrA

The titer was determined and in regularly measurements the strain characteristics were checked. Sterility control performed.
Evaluation criteria:
Positive results
- doubling of the spontaneous mutation rate (control)
- dose response relationship
- reproducibility of the result
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested

Toxicity:

A bacteriotoxic effect (reduced background growth, decrease in the number of revertants, reduction in the titer) was observed in the standard plate test depending on the strain and test conditions from about 500 µg -1,500 µg/plate onward.

In the preincubation assay bacteriotoxicity (reduced background growth, decrease in the number of revertants, reduction in the titer) was observed depending on the strain and test conditions at doses >= 300 µg/plate.

Solubility: No precipitation of the test substance was found.

Mutagenicity:

An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system.

Conclusions:
Thus, under the experimental conditions chosen here, it is concluded that the test substance is not a mutagenic agent in a bacterial reverse mutation test.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-10-27 to 2009-12-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
-stability of the test article was not reported
GLP compliance:
yes
Remarks:
Study conducted in compliance with the US EPA GLP Standards 40 CFR, and the OECD Principle of GLP (C97)186/Final
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 8H306

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light
- Stability under test conditions: the stability of the test article has not been determined by testing facility or the Sponsor.

OTHER SPECIFICS:
- Name of test material (as cited in study report): JEFFAMINE T-403
- Analytical purity: 100%
Target gene:
hypoxanthine-guanine phosphoribosyl transferase locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: medium suplemented with hypoxanthine, aminopterin and thymidine (HAT); F12FBS5-Hx is Ham's F12 medium without hypoxanthine suplemented with 5% dialyzed FBS, 100 units pencilin/mL, 100 ug streptomycin/mL and 2mM L-glutamine/mL; Cloning medium: for selection of the TG-resistant phenotype, the replicates from each treatment condition were trypsinized and replated, in quintuplicate dish in F12FBS5-Hx containing 10 uM 6-thioguanine (TG, 2-amino-6-mercaptopurine). For clonong efficiency determinations at the time of selection, 100 cells/60mm dish were plated in triplicate in medium without TG.
- Properly maintained:yes
- Periodically checked for Mycoplasma contamination:no
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: the CHO-K1-BHR cell line is a proline auxotroph with a modal chromosome number of 20, a population doubling time of 12-14h, and a cloning efficiency of usually greater than 80%
Metabolic activation:
with and without
Metabolic activation system:
Arclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
500, 1000, 2000, 3000, 3500, 4000 and 4400 ug/mL (with and without S9 activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: sterile distilled water was chosen by the Sponsor. The test article was soluble in sterile distilled water at a concentration of 44 mg/mL, the maximum concentration prepared for the preliminary toxicity assay
Untreated negative controls:
yes
Remarks:
medium
Negative solvent / vehicle controls:
yes
Remarks:
sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation (-S9) Migrated to IUCLID6: EMS (ethyl methanesulfonate) was used at a stock concentration of 20 uL/mL, for a final concentration of 0.2 uL/mL
Untreated negative controls:
yes
Remarks:
medium
Negative solvent / vehicle controls:
yes
Remarks:
sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation (+S9) Migrated to IUCLID6: B(a)P (Benzo(a)pyrene: was used at a stock concentration of 400 ug/mL, for a final concentration of 4 ug/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: not applicable
- Exposure duration: 5h
- Expression time (cells in growth medium): 18-24h
- Selection time (if incubation with a selection agent): 7-10 days
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable


SELECTION AGENT (mutation assays): 6-thioguanine (TG)
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): 10% aqueous Giemsa

NUMBER OF REPLICATIONS:2


NUMBER OF CELLS EVALUATED: not applicable


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Other: not applicable


OTHER:no data
Evaluation criteria:
All conclusions were based on scientific judgment, however, the following criteria are presented as a guide to interpretation of the data: -the test article was considered to induce a positive response if there was a concentration-related increase in mutant frequencies with at least tow consecutive concentrations showing mutant frquencies of > 40 mutants per 1E -6 clonable cells; - if a single point above 40 mutants per 1E -6 clonable cells was observed at the highest concentration, the test article was considered suspect; - if no culture exhibited a mutant frequency of > 40 mutants 1E-6 clonable cells, the test article was considered negative
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality: the osmolity of the solvent control was 264 mmol/kg and the osmolality of the highest concentration, 4400 ug/mL , was 269 mmol/kg
- Evaporation from medium: no data
- Water solubility: the test article was soluble in sterile distilled water at a concentration of 44 mg/mL, the maximum concentration prepared for the preliminary toxicity assay
- Precipitation:no test article precipitate at any concentration in treatment medium
- Other confounding effects: no data


RANGE-FINDING/SCREENING STUDIES: Preliminary toxicity assay: the preliminary toxicity assay was used to established the optimal concentrations for the mutagenesis assay and consisted of evaluation of test article effect on cloning efficiency. In the preliminary toxicity assay, the maximum concentration of JEFFAMINE T-403 tested was 4400 ug/mL (10 mM). Selection of concentrations for the mutagenesis assay was based on the cloning efficiency relative to the solvent control. Substantial toxicity, i.e., cloning efficiency ¿50% of the solvent control, was observed at 4400 ug/mL with and without S9 activation. Based on these findings, the concentrations chosen for the mutagenesis assay arnged from 150 to 3000 ug/mL for both the non-activated and S9-activated cultures.
COMPARISON WITH HISTORICAL CONTROL DATA:
The stability of the negative and positive control articles and their mixtures was demonstated by acceptable results that met the criteria for a valid test. The historical control data are presented in Appendix II -see att.background material

ADDITIONAL INFORMATION ON CYTOTOXICITY:
no data
Remarks on result:
other: all strains/cell types tested

Mutagenesis Assay

Relative cloning efficiency was 81% and 14 % at the highest concentration cloned in the non-activated and S9 -activated systems, respectively. Non of the treated cultures exhibited mutant frequencies of greater than 40 mutants per 10^6 clonable cells.

Conclusions:
Under the conditions of this study, the test article was concluded to be negative in the CHO/HGPRT Mutation Assay, with and without metabolic activation.
Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1988-03-29 to 1988-05-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
comet assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 88-006

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the glass bottle receieved from the Sponsor

OTHER SPECIFICS:
- Name of test material (as cited in study report): 5601-96-1
- Analytical purity: 97 %
Species / strain / cell type:
hepatocytes: obtained from male F344 rats
Details on mammalian cell type (if applicable):
- Type and identity of media: WME containing 10% calf serum
Metabolic activation:
without
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- 1.0 E-7 M
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 18-20 hours (10 uCi/ml of 3H-thymidine, specific activity of 50-80 Ci/mM, was added during exposure)


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 evaluated by visual inspection




OTHER: After exposure, the cultures were washed three times with phosphate buffered saline by aspiration. The 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 in light proof slide boxes containing desiccant for one week. Seven days later, autoradiographs were developed.
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 test substance over a wide range of doses, an adequate dose response may have not been attained, and the test substance may have been classified as a "suspect" genotoxicant. To resolve the genotoxic potential of the test 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: rat
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
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 16.7 ug/ml due to excessive toxicity observed at the higher dose levels. In addition to the 16.7 ug/ml dose level, 0.5, 1.67 and 5.0 ug/ml dose levels were also scored.


Remarks on result:
other: all strains/cell types tested
Conclusions:
The results for the test substance 5601-96-1 were negative in the Rat Hepatocyte Primary Culture/DNA Repair Test under the conditions of the assay, without metabolic activation. These findings were based upon the inability of the test substance to produce a mean net nuclear grain count of three or greater than the vehicle control mean net nuclear grain count at dose levels up to the highest non-toxic dose level of 16.7 ug/ml.
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 reliable study was available for this endpoint (Klimisch 2). This study was performed according to OECD guideline 474 and did not observe any genotoxicity.

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:
1988-04-25 to 1988-05-25
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The OECD Guideline 474 states that samples of bone marrow should be taken at least twice, starting not earlier than 24h after treatment, but not extending beyond 48h after treatment with appropriate interval(s) between samples. In this assay, bone marrow samples are taken at 30, 48 and 72h after treatment. 6 Animals died minutes after administrating the dose in the definitive study. However, these animals were replaced.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Principles of method if other than guideline:
The OECD Guideline 474 states: that samples of bone marrow should be taken at least twice, starting not earlier than 24h after treatment, but not extending beyond 48h after treatment with appropriate interval(s) between samples. In this assay, bone marrow samples are taken at 30, 48 and 72h after treatment.
Also, the likelihood that the test substance reach the general circulation or the target tissue has not been discussed.
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Order #88-006, recieved on March 25, 1988

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: no apparent change in its physical state

OTHER SPECIFICS:
- Name of test material (as cited in study report): 5601-96-1
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: 8 weeks
- Weight at study initiation: males: 29-37g, females: 26-32g
- 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: max 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: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70 % humidity
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark, 12 hrs light


IN-LIFE DATES: From: April 1988 To: May 1988
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: deionized water
Details on exposure:
one single intraperitoneal dose per mouse
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.:
2.5 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: 50, 100, 250, 500 and 1000 mg/kg bw .
Pharmacotoxic signs were observed in the 100 mg/kg group and at higher dose levels evaluated. Mortality occurred in the 250 mg/kg bw group (1/4). All animals but one, died within 2 hours post-dose in the 500 and 1000 mg/kg bw dose groups. Therefore, 150 mg/kg bw was selected as dose.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
All mice were sacrified by cervical dislocation.

DETAILS OF SLIDE PREPARATION:
One end of each femur (one proximal, one distal to the iliac end) was opened carefully with 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 a 5ml round bottom culture tube containing 1.0 ml of fetal bovine serum. Each femur was 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 pellet. The pellet was resuspended with a pasteur pipette to assure a homogenous mixture.
A small drop of the cell suspension was spread immediately onto an ethanol pre-cleaned glass slide. The smears were quickly dried on a slide warmer set at approx. 56°C, dipped in absolute methanol (2sec) 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) per animal were scored for the presence of micronuclei, as well as for the number of micronucleated normochromatic erythrocytes (NCE)(mature) present in the optical field containing these 1000 PCE.
Data are expressed as the total number of mean percentage of micronucleated PCE for 10000 PCE per group (1000 PCE per mouse, whenever possible). An additional 1000 erythrocytes (PCE and NCE) were also scored per mouse and the proportion was expressed as the PCE/NCE ratio.
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 0.5% and the positive control is statistically significantly 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 erytrocytes per animal were evaluated by pairwise two-tailed t-test 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:
decreased activity, abnormal gait, body drop and decreased body tone, six animals were dead within minutes of dose administration
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 0.75 - 50 mg/kg
- Clinical signs of toxicity in test animals:
2.5 mg/kg: decreased activity, body drop and abnormal gait (immediately after treatment), no signs (24h), decreased body tone (most animals at 48h), body drop, abnormal gait (1 female, 48h), piloerection, decreased body tone (all, 72h)
1 mg/kg: no signs (immediately to 24h after treatment), decreased body tone (most animals, at 48 and 72h after administration)
0.75 mg/kg: no signs (immediately to 24h after treatment), piloerection, decreased body tone, abnormal gait (all males, 48h), decreased body tone (all, 72h).
2.5 mg/kg was selected for the definitive study

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
8/10000 (30h), 13/10000 (48h), 10/10000 (72h), 11/10000 (neg control), 342/10000 (pos control)
- Ratio of PCE/NCE (for Micronucleus assay):
1.248 (30h), 1.639 (48h), 1.358 (72h), 1.246 (neg control), 0.821 (pos control)
- Appropriateness of dose levels and route: dose level seems appropriate and was based on the range finding study. However, six animals (4 males, 2 females) were dead within minutes of dose administration, indicating that the dose (2.5 mg/kg) was near or at the maximum tolerated dose. Death occurred in 2 males of the 30h group, 2 males of the 48h group, 2 females of the 72h group. Necroscopy for all six mice did not reveal visible lesions. Mice were replaced so that there were 10 mice per dose group.
- Statistical evaluation: OK.
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 between the test substance and the negative control (at none of the sacrifice times).
PCE/NCE ratio was significantly increased for the 48h treated group as compared to the vehicle control. The significance of this positive result is undetermined.
Conclusions:
The test article, administered in single intraperitoneal dose at 2.5 mg/kg bw, did not induce a statistically positive increase in micronuclei in the hemopoietic cells of the mouse bone marrow at the time intervals evaluated under the experimental condition of this assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro:

Ames test according to OECD guideline 471 (Pharmakon Research International, 1982): The results for the test substance were negative in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at 1200, 400, 120, 40 and 12 µg/plate. Cytotoxicity was not determined for Salmonella strains TA1535, TA1537, TA98 and TA100, but was observed for test strains TA1538 and TA100. Positive and solvent controls were tested simultaneously and were within acceptable range of mean historical data.

CHO/HGPRT Mutation Assay according to OECD guideline 476 (BioReliance, 2010): under the conditions of this study, the test substance was concluded to be negative. Cytotoxicity was observed, however no further details were available. Negative, vehicle and positive control data were tested simultaneously and the stability of these controls and their mixtures was demonstrated by acceptable results that met the criteria for a valid test.

Rat Hepatocyte Primary Culture/DNA Repair Test according to OECD guideline 482: Under the conditions of the assay the substance was found negative without metabolic activation. These findings were based upon the inability of the test substance to produce a mean net nuclear grain count of three or greater than the vehicle control mean net nuclear grain count at dose levels up to the highest non-toxic dose level of 16.7 ug/ml. Positive and solvent controls were tested simultaneously and their values of net nuclear grain counts were within the acceptable range of mean historical data.

Genetic toxicity in vivo:

Micronucleus test according to OECD guideline 474 (San Sebastian, 1988): The test substance, administered in single intraperitoneal dose at 2.5 mg/kg bw, did not induce a statistically positive increase in micronuclei in the hemopoietic cells of the mouse bone marrow at the time intervals evaluated under the experimental condition of this assay. Following toxicity was observed: decreased activity, abnormal gait, body drop and decreased body tone, six animals were dead within minutes of dose administration. Vehicle and positive controls were valid.


Justification for classification or non-classification

Based on the available data and the criteria of the CLP Regulation, the substance should not be classified for mutagenicity.