Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: other: Rat bone marrow micronucleus assay by inhalation, in vivo cytogenicity /chromosomal damage
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-12-2003 to 08-12-2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study completed according to GLP principles and in accordance with relevant test guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
The test substance, 2,3-Dichloro-1,3-butadiene (DCBD) was evaluated for its ability to induce micronuclei in bone marrow polychromatic erythrocytes (PCEs) in Crl:CD (SD)IGS BR rats.
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,3-Dichloro-1,3-butadiene (DCBD)
- Physical state: Clear to straw-yellow liquid.
- Unique ID number: H-26034.
- Supplier: DuPont Dow Elastomers, L.L.C., Louisville, Kentucky.
- Lot/batch No.: 10/20/03.
- Stability under test conditions: The test substance is inherently reactive and may spontaneously poylmerise (unless inhibited) or dimerize. Based on purity analyses performed prior to and immediately after study conduct, no evidence for instability of the test substance was detected. Analysis of the liquid test substance however showed the concentrations of mixed dichlorobutadiene-based dimers had increased with time but were less than 1,000 ppm, the maximal level considered acceptable for this study.
- Storage condition of test material: under nitrogen in a freezer maintained at approximately -20°C except when used to generate test atmospheres and when being analysed.

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)IGS BR rats
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina.
- Age at study initiation: Young adult male and females Crl:CD (SD)IGS BR rats. At study start, animals were approximately 8 weeks (55 days) of age.
- Weight at study initiation: The weight variation of selected animals did not exceed ± 20% of the mean weight for each sex.
- Assigned to test groups randomly: yes they were distributed by computerised, stratified randomisation into study groups as designated in the study design, so that there were no statistically significant differences among body weight means within a sex.
- Housing: Individually in stainless steel, wire-mesh cages suspended above cage boards. Each cage rack contained only animals of one sex.
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent Labdiet 5002.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: Upon arrival animals were quarantined for 3 days, identified by tail label, weighed 3 times and observed with respect to weight gain and for any gross signs of disease or injury.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 24 °C.
- Humidity (%): 40 - 60%.
- Photoperiod (hrs dark / hrs light): 12-hour light/ 12-hour dark cycle.

Administration / exposure

Route of administration:
inhalation: gas
Vehicle:
Houseline air was used as the vehicle and the vehicle control.
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body. All exposure groups and the air and positive control groups contained 10 animals, with the exception of the highest concentration groups (220 ppm) which contained 14 animals.

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Constructed of stainless steel and glass (NYU style) with a nominal internal volume of 1.4 m³.
- Method of holding animals in test chamber: Animals were placed in individual stainless steel wire mesh cages.
- Source and rate of air: Nitrogen flows were approximately 0.8, 2, 3 and 5 L/min for the 0, 50, 100 or 200 ppm chambers respectively.
- Method of conditioning air: Nitrogen flows were metered with Brooks model 0154/0154E power supply indicator (with digital display).
- System of generating particulates/aerosols: Chamber atmospheres were generated by bubbling nitrogen through the liquid test substance.
- Temperature, humidity, pressure in air chamber:
- Air change rate: Test atmospheres were generated dynamically using airflows needed to achieve at least 10 air changes per hour in the exposure chamber.
- Treatment of exhaust air: Test atmospheres were discharged directly into an exhaust sack.

TEST ATMOSPHERE
- Brief description of analytical method used: The atmospheric concentration of DCBD was determined by gas chromatography at least hourly during the exposure.
- Samples taken from breathing zone: yes.
Duration of treatment / exposure:
Animals were exposed whole-body to DCBD by inhalation for 6 hours/day for 2 consecutive days.
Frequency of treatment:
Animals were exposed whole-body to DCBD by inhalation for 6 hours/day on 2 consecutive days.
Post exposure period:
Bone marrow was sampled at approximately 24 and 48 hr post treatment from the vehicle control, high, intermediate and low dose groups. Positive controls were sampled concurrently at the 24 hr interval only.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
50 ± 0.11 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
100 ± 0.37 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
200 ± 0.50 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
All exposure groups and the negative control (air) and positive control groups contained 10 animals (equal numbers of male and females), with the exception of the highest concentration groups (220 ppm) which contained 14 animals.
Control animals:
yes, concurrent no treatment
Positive control(s):
The positive control was cyclophosphamide and was administered by gavage.

Examinations

Tissues and cell types examined:
Bone marrow cells were harvested approximately 24 and 48 hours after the completed treatment from animals in all dose groups, and approximately 24 hours after dosing from animals in the positive control groups.
Details of tissue and slide preparation:
PREPARATION OF BONE MARROW SMEARS: Immediately after carbon dioxide asphyxiation, marrow from both femurs of each animal were aspirated into a syringe containing foetal bovine serum (FBS) and transferred to a centrifuge tube containing FBS. Erythrocytes were collected by centrifugation.

DETAILS OF SLIDE PREPARATION: Following centrifugation, most of the supernatant was removed. The pellet was suspended in FBS and a small drop was placed on a pre-cleaned microscope slide. Smears were made using a Mini Prep blood smearing instrument. Slide labels included the animal number followed by the letter 'R' to indicate the test system and a letter (a,b,c etc.). At least 3 slides per animal were prepared. The slides were air-dried and fixed using absolute methanol. Bone marrow smears were stained in acridine orange, a DNA/RNA specific fluorochrome.

SLIDE ANALYSIS: Given the lack of evidence for any sex-related difference in clinical manifestation of systemic toxicity, micronucleus evaluations were conducted on male animals only. Micronucleus evaluations were conducted on 5 animals/group/time point. All groups were evaluated at the 24-hr time point. Only groups (males at 0 and 200 ppm) were initially evaluated at the 48-hour time point. Based on the negative data obtained, male groups (50 and 100 ppm) and all of the female groups were not analysed. To control for bias, slides were coded by labelling each side with a random number. Bone marrow smears from animals in the low and intermediate groups sampled 48 hr after dosing were evaluated only if there was a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (MNPCE's). At the highest concentration, the exposure groups consisted of 7 males and 7 females. Based on survival until the scheduled sacrifice and then based on cage order, 5 males were selected for evaluation. At least 2000 PCE's per animal were scored for the presence of micronuclei (round, bright yellow-green fluorescing bodies). In the event 2000 PCE's were unattainable for a given animal, the animal was excluded from the statistical evaluation of the scoring results. Fluorescent microscopy was used to examine the smears. Colour was used to distinguish PCEs (red-orange) from normochromatic erythrocytes (NCE's) (grey-green). Inclusions in PCE's which were improperly shaped or stained or were not in the focal plane of the cell were considered artefacts and not scored as micronuclei. Cells containing more than one micronucleus were scored as a single micronucleated PCE (MNPCE). The proportion of PCEs among 1000 total erythrocytes (expressed as the PCE/NCE ratio) was determined for each animal. The proportion of PCEs to total erythrocytes in the test substance exposed animals was not less than 20% of the control value.
Evaluation criteria:
Body weight, clinical observation and mortality/moribundity check was carried out at days 0, 1, 2 and 3.
Sacrifice and bone marrow collection was carried out at days 2 and 3 for the test substance treated animals and the negative control animals; and day 2 for the positive control animals.
The bone marrow cells were examined for the presence of micronucleated polychromatic erythrocytes in the animals administered DCBD as compared to the concurrent negative (air) control animals.
Statistics:
Descriptive statistics including mean, standard deviation, and standard error of the mean were used to summarise exposure concentrations and environmental data.
Male and female data were evaluated seperately. Significance was judged at p <0.05.
1. MICRONUCLEUS: Data for the proportion of micronucleated PCEs among 2000 polychromatic erythrocytes and the proportion of PCEs among 1000 erythrocytes (MNPCE and PCE frequency, respectively) were transformed prior to analysis using an arcsine square root or Freeman-Tukey function. This transformation is appropriate for proportions since the distribution of the transformed data more closely approximates a normal distribution than does the nontransformed proportion. Transformed data for PCE and MNPCE frequencies were analysed seperately for normality of distribution and equal variance using the Shapiro-Wilk and Levene's tests, respectively. Data from the positive control group was not included in evaluating normality or variance homogeneity of distribution. For those data that are normally distributed and have equal variance, parametric statistics (eg ANOVA and Dunnett's test) were performed using the transformed data. For those data that are normally distributed but have unequal variance, a robust ANOVA and unequal-variance Dunnett test was done. For those data that are not normally distributed, nonparametric data was performed. The individual animal was considered the experimental unit. All data analyses was one-tailed and conducted at a significance level of 5%.
2. BODYWEIGHTS: If the Shapiro-Wilk test was not significant but Levene's test was significant, a robust version of Dunnett's test was used. If the Shapiro-Wilk test was significant, Kruskal-Wallis test was followed with Dunn's test.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
The exposure levels were chosen based on previous acute and repeated-exposure inhalation studies with DCBD. Based on acute studies, the 4 -hour inhalation LC50 value for male rats was 410 ppm DCBD in air. One death was observed at each of the exposures conducted at 230, 340 and 370 ppm; no deaths occurred after an exposure at 212 ppm. Deaths were delayed, occurring 1 to 9 days after exposure. In a range finding study conducted to support exposure level selection in a developmental toxicity study, body weight losses, lethargy, closed eyes and salivation were observed from the initial exposure to 50, 100 and 150 ppm DCBD. Based on these observations, exposure levels of 50, 100 and 200 ppm were selected.

RESULTS OF DEFINITIVE STUDY
BODYWEIGHTS: Slight to severe concentration-dependent decreases in mean terminal body weight were observed in both males and females. Relative to air-exposed controls, these changes were statistically significant in males at all exposure concentrations and in females at 200 ppm. Overall body weight gains during the study were significantly reduced in males at 200 ppm DCBD throughout the study and on test days 0-1 and 1-2 at 100 ppm. The mean body weight gains were significantly reduced on test days 0-1 and 1-2 in females exposed to 100 and 200 ppm DCBD, and on test days 0-1 at 50 ppm. These body weight reductions were considered biologically relevant, test-substance related signs of systemic toxicity.

CLINICAL OBSERVATIONS AND MORTALITY: During the exposures, the acoustic startle responses was determined once prior to the exposure, 3 times during the exposure (within the first 30 minutes, and after approximately 2 and 4 hours), and then again after the exposure was completed. During both exposures, male and female rats from the 50, 100 and 200 ppm exposure groups displayed a diminished startle response after approximately 2 and 4 hours. The startle response was normal for all exposure groups immediately after the exposures. No clinical signs of toxicity were observed either post-exposure or during the 2-day recovery period. No mortality or morbidity was observed in any dose group in either male or female animals.

MICRONUCLEUS EVALUATION: Based on the absence of evidence of DCBD-related sex differences, the micronucleus evaluation was conducted only in male animals. There were no statistically significant or biologically relevant increases in MNPCE frequency at any time point in male rats exposed to DCBD. All obtained values were also within the historical control range for this laboratory. As expected, statistically significant increases in MNPCE frequency were found in CP-treated male rats. Test substance-related depression in the PCE/NCE ratio was observed in male rats exposed to 200 ppm DCBD relative to the control mean. The magnitude of this statistically significant (p < 0.05) decrease was approximately 37% in the 200 ppm dose group at the 24-hour time point. A smaller, non-significant decrease of approximately 10 % was observed at the 48-hour time point. Similar decreases were seen in the ratios of PCE to total erythrocytes at 200 ppm. These decreases are indicative of bone marrow toxicity, and that the test substance had reached the target tissue.

Any other information on results incl. tables

No additional information.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this study, DCBD did not induce a statistically significant increase in micronucleated polychromatic erythrocytes in rat bone marrow. The highest exposure concentration in the study was 200 ppm, a level producing clear evidence of bone marrow and systemic toxicity. Therefore, the test substance was negative in this in vivo assay.
Executive summary:

Under the conditions of this study, DCBD did not induce a statistically significant increase in micronucleated polychromatic erythrocytes in rat bone marrow. The highest exposure concentration in the study was 200 ppm, a level producing clear evidence of bone marrow and systemic toxicity. Therefore, the test substance was negative in this in vivo assay.