Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an Ames Test according OECD TG 471 the test substance is mutagenic in the Salmonella typhimurium strains TA 1537 and TA 100 in the presence and absence of metabolic activation. Chromosome aberrations have been reported in CHO cells after exposure to the test item in presence and absence of metabolic activation. The substance was mutagenic in the L5178Y mouse lymphoma cell forward mutation assay in presence and absence of S9 mix.

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:
1989-07-14 to 1989-07-20
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only 4 strains used
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: rat liver S9 mix
- method of preparation of S9 mix: 5 male Sprague-Dawley rats (200 - 300 g) receive a single intraperitoneal injection of 500 mg Aroclor 1254 (as a 20% solution in peanut oil - w/v) per kg body weight 5 days before sacrifice. During this time the animals are housed in Makrolon cages in air-conditioned rooms. The day/night rhythm is 12 hours (light period from 6.00 - 18.00 hours and dark period from 18.00 - 6.00 hours). Standardized pelleted feed and tap water from bottles are available ad libitum. 5 days after administration the rats are sacrificed, and the livers are prepared (all preparation steps for obtaining the liver microsome enzymes are carried out using sterile solvents and glassware at a temperature of +4°C). The livers are weighed and washed in an equivalent volume of a 150 mM KCl solution (1 mL = 1 g wet liver), then cut into small pieces and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5-ml portions of the supernatant (so-called S-9 fraction) are quickly deep-frozen in dry ice and stored at -70°C to -80°C for 2 months at the most. The S-9 mix is prepared freshly prior to each experiment. For this purpose, a sufficient amount of S-9 fraction is thawed at room temperature and 3 volumes of S-9 fraction are mixed with 7 volumes of S-9 supplement (cofactors). This preparation was kept on ice until used.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL
- quality controls of S9: no data
Test concentrations with justification for top dose:
20, 100, 500, 2500 and 5000 µg/plate (1st experiment with and without S9 mix)
100, 500, 2500, 5000 and 7500 ug/plate (2nd experiment with and without S9 mix)
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
100 µg, without S9 mix (TA 1537)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
10 µg, without S9 mix (TA 98)
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-nitroso- guanidine (MNNG)
Remarks:
5 µg, without S9 mix (TA 100, TA 1535)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
10 µg, with S-9 mix (TA 100, TA 98, TA 1537, TA 1535)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- 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: reduced background growth
Evaluation criteria:
In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.
Statistics:
none
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
not examined
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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
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:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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: no data

STUDY RESULTS
Tests without S-9 mix:
TA 1535: Weakly positive reaction at 2500 µg/plate (factor 3.3) and at 5000 µg/plate (factor 3.4).
TA 100: Slight increase in the number of revertant colonies at 2500 µg/plate (factor 1.7 - 1.9) - 7500 µg/plate (factor 3.1).
TA 1537: No increase in the number of revertants.
TA 98: No increase in the number of revertants.

Tests with S-9 mix:
TA 1535: Weakly positive reaction at 2500 µg - 5000 µg/plate (factor 1.9 - 3.3)
TA 100: Slight increase in the mutation rate at 5000 µg and 7500 µg/plate (factor 1.8 - 2.6).
TA 1537: No increase in the number of revertants.
TA 98: No increase in the number of revertants.

-Concurrent vehicle negative and positive control data: see attached tables

- Signs of toxicity: No bacteriotoxic effect (reduced his- background growth) was observed.
- Individual plate counts: see attached tables

HISTORICAL CONTROL DATA: no data
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
no guideline followed
Principles of method if other than guideline:
The substance was tested for its mutagenic potential in the L5178Y tk+/tk- mouse lymphoma cell forward mutation assay, by means of procedures based upon those described by Clive and Spector (Mutat Res 44:269-278, 1975) and Clive et al (Mutat Res 59:61-108, 1979).
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
thymidine kinase locus (tk) in a mammalian cell
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: The tk+/tk- -3.7.2C heterozygote of L5178Y mouse lymphoma cells was obtained from Dr. D. Clive, Burroughs Wellcome Co. (Research Triangle Park, NC)
- Absence of Mycoplasma contamination: Laboratory cultures were confirmed as free from mycoplasma by cultivating or Hoechst staining techniques.
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media:
Fischer's medium was supplemented with 2 mM L-glutamine, sodium pyruvate, 110 µg/mL, 0.05% pluronic F68, antibiotics, and 10% heat-inactivated donor horse serum (v/v)
- Cell were maintained at 37°C
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: rat liver S-9 mix
- method of preparation of S9 mix: Post-mitochondrial supernatant fractions of liver homogenates (S9) were prepared from male, 200-g, Fischer 344 rats, obtained from the Frederick Cancer Research Center (Frederick, MD). To prepare induced S9, the rats were injected intraperitoneally with Aroclor 1254 (500 mg/kg) in corn oil five days before they were killed. Uninduced S9 was prepared from untreated rats. Livers were removed and washed in cold 0.15 M KCI, then homogenized in 3 volumes 0.15 M KCI. The homogenate was centrifuged for 10 min at 10000 rpm (9000 g ). The supernatant fluid was mixed and then stored in liquid nitrogen (-196°C) until used.
- concentration or volume of S9 mix and S9 in the final culture medium: 10%
Test concentrations with justification for top dose:
50 , 100, 200, 400, 800 µg/mL
Vehicle / solvent:
- Vehicle/solvent used: destilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
250 µg/ mL, without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
2.5 µg/mL, with S9 mix
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hours

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days
Cell suspensions (3 x 10E5 cells/ mL) were incubated for a 2-day expression period, the cell population density being adjusted back to 20 mL of 3 x 10E5 cells/mL after 24 hr. After 48 hr, the cell population densities were estimated and culture volumes containing 3 x 10E6 cells, giving a cell population density of 2 x 10E5 cells/mL.
- Method used: agar plates
- Cloning efficiency: A 0.1 mL sample of the cell suspension was withdrawn and diluted 1:100. Three 0.1 mL samples (200 cells) of the diluted cultures were transferred to 30-mL tubes, mixed with 25 mL cloning medium (Fischer's medium containing 20% heat-inactivated horse serum) containing 0.35% Noble agar, and poured into 90-mm petri plates.
- Selective agent: 3 µg trifluorothymidine/mL
Three aliquots (each containing 10E6 cells) of the culture were distributed to 30-mL tubes, mixed with 20 mL cloning medium to give final concentrations of 0.35 % Noble agar and 3 µg trifluorothymidine/mL, then poured into 90-mm petri plates.
- Selection time: 11-14 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Toxicity was expressed as eithera reduction of cell population growth in suspension during the expression period or a reduction in cloning efficiency. A measure of the overall toxicity was the relative total growth (RTG).
With any chemical, the first experiment was a toxicity test in which cell population expansion was measured. Tenfold differences in test compound concentrations were used in the toxicity test, the highest being 5 mg/mL unless a much lower concentration was indicated by the poor solubility of a compound. This test was followed by at least two experiments in the absence of S9 mix. If no clear positive response was observed, then two experiments were performed in the presence of S9 mix.
Evaluation criteria:
Compliance with predetermined quality control criteria was required before the response of a cellular population to the test chemical was evaluated. The basis for this choice of criteria is described elsewhere (Caspary et al, manuscript under review for Environmental Mutagenesis) and is the outcome of evaluation of historical data in two laboratories (Litton Bionetics, Inc., and SRI International). Four response categories were defined. Primary judgments were made at the level of individual experiments, but judgment on the mutagenic potential of a chemical was made on a basis of concensus of all valid experimental results.
Statistics:
The statistical analysis was based upon the mathematical model proposed for this system [Lee and Caspary, 1983] and consisted of a dose-trend test [Barlow et al, 1972, p215] and a variance analysis of pair-wise comparisons of each dose against the vehicle control. Where a statistically significant response occurred, the lowest observed effective dose (LOED) was noted.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
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:
STUDY RESULTS
Significant mutagenic responses were obtained in both the presence and absence of S9 mix in a total of four experiments. The LOED was 200 µg/mL in the presence and absence of S9 mix in one experiment with each activation condition and 400 µg/mL in the other. At these concentrations, the RTG values were usually greater than 50%. The increases in mutation frequency at 400 µg/mL were 1.9x and 2.4x the vehicle control in the presence of S9 mix and 1.6x and 4.0x the vehicle control in the absence of S9 mix. The positive control cloning efficiencies exceeded the permitted levels in trial 2 with S9 mix, but the data from 1,2-epoxybutane-treated cultures fully supported the results from trial 1.

HISTORICAL CONTROL DATA
- no data
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
no guideline followed
Principles of method if other than guideline:
A Chromosome aberration test was performed in CHO cells.
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: CHO cells were cloned at Litton Bionetics Inc.

For cell lines:
- Absence of Mycoplasma contamination: Cells were routinely checked for mycoplasma contamination; the results of these analyses disclosed no evidence of contamination.
- Number of passages: Cells were not used beyond 15 passages after cloning.

MEDIA USED
Cells for experiments were thawed and grown in McCoys 5A medium suppleinented with antibiotics and 10% fetal calf serum at 37°C using 5% CO2.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: rat liver S-9 mix
- method of preparation of S9 mix: supernatant from the 9000 g fraction of the livers from Aroclor 1254-induced male Sprague Dawley rats.
- concentration or volume of S9 mix and S9 in the final culture medium: The final concentration of S9 in the medium was either 15 or 20 µL/mL.
- quality controls of S9: no data
Test concentrations with justification for top dose:
16, 50, 160 and 500 µg/mL (with and without S9 mix)
Vehicle / solvent:
- Vehicle/solvent used: DMSO

- Justification for percentage of solvent in the final culture medium: The concentration of the solvent in the treatment flasks did not exceed 1 % and no adverse effects on cells is expected from this low concentration.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 mix
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density: Approximately 24 hr before treatment, cells were initiated at a density of 1 .2-1.75 x 10-6 cells/75 cm2 flask.
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: For the trials without S9, the cells were incubated with the appropriate control or chemical for 8 hr. The cells were then washed and Colcemid added for a 2-2.5 h exposure. For the trials with S9, the cells were treated with S9 and test chemical in serum-free medium for 2 h, washed, resuspended in medium containing serum, and incubated for an additional 8-10 h, with Colcemid present for the final 2 h.

FOR CHROMOSOME ABERRATION:
- Spindle inhibitor: colchicine incubated 2 h on cells

- Methods of slide preparation and staining technique used including the stain used: Cells were harvested by mitotic shake-off and stained with Giemsa.

- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Cells with good morphology and with a chromosome number of 21 +/- 2 were selected for analysis. With the exception of the high-dose positive controls, all slides were scored coded. 100 cells per dose were scored. Fewer cells were scored if a strong response was observed or a dose was very toxic. For the assay, cells were scored for "simple" (chromatid gaps and breaks, fragments, deletions, chromosome gaps and breaks, and double minutes), "complex" (interstitial deletions, triradials, quadriradials, rings, and dicentrics), and "other' (pulverized, polyploids, and endoreduplications) aberrations. These categories were combined to form the category "total". Galloway et al . [1987] reported that analyzing the individual "simple," "complex," and "other" categories rarely added to the information obtained by analyzing total aberrations alone; therefore, statistical analysis was conducted only on "total" aberrations. The percent of cells with aberrations, rather than aberrations per cell, were analyzed so as to avoid distorting the data for cases where a small number of cells had a large number of aberrations. Gaps and endoreduplications were scored but not tabulated in the totals or included in the statistical analyses.

- Determination of polyploidy: yes

- Determination of endoreplication: yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: no data
Evaluation criteria:
A complete description of the procedures used for data evaluation are presented in Galloway et al . [1987] and Margolin et al . [1986].
Statistics:
Statistical analysis was conducted only on "total" aberrations. The percent of cells with aberrations, rather than aberrations per cell, were analyzed so as to avoid distorting the data for cases where a small number of cells had a large number of aberrations. Gaps and endoreduplications were scored but not tabulated in the totals or included in the statistical analyses.

A binomial sampling assumption was used to evaluate an absolute increase in aberrations over the solvent control. Dose points with P values adjusted by Dunnett's method were considered significant if < 0.05, whereas a trend of P < 0.003 was significant. If a trial had a positive trend and no significant doses, or if there was no trend and only one significant dose, the trial was judged equivocal (?); if a trial had significant trend and one significant dose it was judged weak positive (+W); and if the trial had two significant doses it was judged positive (+), whether or not a positive trend was obtained. In the trials, if only one dose was significant and the increase over the control was P < 0.0005 the trial was denoted (+W*). The term "weak positive" (+ W) refers to the strength of the evidence for the positive call, not to the potency of the response. In general, positive responses were repeated , whereas repeats were not required for the trials concluded to be negative. If positive responses were obtained both with and without S9, the laboratories were required to repeat only one activation condition. When combining the conclusions of individual trials, more emphasis was given to trials in which experimental conditions were optimized.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
positive
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:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
ambiguous
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
- no data

STUDY RESULTS
- see tables

HISTORICAL CONTROL DATA
- no data
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Negative results have been observed in a chromosome aberration assay (similar OECD TG 475). The test substance was given via inhalation at 250 and 1000 ppm (7 single exposures for 5 consecutive days) to male and female rats (NIOSH, 1981). In a dominant-lethal test similar to OECD TG 478, butylene oxide had no adverse effect on male rats when given in doses of 250 and 1000 ppm via inhalation (NIOSH, 1981). Apurinic/apyrimidinc (AP) sites were not increased in tissues of mice and rats after inhalation exposure to the structurally similar substance 1,2-Epoxypropane (CAS 75-56-9) after 4 weeks.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
The tandem mass spectrometric detection was done in the Selected Reaction Monitoring (SRM) mode. The SRM transitions used were m/z 210 to 152 and m/z 214 to 156 for hydroxypropylguanine (N7-HPG) and its [13C4]–labeled internal standard, respectively. Apurinic/apyrimidinc (AP) sites in DNA were measured following a procedure reported by: Nakamura, J, and Swenberg, JA. 1999. Endogenous apurinic/apyrmidinic sites in genomic DNA of mammalian tissues. Cancer Res. 59:2522-2526.
GLP compliance:
yes (incl. QA statement)
Remarks:
The Dow chemical company Midland, Michigan 48674
Type of assay:
other: DNA adducts and Apurinic/apyrimidinc (AP) sites in DNA
Species:
mouse
Strain:
B6C3F1
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Lab. (Raleigh, North Carolina)
- Age at study initiation: 9-10 weeks
- Assigned to test groups randomly: yes
- Housing: one per cage
- Diet : ad libitum
- Water: ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25
- Humidity (%): 40-68
- Air changes (per hr): 12-15 times
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Vehicle:
none
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Whole body exposure chambers (Rochester-style):
The various concentrations of PO were generated using the glass J-tube method (Miller et al., 1980). Liquid test material was pumped into the glass J-tube assemblies (1 per exposure chamber) and vaporized by the flow of nitrogen gas passing through the bead bed of the glass J-tube. The nitrogen was heated as needed with a flameless heat torch (FHT-4, Master Appliance Corporation, Racine, Wisconsin) to the minimum extent necessary to vaporize the test material. All chambers, including the 0 ppm (control) chamber received the same amount (20 liters per minute) of supplemental nitrogen (carrier gas). The minimum amount of nitrogen necessary to reach the desired chamber concentrations was used. The generation system was electrically grounded and the J-tubes were changed as needed. The vaporized test material and carrier gas were mixed and diluted with supply air to achieve the desired test chamber concentration.

The animals were exposed to filtered air or PO vapors in 4 cubic meter stainless steel and glass Rochester-type whole-body exposure chambers [1.5 meters (m) x 1.5 m wide x 1.3 m deep with a pyramidal top and bottom]. Chamber airflow was maintained at a flow rate sufficient to provide the normal concentration of oxygen to the animals and 9-11 calculated air changes per hour. The chambers were operated at a slightly negative pressure, relative to the surrounding area. Animals were singly housed to minimize crowding during the exposure.

The chamber concentrations of PO, measured approximately in the center of the breathing zone of the animals, were determined at least once per hour with a Miran 1A infrared (IR) spectrophotometer (Foxboro/Wilks, South Norwalk, Connecticut) and reported by a strip chart recorder. The IR spectrophotometer was calibrated and a standard curve was compiled prior to and at the end of the study, using air standards prepared by vaporizing measured volumes of PO into Tedlar® sample bags (Series 233, SKC, Eighty Four, Pennsylvania) along with the metered volumes of dry, compressed air.
Duration of treatment / exposure:
6 hr/day
Frequency of treatment:
5 days/ week for 4 consecutive weeks
Post exposure period:
none
Dose / conc.:
50 ppm (nominal)
Dose / conc.:
100 ppm (nominal)
Dose / conc.:
200 ppm (nominal)
Dose / conc.:
400 ppm (nominal)
No. of animals per sex per dose:
16/exposure level
Control animals:
yes, concurrent no treatment
Tissues and cell types examined:
nose, respiratory epithelium, liver, spleen and lung.
Details of tissue and slide preparation:
DNA was isolated from liver, spleen, and lung by the Gentra Puregene System.
The DNA from nasal respiratory epithelium was isolated by phenol/chloroform extraction
Evaluation criteria:
Quantitation was based on comparisons to internal standard DNA containing known amounts of AP sites, calibrated with DNA from Kubo et al. (6).

Calibration curves were generated by using the standard solutions prepared by spiking varying amounts of 7-HPG into the solutions that contained a constant amount of internal standard. Solvent control was applied per sample set in order to avoid contamination of regents.
Statistics:
AP site measurement based on Dunnett’s multiple comparisons analysis.
Sex:
male
Genotoxicity:
other: A dose-related increase for N7-HPG (a biomarker of exposure) was seen from 50 ppm onward, the higets levels in the nasal repiratory epithelium.
Toxicity:
yes
Remarks:
reduced bw gain at the highest dose only; 400 ppm
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Sex:
male
Genotoxicity:
other: no significant differences in the number of AP sites in DNA from nasal respiratory epithelium
Toxicity:
yes
Remarks:
reduced bw gain at the highest dose only; 400 ppm
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Additional information on results:
The 7-HPG adducts form 50 ppm onward PO exposure concentration and the linear dose-response relationship was noted.

AP site determination found that there were no significant differences in the number of AP sites in DNA from nasal respiratory epithelium, across the dose groups.

Clearly the target tissue, nasal respiratory epithelium, receives the highest dose of N7-HPG, as expected for the site-of-contact tissue.


The N7-HPG levels in other tissues examined were slightly higher in mice than in rats exposed to the same air concentrations at the high exposures (200 and/or 400 ppm).


Exposure did not cause an increase in AP sites in respiratory nasal mucosa, despite the high number of N7-HPG adducts. HPG is not considered to be a promutagenic DNA adduct. However, it is chemically unstable, resulting in chemical depurination and the formation of an AP site. The fact that AP sites were not increased with increasing exposure demonstrates that AP sites resulting from such chemical depurination do not lead to unbalanced DNA repair.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
The tandem mass spectrometric detection was done in the Selected Reaction Monitoring (SRM) mode. The SRM transitions used were m/z 210 to 152 and m/z 214 to 156 for hydroxypropylguanine (N7-HPG) and its [13C4]–labeled internal standard, respectively. Apurinic/apyrimidinc (AP) sites in DNA were measured following a procedure reported by: Nakamura, J, and Swenberg, JA. 1999. Endogenous apurinic/apyrmidinic sites in genomic DNA of mammalian tissues. Cancer Res. 59:2522-2526.
GLP compliance:
yes (incl. QA statement)
Remarks:
The Dow chemical company Midland, Michigan 48674
Type of assay:
other: DNA adducts and Apurinic/apyrimidinc (AP) sites in DNA
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Lab. (Raleigh, North Carolina)
- Age at study initiation: 9-10 weeks
- Assigned to test groups randomly: yes
- Housing: one per cage
- Diet : ad libitum
- Water: ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25
- Humidity (%): 40-68
- Air changes (per hr): 12-15 times
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Vehicle:
none
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Whole body exposure chambers (Rochester-style):
The various concentrations of PO were generated using the glass J-tube method (Miller et al., 1980). Liquid test material was pumped into the glass J-tube assemblies (1 per exposure chamber) and vaporized by the flow of nitrogen gas passing through the bead bed of the glass J-tube. The nitrogen was heated as needed with a flameless heat torch (FHT-4, Master Appliance Corporation, Racine, Wisconsin) to the minimum extent necessary to vaporize the test material. All chambers, including the 0 ppm (control) chamber received the same amount (20 liters per minute) of supplemental nitrogen (carrier gas). The minimum amount of nitrogen necessary to reach the desired chamber concentrations was used. The generation system was electrically grounded and the Jtubes were changed as needed. The vaporized test material and carrier gas were mixed and diluted with supply air to achieve the desired test chamber concentration.

The animals were exposed to filtered air or PO vapors in 4 cubic meter stainless steel and glass Rochester-type whole-body exposure chambers [1.5 meters (m) x 1.5 m wide x 1.3 m deep with a pyramidal top and bottom]. Chamber airflow was maintained at a flow rate sufficient to provide the normal concentration of oxygen to the animals and 9-11 calculated air changes per hour. The chambers were operated at a slightly negative pressure, relative to the surrounding area. Animals were singly housed to minimize crowding during the exposure.

The chamber concentrations of PO, measured approximately in the center of the breathing zone of the animals, were determined at least once per hour with a Miran 1A infrared (IR) spectrophotometer (Foxboro/Wilks, South Norwalk, Connecticut) and reported by a strip chart recorder. The IR spectrophotometer was calibrated and a standard curve was compiled prior to and at the end of the study, using air standards prepared by vaporizing measured volumes of PO into Tedlar® sample bags (Series 233, SKC, Eighty Four, Pennsylvania) along with the metered volumes of dry, compressed air.
Duration of treatment / exposure:
6 hr/day
Frequency of treatment:
5 days/ week for 4 consecutive weeks
Post exposure period:
none
Dose / conc.:
50 ppm (nominal)
Dose / conc.:
100 ppm (nominal)
Dose / conc.:
200 ppm (nominal)
Dose / conc.:
400 ppm (nominal)
No. of animals per sex per dose:
16/exposure level
Control animals:
yes, concurrent no treatment
Tissues and cell types examined:
nose, respiratory epithelium, liver, spleen and lung.
Details of tissue and slide preparation:
DNA was isolated from liver, spleen, and lung by the Gentra Puregene System.
The DNA from nasal respiratory epithelium was isolated by phenol/chloroform extraction
Evaluation criteria:
Quantitation was based on comparisons to internal standard DNA containing known amounts of AP sites, calibrated with DNA from Kubo et al. (6).

Calibration curves were generated by using the standard solutions prepared by spiking varying amounts of 7-HPG into the solutions that contained a constant amount of internal standard. Solvent control was applied per sample set in order to avoid contamination of regents.
Statistics:
AP site measurement based on Dunnett’s multiple comparisons analysis.
Sex:
male
Genotoxicity:
other: A dose-related increase for 7-HPG (a biomarker of exposure) was seen from 50 ppm onward, the highest levels in the nasal repiratory epithelium.
Toxicity:
yes
Remarks:
reduced bw gain at the highest dose
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Sex:
male
Genotoxicity:
other: no significant differences in the number of AP sites in DNA from nasal respiratory epithelium
Toxicity:
yes
Remarks:
reduced bw gain at the highest dose
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Additional information on results:
For rats, the N7-HPG adducts at the 50 ppm PO exposure concentration and the linear dose-response relationship was noted.

AP site determination found that there were no significant differences in the number of AP sites in DNA from nasal respiratory epithelium, across the dose groups (Table 2 p>0.05).

Clearly the target tissue, nasal respiratory epithelium, receives the highest dose of N7-HPG, as expected for the site-of-contact tissue.


Exposure did not cause an increase in AP sites in respiratory nasal mucosa, despite the high number of N7-HPG adducts. HPG is not considered to be a promutagenic DNA adduct. However, it is chemically unstable, resulting in chemical depurination and the formation of an AP site. The fact that AP sites were not increased with increasing exposure demonstrates that AP sites resulting from such chemical depurination do not lead to unbalanced DNA repair.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
GLP compliance:
not specified
Type of assay:
rodent dominant lethal assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (U.K.) Limited, Manston, Kent
- Age at study initiation: 8-10 weeks
- Weight at study initiation: no data
- Housing: housed individually in cages
The rats were housed in suspended polycarbonate cages measuring 24 x 18 x 41 cm with steel mesh tops and bottoms. The cages were suspended over trays lined with absorbent paper.
- Diet: ad libitum, Spratts-Spillers No.1.
- Water: ad libitum
Animals were not allowed access to food or water during the exposure period.
- Acclimation period: none

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 22 (with extreme limits of 23.5°C and 17°C)
- Humidity (%): ca. 50 (with extreme limits of 60% and 32%)
- Air changes (per hr): approx.10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Vehicle:
air
Details on exposure:
TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
The test atmospheres were produced by bubbling dry, oxygen-free nitrogen (BOC Limited) through a liquid reservoir of butylene oxide contained in a glass washing, or Drechsel bottle immersed in a temperature controlled water bath at 28°C. The nitrogen/butylene oxide vapour mixture so generated was ducted through a vertical stainless steel piping approximately 1.5 m long to a glass mixing vessel and diluted with filtered, compressed air. The resulting mixture of butylene oxide/air was ducted through a 3/4" ID stainless steel tube to the apex of the exposure chamber.
The atmospheres in the exposure chambers were dynamic in that they were continuously generated for a single pass through the animal holding zone, before being extracted from the bottom and ducted away for 'scrubbing'. The required atmospheric concentrations within the exposure chambers were maintained by finely regulating the flow of nitrogen and diluting air into the mixing vessels, by means of adjustable flow meters.
The chamber temperature and relative humidity were recorded at hourly intervals through the exposure period. The animals were also observed at regular intervals for the appearance of clinical signs or adverse reactions to treatment. On completion of the exposure period and purging of the chamber of test compound, the animals were removed from the exposure chamber and returned to the animal holding area. The animals were then removed from their individual compartments, observed for clinical signs, ear numbers checked, body weights recorded and returned to their cages.

Measurement of Chamber Concentrations
Atmospheric concentrations of butylene oxide were monitored continuously during the 7 h exposure period from the breathing zone of the animals. A separate monitoring system was used for each concentration level. Stainless steel sampling lines, fitted with a particulate filter (Whatman Mini Filter, Grade 80) and positioned on a central reference point in each exposure chamber, were connected to the infrared gas analysers. The sampling flow rate was approximately 4 L/min.
Duration of treatment / exposure:
7 h per day for 5 consecutive days
Frequency of treatment:
daily
Post exposure period:
no
Dose / conc.:
250 ppm (nominal)
Remarks:
0.75 mg/L (nominal)
Dose / conc.:
1 000 ppm (nominal)
Remarks:
3 mg/L (nominal)
No. of animals per sex per dose:
10
Control animals:
other: yes; air control group
Positive control(s):
Ethyl methanesulphonate (EMS)
EMS was administered orally by gavage to the rodents at a constant dose volume of 10 mL/kg on each day that dosing was required.
Details of tissue and slide preparation:
METHOD OF ANALYSIS
Ovaries and uteri of the killed rats were removed and the ovaries examined for corpora lutea graviditatis, which were counted and this result recorded. Uteri were then opened, examined for live implantations, early deaths and late deaths. These data and any observed abnormalities were recorded. Live implantations were recognised as rat foetuses normally developed for approximately Day 14 of gestation and with a vasculature which had clearly been functioning until at least maternal death. A late death was diagnosed as a foetus where organogenesis had occurred, but was now bloodless due to death of the foetus within the last 2 days of intra-uterine existence.
Statistics:
Freeman-Tukey Poisson transformation, Freeman-Tukey binomial transformation
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
CLINICAL OBSERVATIONS AND BODY WEIGHTS
There was little evidence of toxicity in rats exposed to 250 ppm butylene oxide atmospheres, but at 1000 ppm there were marked effects. Rats were subdued by the end of each exposure period and showed little response to auditory stimuli. Body weights were decreased in both male and female rats exposed to 1000 ppm butylene oxide. Reductions in body weights were also seen in rats dosed with the positive control EMS.

RESULTS OF DEFINITIVE STUDY
Pregnancy frequency was calculated in 2 ways: firstly, by considering as pregnant femalas with Corpora lutea gravidotatis and secondly and more reliably , by considering as pregnant only females with implantations. With neither method was there any effect upon pregnancy frequency due to butylene oxide treatment, but there were reductions in Weeks 2 and 3 in the positive control group.
Corpora lutea graviditatis counts were not reduced in either of the butylene oxide treated groups; these counts were greatly reduced, however, in Weeks 1-3 of the positive control group. Implantations per pregnancy were unaffected by butylene oxide treatment except in Week 4, 1000 ppm exposure group where there was a small, but significant reduction. There were substantial reductions in Weeks 1 - 4 of the positive control group. The apparent lack of statistical significance in Week 2 is a result of the low number of degrees of freedom, there being only 2 pregnancies in this week-group. The frequencies of live implantations and live implantations + late deaths followed very closely the pattern of total implantations per pregnancy. However, there were no significant reductions at all in these parameters in butylene oxide treated groups compared with the air control group. Analysis of the proportions of early deaths by various statistical methods did not indicate any effects attributable to butylene oxide treatment. There were marked increases in early deaths, compared with the air control groups, in Weeks 1 - 4 of the positive control group.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
GLP compliance:
not specified
Type of assay:
mammalian bone marrow chromosome aberration test
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (U.K.) Limited, Manston, Kent
- Age at study initiation: 11-12 weeks
- Weight at study initiation: no data
- Housing: housed individually in cages
The rats were housed in suspended polycarbonate cages measuring 24 x 18 x 41 cm with steel mesh tops and bottoms. The cages were suspended over trays lined with absorbent paper.
- Diet: ad libitum, Spratts-Spillers No.1.
- Water: ad libitum
Animals were not allowed access to food or water during the exposure period.
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 22 (with extreme limits of 23.5°C and 17°C)
- Humidity (%): ca. 50 (with extreme limits of 60% and 32%)
- Air changes (per hr): approx.10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Vehicle:
air
Details on exposure:
TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
The test atmospheres were produced by bubbling dry, oxygen-free nitrogen (BOC Limited) through a liquid reservoir of butylene oxide contained in a glass washing, or Drechsel bottle immersed in a temperature controlled water bath at 28°C. The nitrogen/butylene oxide vapour mixture so generated was ducted through a vertical stainless steel piping approximately 1.5 m long to a glass mixing vessel and diluted with filtered, compressed air. The resulting mixture of butylene oxide/air was ducted through a 3/4" ID stainless steel tube to the apex of the exposure chamber.
The atmospheres in the exposure chambers were dynamic in that they were continuously generated for a single pass through the animal holding zone, before being extracted from the bottom and ducted away for 'scrubbing'. The required atmospheric concentrations within the exposure chambers were maintained by finely regulating the flow of nitrogen and diluting air into the mixing vessels, by means of adjustable flow meters.
The chamber temperature and relative humidity were recorded at hourly intervals through the exposure period. The animals were also observed at regular intervals for the appearance of clinical signs or adverse reactions to treatment. On completion of the exposure period and purging of the chamber of test compound, the animals were removed from the exposure chamber and returned to the animal holding area. The animals were then removed from their individual compartments, observed for clinical signs, ear numbers checked, body weights recorded and returned to their cages.

Measurement of Chamber Concentrations
Atmospheric concentrations of butylene oxide were monitored continuously during the 7 h exposure period from the breathing zone of the animals. A separate monitoring system was used for each concentration level. Stainless steel sampling lines, fitted with a particulate filter (Whatman Mini Filter, Grade 80) and positioned on a central reference point in each exposure chamber, were connected to the infrared gas analysers. The sampling flow rate was approximately 4 L/min.
Duration of treatment / exposure:
7 single exposures for 5 consecutive days
Frequency of treatment:
daily
Post exposure period:
no
Dose / conc.:
250 ppm (nominal)
Remarks:
0.75 mg/L (nominal)
Dose / conc.:
1 000 ppm (nominal)
Remarks:
3.0 mg/L (nominal)
No. of animals per sex per dose:
10
Control animals:
other: yes; air control group
Positive control(s):
Ethyl methanesulphonate (EMS)
EMS was administered orally by gavage to the rodents at a constant dose volume of 10 mL/kg on each day that dosing was required.
Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES
Each rat was injected i.p. with 3 mg/kg colchicine dissolved in Hank's Balanced Salt Solution (HBSS) 4 h after the last dose was given. Two hours later the rats were killed by neck dislocation. One femur from each animal was dissected out, cleaned of adherent tissue and the marrow aspirated into a 10 mL plastic blood sample tube containing 4 mL HBSS at ambient temperature and lithium heparin (250 IU). Each tube was labelled with the appropriate random number from a slide coding sheet. Hence, from this time until the completed result sheets were de-coded, the rat number and group were unknown to the scientists and technicians. The cell suspension was centrifuged at 1500 rpm. for 5 min, the supernatant fluid discarded and replaced with 4 mL fresh HBSS. The cells were suspended, then centrifuged again and the supernatant fluid discarded.

DETAILS OF SLIDE PREPARATION
4-5 ml 0.075 M-KCl pre-heated to 37°C was added to the cells while they were agitated on a vortex mixer. Following incubation for 20 min in a 37°C water bath, the cells were centrifuged, the supernatant fluid decanted and the cells fixed in 4 mL freshly prepared fixative (methanol:glacial acetic acid; 3:1). The fixative was removed after centrifugation and replaced with 2 mL fresh fixative. Tubes containing fixed cells were stored in a 4°C refrigerator overnight. The following morning (or later, up to 3 days) the fixative was changed and cell suspensions dropped onto clean slides labelled with the same number as the tube and allowed to dry thoroughly. Each rat was injected i.p. with 3 mg/kg colchicine dissolved in Hank's Balanced Salt Solution (HBSS) 4 h after the last dose was given. Two hours later the rats were killed by neck dislocation. One femur from each animal was dissected out, cleaned of adherent tissue and the marrow aspirated into a 10 mL plastic blood sample tube containing 4 mL HBSS at ambient temperature and lithium heparin (250 IU). Each tube was labelled with the appropriate random number from a slide coding sheet. Hence, from this time until the completed result sheets were de-coded, the rat number and group were unknown to the scientists and technicians. The cell suspension was centrifuged at 1500 rpm. for 5 min, the supernatant fluid discarded and replaced with 4 mL fresh HBSS. The cells were suspended, then centrifuged again and the supernatant fluid discarded.
(Gurr) diluted with 10 parts distilled water for 30 min, rinsed briefly in distilled water, dehydrated in alcohol, cleared in xylene and mounted in DePeX.

METHOD OF ANALYSIS
Leitz binocular microscopes were used for this purpose. Magnification was nominally x 1000 using x 10 magnification eye pieces and x 100 objectives.
Wherever possible, for each animal 50 cells with a minimum of 41 well spread chromosomes were examined and scored. The location of all spreads examined was recorded using the microscope stage vernier. The slide number was always located on the right hand side. The number of abnormalities was recorded on sheets of the design. Abnormalities looked for were: gaps, breaks, fragments, dicentrics, translocations (within the limitations of the staining methods) pulverisation.
Statistics:
A one-sided Student's t test was used on the transformed values.
This analysis was performed (a) including all abnormalities and (b) excluding cells only exhibiting gaps.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
CLINICAL OBSERVATIONS AND BODY WEIGHTS
There was little evidence of toxicity in rats exposed to 250 ppm butylene oxide atmospheres, but at 1000 ppm there were marked effects. Rats were subdued by the end of each exposure period and showed little response to auditory stimuli. Body weights were decreased in both male and female rats exposed to 1000 ppm butylene oxide. Reductions in body weights were also seen in rats dosed with the positive control EMS.

RESULTS OF DEFINITIVE STUDY
In the multiple exposure cytogenetic test, there were no indications of induction of chromosomal damage in either male or female rats exposed to 250 ppm or 1000 ppm butylene oxide atmospheres.
On the other hand, treatment with 100 mg EMS/kg/day for 5 days induced large increases in the frequencies of cells with chromatid damage. In both male and female rats this damage included gaps as well as chromatid breaks with fragments.
In the single exposure test rats treated with ethyl methanesulphoate, there were significant increases in aberrant cell frequencies in males and females at the 6 h and 24 h sampling times and in the females at the 48 h sampling time. There was no significant response in the 48 h sampling time males. These responses were significant if all aberrant cells were analysed or if cells containing only gaps were discarded. The damage was mainly to chromatids and there was a tendency for damage other than gaps to appear in the 24 h samples.
Butlyene oxide exposure groups did not show any increase in the frequency of aberrant cells except in the 6 h sample time males exposed to 1000 ppm butylene oxide. If cells only containing gaps were excluded from the analysis, then the statistical significance of the difference from the air control group was reduced.
This result with butylene oxide is probably insufficient evidence on which to base a conclusion for the clastogenic potential of this compound in vivo: the effect was small and was not observed in female rats.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro


The substance was tested for mutagenicity according to OECD TG 471 in the Ames test (standard plate test) both in the presence and in the absence of a metabolizing system obtained from rat liver (S9 mix) using the strains TA 1535,TA 100, TA 1537 and TA 98. In the first experiment 0, 20, 100, 500, 2500 and 5000 µg/plate was tested in all 4 strains. In the second experiment 0, 100, 500, 2500, 5000 and 7500 µg/plate was tested in TA 100. Parallel with each experiment with and without S9 mix, a negative control (solvent control, sterility control) was carried out for each tester strain in order to determine the spontaneous mutation rate. Furthermore, 2-aminoanthracene was used as positive control with S9 mix and N-methyl-N'-nitro-N-nitroso-guanidine, 4-nitro-o-phenylenediamine and 9-aminoacridine chloride were used as positive controls without S9 mix. The substance showed a weakly positive reaction at 2500 µg/plate (factor 3.3) and at 5000 µg/plate (factor 3.4) in the tester strain TA 1535 without S9 mix. There was also a weakly positive reaction at 2500 µg to 5000 µg/plate (factor 1.9-3.3) in the absence of S9 mix in the TA 1535 strain reported. A slight increase in the number of revertant colonies at 2500 µg/plate (factor 1.7 - 1.9) to 7500 µg/plate (factor 3.1) was shown for the strain TA 100 in the absence of S9 mix. In the presence of S9 mix a slight increase in the mutation rate at 5000 µg and 7500 µg/plate (factor 1.8 - 2.6) was reported for TA 100. The strains TA 1537 and TA 98 showed no increase in the number of revertants. No bacteriotoxic effect (reduced his- background growth) was observed. The positive controls showed the expected increases in revertants. According to the results of the present study, the test substance is weakly mutagenic in the Ames test under the experimental conditions chosen (BASF, 1989).


The substance was tested for its ability to induce chromosome aberrations in Chinese hamster ovary cells (CHO). Tests were performed with and without the addition of an exogenous metabolic activation (S9) mixture (from Aroclor 1254-induced male rat liver). For the trials without S9, the cells were incubated with the test substance (16, 50, 160 and 500 µg/mL) for 8 hr. For the trials with S9 mix, the cells were treated with the test substance (16, 50, 160 and 500 µg/mL) in serum-free medium for 2 hr, washed, resuspended in medium containing serum, and incubated for an additional 8-10 hr. Concurrent solvent and positive controls were included in all trials. Mitomycin C (MMC) was the positive control in the trials without S9 and cyclophosphamide (CPA) was used in the trials with S9. Colcemid was used as spindle inhibitor. Cells with good morphology and with a chromosome number of 21 +/- 2 were analysed and 100 cells per dose were scored. A positive result was reported without S9 mix at the highest dose (500 µg/mL), and an equivocal result with S9 mix in Chinese hamster ovary cells (Anderson et al., 1990).


Furthermore, the substance was tested for its mutagenic potential in the L5178Y mouse lymphoma cell forward mutation assay. Cultures were exposed to the test item in concentrations of  50, 100, 200, 400, 800 µg/mL for 4 hours, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT) for 11 to 14 days selection time. Experiments have been performed in presence and absence of metabolic activation (S9 mix from Aroclor 1254-induced rat liver). 3-Methylcholanthrene (with S9 mix) and ethyl methanesulphonate (without S9 mix) were used as positive controls. Significant mutagenic responses were obtained in both the presence and absence of S9 mix in a total of four experiments. The lowest effect dose was 200 µg/mL in the presence and absence of S9 mix in one experiment with each activation condition and 400 µg/mL in the other. At these concentrations, the relative total growth values were usually greater than 50%. The increases in mutation frequency at 400 µg/mL were 1.9x and 2.4x the vehicle control in the presence of S9 mix and 1.6x and 4.0x the vehicle control in the absence of S9 mix. The positive controls showed the expected mutagenic responses. Therefore, the mammalian gene mutation assay with mouse lymphoma L5178Y cells was found positive with and without S9 mix (McGregor et al. 1987).


A supporting Ames test equivalent or similar to OECD TG 471 is available. The study was conducted with Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538.  In this assay the bacterial strains were exposed to vapor concentrations of 39, 74, 149, 298, 599, 1198 ppm in airtight dessicator jars. The assay was performed with and without S9 mix (from Aroclor 1254 induced rat liver). A positive effect with and without metabolic activation was found for the S. typhimurium strains TA 100 and TA 1535, but not for the other strains (Dow, 1980).


In a further bacterial gene mutation assay using S. typhimurium strains TA1535, TA1537 and TA1538 positive results were found using TA 1535 in presence and absence of S9 mix (plate test and suspension test). A negative result was shown for TA 1537 and TA1538 in the plate test and suspension test. Concentrations ranged from 0.05 - 0.2%. This test was done with and without metabolic activation (Dow, 1975). In the same study report it was shown that the test substance was not active in producing frameshift mutations or mitotic gene conversions in the yeast Saccharomyces cerevisiae. Concentrations used were 0.25, 0.5 and 1% and the test was done with and without metabolic activation.


In vivo


A study similar to OECD TG 475 was conducted in male and female Sprague-Dawley rats (10/sex/group). Animals have been exposed to 250 or 1000 ppm test item vapours a via inhalation for 7 h per day for 5 consecutive days. Another group of male and female rats received the same doses as single exposures for 7 h duration. Control animals have been exposed to air. The positive control group received 100 mg/kg bw/day Ethyl methanesulphonate (EMS) orally by gavage. Each rat was injected i.p. with 3 mg/kg colchicine 4 h after the last dose was given. One femur from each animal was dissected and the marrow cells were collected for analysis of chromosome aberrations. Wherever possible, for each animal 50 cells with a minimum of 41 well spread chromosomes were examined and scored. The number of abnormalities including gaps, breaks, fragments, dicentrics, translocations (within the limitations of the staining methods) and pulverisation was recorded. Rats were subdued by the end of each exposure period and showed little response to auditory stimuli. Body weights were decreased in both male and female rats exposed to 1000 ppm butylene oxide. Reductions in body weights were also seen in rats dosed with the positive control EMS. In the multiple exposure cytogenetic test, there were no indications of induction of chromosomal damage in either male or female rats exposed to 250 ppm or 1000 ppm butylene oxide atmospheres. Treatment with the positive control EMS for 5 days induced large increases in the frequencies of cells with chromatid damage. In both male and female rats this damage included gaps as well as chromatid breaks with fragments. In the single exposure test rats treated with ethyl methanesulphonate, there were significant increases in aberrant cell frequencies in males and females at the 6 h and 24 h sampling times and in the females at the 48 h sampling time. There was no significant response in the 48 h sampling time males. These responses were significant if all aberrant cells were analysed or if cells containing only gaps were discarded. The damage was mainly to chromatids and there was a tendency for damage other than gaps to appear in the 24 h samples. Butlyene oxide exposure groups did not show any increase in the frequency of aberrant cells except in the 6 h sample time males exposed to 1000 ppm butylene oxide. If cells only containing gaps were excluded from the analysis, then the statistical significance of the difference from the air control group was reduced. This result with butylene oxide is probably insufficient evidence on which to base a conclusion for the clastogenic potential of this compound in vivo: the effect was small and was not observed in female rats (NIOSH, 1981).


A dominant lethal assay similar to OECD TG 478 was conducted in male rats. Sprague-Dawley rats have been exposed to 250 or 1000 ppm test item vapours a via inhalation for 7 h per day for 5 consecutive days. Control animals received air and the positive control group received 100 mg/kg bw/day Ethyl methanesulphonate (EMS) orally by gavage. After the exposure period male rats were mated with virgin females. Females were killed and examined for pregnancy and dominant lethal effects. Ovaries and uteri were removed and the ovaries examined for corpora lutea graviditatis. Uteri were examined for live implantations, early deaths and late deaths. There was little evidence of toxicity in rats exposed to 250 ppm butylene oxide atmospheres, but at 1000 ppm there were marked effects. Rats were subdued by the end of each exposure period and showed little response to auditory stimuli. Body weights were decreased in both male and female rats exposed to 1000 ppm butylene oxide. Reductions in body weights were also seen in rats dosed with the positive control EMS. There was no effect upon pregnancy frequency due to butylene oxide treatment, but there were reductions in Weeks 2 and 3 in the positive control group. Corpora lutea graviditatis counts were not reduced in either of the treated groups; these counts were greatly reduced, however, in Weeks 1-3 of the positive control group. Implantations per pregnancy were unaffected by butylene oxide treatment except in Week 4, 1000 ppm exposure group where there was a small, but significant reduction. There were substantial reductions in Weeks 1 - 4 of the positive control group. The apparent lack of statistical significance in Week 2 is a result of the low number of degrees of freedom, there being only 2 pregnancies in this week-group. The frequencies of live implantations and live implantations plus late deaths followed very closely the pattern of total implantations per pregnancy. However, there were no significant reductions at all in these parameters in butylene oxide treated groups compared with the air control group. Analysis of the proportions of early deaths by various statistical methods did not indicate any effects attributable to butylene oxide treatment. There were marked increases in early deaths, compared with the air control groups, in Weeks 1 - 4 of the positive control group. There were no effects attributable to butylene oxide in the dominant lethal test on pregnancy frequency, numbers of corpora lutea or implantations, or the frequency of early deaths (NIOSH, 1981).


Male Fisher rats were exposed whole body to vapours of the structurally similar substance 1,2-Epoxypropane (CAS 75-56-9) for 4 weeks (6 h/day, 5 days/week). Concentrations of 50, 100, 200 and 500 ppm were tested. Control animals were exposed to air only. After the end of the exposure period, DNA was isolated from liver, spleen, and lung by the Gentra Puregene System. The DNA from nasal respiratory epithelium was isolated by phenol/chloroform extraction. The accumulation of N7-(2-hydroxypropyl)guanine (N7-HPG) in tissues was measured by gas chromatography-high resolution mass spectrometry (GC-HRMS). Apurinic/apyrimidinc (AP) sites in DNA were measured. Clearly the target tissue, nasal respiratory epithelium, receives the highest dose of N7-HPG, as expected for the site-of-contact tissue. Exposure did not cause an increase in AP sites in respiratory nasal mucosa, despite the high number of N7-HPG adducts. HPG is not considered to be a promutagenic DNA adduct. However, it is chemically unstable, resulting in chemical depurination and the formation of an AP site. The fact that AP sites were not increased with increasing exposure demonstrates that AP sites resulting from such chemical depurination do not lead to unbalanced DNA repair (Dow, 2009).


Conclusion


The substance is genotoxic in in vitro studies (Ames test, chromosome aberration, gene mutation) and in vivo studies have been performed for further evaluation. The substance shows no genotoxic effects in vivo in a chromosome aberration test and in a dominant lethal assay after inhalation exposure, which is the predominant exposure route. It can be assumed that the substance is rapidly metabolized in vivo via conjugation with GSH and therefore, only insufficient concentrations of the parent substance are present anymore. Also in tissues, in which the local concentration is high due to direct contact (epithelia of respiratory tract), no effects have been reported for structurally similar substance 1,2-Epoxypropane (CAS 75-56-9). Here, studies of DNA adduct formation in nasal respiratory epithelium in rats and mice indicate that N7-HPG DNA adducts, a marker of exposure, have been observed especially at the site-of-contact. However, AP sites have not been increased indicating that the DNA repair was not affected. The available in vivo study regarding DNA repair for the source substance together with the negative in vivo genotoxicity studies for the target substance, which is in theory considered even more reactive than the target substance due to a shorter carbon chain lenght, cover the endpoint in vivo genotoxicity appropriately and no classification for genotoxicity in warranted. Please refer to the Read-Across Justification in IUCLID chapter 13 for more details.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008


The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data the test item is not classified for genetic toxicity according to Regulation (EC) No 1272/2008 (CLP).