Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Six in vitro Ames studies are available for this substance:

Barber E.D., Donish W.H. and Meuller K.R. (1981): Positive for S. typhimurium TA 1535 and TA 100

Lehrer. S (1981): Negative for all strains tested

Décharioux. H (1993): Negative for all strains tested

Moon. Y.H. et al (1998): Negative for all strains tested (including E.coli)

USA National Institute of Health (2011): Negative for all strains tested

USA National Institute of Health (2011): Negative for all strains tested (including E.coli)

An in vitro mouse lymphoma study is available for this substance:

Molinier. B (1996): Positive

Three in vivo Mouse Micronucleus studies are available for this substance:

Molinier. B (1995): 2 intraperitoneal doses - Negative

Moon. Y.H. et al (1998): 13-week inhalation exposure - Negative

USA National Institutes of Health (2011): 3-month inhalation exposure - Negative

Two in vivo Dominant Lethal Assay are available for this substance:

Saito-Suzuki R., Teramoto S. and Shirasu Y. (1982): Negative

Yu W. J. et al (2008): Negative

One in vivo Transgenic Rodent Somatic Cell Gene Mutation Assay in female mice is available for this substance:Young R. (2016): Negative

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

Genetic toxicity in vivo

Description of key information

Three in vivo Mouse Micronucleus studies are available for this substance:

Molinier. B (1995): 2 intraperitoneal doses - Negative

Moon. Y.H. et al (1998): 13-week inhalation exposure - Negative

USA National Institutes of Health (2011): 3-month inhalation exposure - Negative

Two in vivo Dominant Lethal Assay are available for this substance:

Saito-Suzuki R., Teramoto S. and Shirasu Y. (1982): Negative

Yu W. J. et al (2008): Negative

One in vivo Transgenic Rodent Somatic Cell Gene Mutation Assay in female mice is available for this substance:Young R. (2016): Negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: gene mutation
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The study was designed to investigate the possibility for a mutagenic mode of action for tumor formation, primarily in the lungs, at the request of ECHA (European Chemicals Agency) under the REACH Regulation
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 488 (2013)
Version / remarks:
The in-life and post-mortem portions of the study were conducted at WIL Research. The Big Blue® mutation assay portion of the study was conducted at BioReliance Corporation. Due to the acquisition of WIL Research by Charles River, the name of the WIL Research facility in Ashland, OH has been changed to Charles River Laboratories Ashland, LLC, 1407 George Road, Ashland, OH 44805, USA. Study documents may contain both names and both names are considered equivalent and may be used as the name WIL Research transitions to Charles River.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
transgenic rodent mutagenicity assay
Specific details on test material used for the study:
The test substance, n-propyl bromide (also known as 1-bromopropane; CAS no. 106-94-5), Commercial batch no. 864160042, exp. date: 10-Feb-2018.
The purity of the test substance was 99.90%. The test substance was stored at room temperature and was considered stable under this condition.
Species:
mouse
Strain:
other: Big Blue® B6C3F1 heterozygous female transgenic mice [Taconic nomenclature: B6C3F1-Tg(TacLIZa)A1Jsh]
Details on species / strain selection:
Details on species:
Big Blue® B6C3F1 heterozygous female transgenic mice (Taconic nomenclature: B6C3F1 Tg[TacLIZa]A1Jsh), obtained by WIL Research from BioReliance Corporation, Rockville, MD, USA and bred by Taconic Biosciences, Inc., Germantown, NY, USA on behalf of BioReliance, were used as the test system on this study. Each animal was uniquely identified by a Monel® metal ear tag displaying the permanent identification number. The animals were approximately 10 weeks old and within the age range of 8 to 12 weeks old at initiation of exposure, as specified in OECD TG 488. The individual body weights ranged from 16.2 g to 21.3 grams at the initiation of exposure.

Justification for Selection of Species and Strain:
Mice have been used historically in safety evaluation and genotoxicity studies and are recommended by regulatory agencies. Because this study was conducted in accordance with regulatory guidelines and regulatory agency request, alternatives could not be considered.

The Big Blue® B6C3F1 mouse was an appropriate species and strain to use in this in vivo mutation assay being performed to follow up on positive tumor findings in a 2-year rodent carcinogenicity study performed using B6C3F1 mice. Females were the appropriate sex for this study since only females demonstrated a significant increase in tumor response (NTP, 2013).

The Big Blue® in vivo mutation assay is a Transgenic Rodent (TGR) Mutation assay described in OECD TG 488 (OECD, 2013). TGR assays in general and the Big Blue® assay in particular have been reviewed (OECD, 2009 and 2011a) and identified in OECD TG 488 (OECD, 2011b and 2013) as being appropriate to investigate a potential mutagenic mode of action associated with tumor formation.
Sex:
female
Details on test animals or test system and environmental conditions:
Animal Receipt and Acclimation: Each animal was inspected by a qualified technician upon receipt. Animals judged to be in good health were placed in acclimation for a minimum of 7 days.

Animal Housing: All animals were housed in clean, solid-bottom cages with bedding material or other suitable material in an environmentally controlled room. All animals weremaintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011).
Environmental Conditions:
Environmental controls in the animal room were set to maintain a temperature of 22 ± 3oC and relative humidity at 50 ± 20%. Fluorescent lighting provided illumination for a 12-hour light/dark photoperiod. The ventilation rate was set at a minimum of 10 room air changes per hour, 100% fresh air.

Drinking Water: Reverse osmosis-treated water was available ad libitum. Water samples are routinely analyzed for environmental contaminants. Results are maintained in the facility records.

Basal Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 meal was offered ad libitum during the study. Each lot utilized will be identified and recorded.

Environmental Enrichment: Enrichment devices were provided to each animal for environmental enrichment and to aid in maintaining the animals' oral health, beginning during acclimation and continuing throughout the course of the study.

Veterinary Care: Animals were monitored by the technical staff for any condition requiring possible veterinary care. Animals were examined and approved for use by the Veterinarian, or designee, prior to randomization and this were documented in the raw data.
Route of administration:
inhalation: aerosol
Vehicle:
Not applicable
Details on exposure:
Inhalation Exposure and Exposure Atmosphere Generation Methods:
Exposures were conducted in 4 approximately 1000-L stainless steel and glass whole-body inhalation chambers. One chamber was dedicated for each group for the duration of the study. Chamber supply air was provided from the Charles River Inhalation Department breathing-quality, in-house compressed air source and charcoal filtered, temperature-, and humidity-controlled source. All control and test substance atmosphere chamber exhaust passed through the facility exhaust system, which consisted of redundant exhaust blowers preceded by activated-charcoal and HEPA filtration units. Animals were housed in a normal animal colony room during non exposure hours. Prior to each exposure, the animals were placed into the exposure batteries, transported to the exposure room, placed into the exposure chambers, exposed for the requisite duration, and then returned to their home cages in the animal colony room. Animals were housed individually in standard exposure batteries of appropriate size for the whole-body exposure chambers used during exposure periods. Food, but not water, was withheld during all animal exposure periods.

Temperature, relative humidity, airflow rate, and negative pressure within the exposure chambers were continually monitored and recorded approximately every 45 minutes during the exposure periods (see Section 5 - Phase Plan Deviations). The mean temperature and mean relative humidity were to be between 20°C to 26°C and 30% to 70% (see Section 5 - Phase Plan Deviations), respectively. Oxygen content was measured during the method development phase and was 20.9% for all chambers. In addition, during the method development phase the high concentration chamber was monitored for aerosol formation using a Microdust Pro aerosol monitoring system. No aerosol was detected.

The control exposure system was operated as follows. Humidified air was delivered to the whole-body chamber system using a rotameter-type flowmeter connected to the Charles River Inhalation Department supply air source.

Each test substance exposure system was operated as follows. Test substance vapors were generated using a bubbler-type vaporization system filled with an appropriate amount of liquid test substance. Dry, compressed air from an Inhalation Department in-house system was metered into the inlet stem of each gas washing bottle. Air bubbled through the fritted disc and the liquid test substance to produce concentrated vapors of the test substance. Compressed air was metered to the gas washing bottle using a regulator and was controlled using a needle valve and flowmeter.

The concentrated vapors were delivered to the exposure chamber inlet and were diluted to the desired exposure concentration by mixing with the chamber supply air prior to entering the chamber.
Duration of treatment / exposure:
6 hours/Day for 28 consecutive days
Frequency of treatment:
Daily for 28 days followed by 2 days postdosing period..
Post exposure period:
2 days
Dose / conc.:
62.5 ppm (nominal)
Dose / conc.:
125 ppm (nominal)
Dose / conc.:
250 ppm
No. of animals per sex per dose:
6 females per dose group.
Control animals:
yes, concurrent no treatment
Positive control(s):
Yes. Liver and lung tissue samples were processed for DNA isolation from frozen tissues from 5 positive control-treated animals, collected as part of BioReliance Corporation Study Number AE34AA.170.BTL. This use of “packaging controls” is permitted by OECD TG 488. The goal of the positive control group was to demonstrate the ability to recover induced mutants from the study target tissues. ENU is a potent direct acting mutagen, demonstrated to be mutagenic in the target tissues.
Tissues and cell types examined:
Extraction of Genomic DNA

Liver and lung samples from the first 5 test or control substance-treated animals/group were processed for DNA isolation. Tissues from the sixth animal per group were retained frozen in reserve and not processed further, unless needed. After initial analysis of the lung DNA, it was apparent that one animal in both Groups2 (62.5 ppm, low dose) and 3 (125 ppm, mid-dose) had an elevated mutant frequency, possibly consistent with a “jackpot” mutation (The presence of a pre-existing mutation will show up as an elevated mutant frequency). An animal with a ‘jackpot mutation’ is generally identified because the mutant frequency of that animal is an ‘outlier’ from the rest of the animals in the group – or something like this – make it sound more scientific!) For that reason, the sixth (replacement) animal from Groups 2 and 3 was also extracted and analyzed. DNA was extracted following BioReliance SOP OPGT9030, which was based on methods described for Agilent product RecoverEase (Agilent, 2009a) for somatic tissues. Isolated DNA samples were stored at 2 to 8°C.
Statistics:
Statistical Analysis for In-Life Parameters
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group.
Body weight, body weight change, food consumption, and organ weight data were subjected to a parametric one way ANOVA to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance treated groups to the control group.
Key result
Sex:
female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
All animals survived to the scheduled necropsy. There were no test substance-related clinical observations or effects on food consumption or liver and lung weights. There were no gross lesions noted for any animals at the scheduled necropsy. Body weights were unaffected by exposure to the test substance. Body weight losses were noted in all test substance-treated groups from study day 6-13; however, the changes were small in magnitude, did not occur in a dose-related manner and were, therefore, not considered related to exposure to the test substance.

Mortality and In-life Observations

 Summary and individual animal data are presented in Appendix C (attached).

 All animals survived to the scheduled necropsy. There were no test substance-related clinical observations.

   

 In-life Measurements

 Summary and individual body weights and food consumption data are presented in Appendix C (attached).

 Body weights were unaffected by exposure to the test substance. Body weight losses were noted in all test substance-treated groups from Days 6 to 13 (relative to Test Site Study Day 0); however, the changes were small in magnitude, did not occur in a dose-related manner and were, therefore, not considered related to exposure to the test substance. There were no test substance-related effects on food consumption.

Post-life Data

Summary and individual animal data are presented in Appendix C (attached).

 There were no gross lesions noted for any animals at the scheduled necropsy, and there were no test substance- related effects on liver and lung weights. 

Mutant Frequency Data

Summaries of mutant frequency data are presented in Table 1 (liver) and Table 2 and Table 3 (lungs) (attached).

 Individual mutant frequency data are presented in Table 4 (liver) and Table 5 (lungs). Historical control mutant frequency data are presented in Appendix B (attached).

 

Sufficient quantity and quality of DNA was obtained to permit 2 to 6 packagings of each DNA sample, yielding more than the OECD-specified minimum of 125,000 phage/tissue/animal. Since the experimental unit is the animal, the total number of plaque-forming units, the number of mutants and the mutant frequency for each tissue per animal are reported. In addition, the number of packaging cycles used to achieve the reported data is reported for each tissue from each animal.

 Liver

 For liver, the Big Blue®B6C3F1 mouse mutation assay gave background mutant frequencies in the filtered air control (Group 1) of 60.5 ± 9.8 x 10-6(mean ± standard deviation) (Table 1). This was comparable to historical experience of45.5 ± 21.8 x 10‑6for liver(Appendix B, Table 6).

 MeancIImutant frequencies in liver fromn-propyl bromide treated animals were 65.4 ± 27.2 x 10‑6, 48.8 ± 11.4 x 10‑6, and 75.6 ± 39.1 x 10‑6(Groups 2, 3 and 4, respectively,Table 1). Individual animal liver mutant frequencies in animals exposed to n-propyl bromide (Table 4) were within the historical range for liver vehicle control animals but did exhibit some variation common to biological systems. While one high dose animal slightly exceeded the 99% Control Limit (Mean ± 3 times the Standard Deviation) for vehicle control animals, the mutant frequency was within the historic range for vehicle control animals and is consistent with normal animal to animal variation (Appendix B, Table 6).

 

Statistical analysis of the n-propyl bromide treated groups relative to the filtered air control by 1-Way ANOVA revealed that the mean mutant frequency of the test substance-treated groups was not significantly different than the control group (p = 0.701).  The normality test performed on the residuals (p > 0.100) and the equal variance test (p = 0.325) confirmed that the ANOVA criteria were met, therefore the analysis was considered adequate.

 

Results from liver DNA from Positive Control treated animals (Group 5) demonstrated reproducible mutant frequencies of 191.6 ± 51.6 x 10-6(Table 1). The ENU Positive Control mutant frequencies were comparable to historical experience of 183.3 ± 50.2 x 10‑6for liver (Appendix B, Table 6).1-Way ANOVA revealed that the mean mutant frequency of the ENU-treated group was significantly elevated over the control group (p < 0.001).  However, the normality test performed on the residuals failed (p < 0.010), therefore the data were not normally distributed, and the ANOVA test was not considered appropriate.  Data was further analyzed using the Kruskal-Wallis test (non‑parametricalternative to ANOVA); this test confirmed that the median MF of the ENU-treated group was significantly elevated over the control group (p = 0.009).

 

 Lung

For lung, the Big Blue®B6C3F1 mouse mutation assay gave background mutant frequencies in the control (Group 1) of 71.4 ± 26.7 x 10-6(Tables 2and3). The control mutant frequencies were comparable to historical experience of60.7 ± 16.0 x 10‑6for lung(Appendix B, Table 7).

 

Lung DNA from twon-propyl bromide treated animals had elevated mutant frequencies. These occurred in low dose animal number 41138 (Group 2) and mid-dose animal number 41156 (Group 3) and had mutant frequencies of 186.8 x 10-6and 366.1 x 10-6, respectively (Table 5). In addition, one filtered air control animal, number 41153 (Group 1) had a mutant frequency of 112.4 x10-6, a value just outside the historic background range and 99% Control Limit (Table 5). This last value was consistent with normal animal to animal variation in background mutant frequency seen in lung and other tissues such as liver (Appendix B). The mutant frequencies from animals 41138 and 41156 were outside normal animal to animal variability and greatly exceeded the upper 99% Control Limit and past historical experience for background mutant frequency. To help provide additional power to the analysis of these dose groups, the additional sixth animal in Groups 2 and 3 was extracted and analyzed providing a group size of 6 for these two groups. The pattern of a single animal in each group with an elevated mutant frequency and other animals at control levels is suggestive of a possible pre-existing jackpot mutation in the tissue. As these were test substance exposed animals, a conservative approach was taken and all animals were initially analyzed as a group including the ones with elevated mutant frequencies (Table 2 and Table 5) followed by analysis excluding animal numbers 41138 and 41156 in Groups 2 and 3 (Table 3 and Table 5).

 MeancIImutant frequencies in lungs fromn-propyl bromide treated animals were 83.1 ± 52.9 x 10‑6, 112.9 ± 128.4 x 10‑6, and 43.2 ± 14.4 x 10‑6(Groups 2, 3 and 4, respectively,Table 2). This includes both outlier animals and replacement animals in Groups 2 and 3. Exclusion of outlier animals (animal 41138 in Group 2 and animal 41156 in Group 3) resulted in group meancIImutant frequencies of 62.4 ± 16.7 x 10‑6, 62.2 ± 37.1 x 10‑6, and 43.2 ± 14.4 x 10‑6(Groups 2, 3 and 4, respectively,Table 3).

Conclusions:
n-propyl bromide is considered not mutagenic in the mouse transgenic rodent mutation assay.

Treatment with n-propyl bromide did not cause statistically elevated mutant frequencies at the cII gene in liver and lungs of Big Blue® female mice. The positive control treatment with ENU produced statistically significant increases in mutant frequencies for both tissues tested, demonstrating the utility of the test system to detect and quantify induced mutants following exposure to a known direct acting mutagen. The study design and results obtained met protocol-specified assay acceptance criteria and were consistent with the study requirements of OECD TG 488 for transgenic rodent mutation assays, supporting the conclusion that n-propyl bromide does not cause significantis negative for the induction of cII mutants in liver and lungs of Big Blue® female mice under the conditions of testing. Therefore, n-propyl bromide is considered not mutagenic in the mouse transgenic rodent mutation assays.
Executive summary:

 SUMMARY

 The purpose of this study was to determine the effect of the test substance, n-propyl bromide (also known as 1‑bromopropane), on mutant frequency at the cII gene in liver and lung from female transgenic Big Blue® B6C3F1 mice following whole-body inhalation exposure for 6 hours per day on a 7 day per week basis for 4 weeks (28 exposures for each animal).

The Big Blue® Assay is a Transgenic Rodent (TGR) mutation assay, described in OECD Test Guideline (TG) 488 (OECD, 2013).

The in-life and post-mortem portions of the study were conducted at WIL Research. The Big Blue® mutation assay portion of the study was conducted at BioReliance Corporation. Due to the acquisition of WIL Research by Charles River, the name of the WIL Research facility in Ashland, OH has been changed to Charles River Laboratories Ashland, LLC, 1407 George Road, Ashland, OH 44805, USA. Study documents may contain both names and both names are considered equivalent and may be used as the name WIL Research transitions to Charles River.

The test substance, n-propyl bromide, was administered via whole-body inhalation exposure for 6 hours per day for 28 consecutive days to 3 groups (Groups 2, 3 and 4) of female Big Blue® B6C3F1 mice. Target exposure concentrations were 62.5, 125 and 250 ppm for Groups 2, 3 and 4, respectively. A concurrent control group (Group 1) was exposed to humidified, filtered air on a comparable regimen. Each group consisted of 6 female animals. The first day of exposure for this study report was designated as Day 1. On Day 31, the third day after the last dose administration, all animals were euthanized. The first day of inhalation exposure is defined by the Test Site (WIL Research) as Day 0, while the Testing Facility (BioReliance) defines the first day of exposure as Day 1.

All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed twice daily on the days of exposure, prior to exposure and at 0-1 hour (+ 0.25 hour) [presented as 1 hour post-exposure for report presentation purposes] following exposure, and once daily on non-exposure days. Detailed physical examinations were performed and individual body weights (non-fasted) were recorded within 4 days of receipt, on the day of randomization, weekly (± 1 day) during the study period, and on the day of the scheduled necropsy. Individual food weights were recorded once weekly (± 2 days) beginning after randomization and throughout the study period, including the day of the scheduled necropsy. In order to minimize the in situ degradation of the DNA, all animals were euthanized by carbon dioxide inhalation, and the liver and lungs were weighed (for prediction of the number of DNA extractions possible from a tissue), flash frozen in liquid nitrogen, and stored at approximately -80°C. The tissues were shipped on dry ice via overnight courier to BioReliance, Rockville, MD, for cII mutant analysis.

As specified in the study protocol, the liver and lungs from 5 animals per group were processed for DNA isolation and analysis of cII mutants, following BioReliance SOP’s. Due to the elevated mutant frequency in lung DNA from one animal in both Groups 2 and 3, the sixth animal in each of these groups was also extracted and analyzed for cII mutants.

In addition, liver and lung tissue samples were processed for DNA isolation from frozen tissues from 5 positive control-treated animals, collected as part of BioReliance Corporation Study Number AE34AA.170.BTL. This positive control group is identified in post-life data as Group 5. This use of “packaging controls” is permitted by OECD TG 488. The goal of the positive control group was to demonstrate the ability to recover induced mutants from the study target tissues. Positive control tissues from target organs were collected on Testing Facility Study Day 31 after the start of dosing (Day 1 was defined by Testing Facility as the first day of dosing) from Big Blue® B6C3F1 female mice exposed by oral gavage to 40 mg/kg/dose of ethyl nitrosourea (ENU) on Study Days 1, 2 and 3. ENU is a potent direct acting mutagen, demonstrated to be mutagenic in the target tissues.

The study was designed to investigate the possibility for a mutagenic mode of action for tumor formation, primarily in the lungs, at the request of ECHA (European Chemicals Agency) under the REACH Regulation. The target exposure concentrations and the exposure regimen (6 hours/day for 7 days/week for a 28-day period) were selected by the Sponsor’s Representative and are consistent with those recommended in OECD Test Guideline 488 (OECD, 2013). Since the request for the TGR assay was based on the National Toxicology Program (NTP) carcinogenicity study in B6C3F1 mice, (NTP, 2013), tumorogenic dose levels (ranging from 62.5 to 250 ppm) and exposure conditions (inhalation) were established to match those of the NTP study design. The only modification was the use of a 6 hour/day, 7 day/week dosing regimen for 28 days. The modification of exposure for 7 days per week was used to be complient with the OECD TG 488 which specifies a 7 day/week exposure for 28 days, which represented the worst case. The design is sufficient to permit genetic damage and fixation of the damage into detectable mutants if n-propyl bromide carcinogenicity is due to a mutagenic mode of action.

Treatment with n-propyl bromide did not cause statistically elevated mutant frequencies at the cII gene in liver and lungs of Big Blue® female mice. The positive control treatment with ENU produced statistically significant increases in mutant frequencies for both tissues tested, demonstrating the utility of the test system to detect and quantify induced mutants following exposure to a known direct acting mutagen. The study design and results obtained met protocol-specified assay acceptance criteria and were consistent with the study requirements of OECD TG 488 for transgenic rodent mutation assays, supporting the conclusion that n-propyl bromide is negative for the induction of cII mutants in liver and lungs of Big Blue® female mice under the conditions of testing. Therefore, n-propyl bromide is considered not mutagenic in the mouse transgenic rodent mutation assay.

 

Key result
Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The test material was administered once daily by gavage to male mice for 10 consecutive days before mating. Positive and vehicle controls were run concurrently for comparison. After completion of the dosing period males were mated with untreated females during six sequential mating periods of a week each. Males were sacrificed at the end of mating and so were the pregnant females on days 15–17 of gestation. Clinical signs, gross findings, mating index, gestation index, the numbers of corpora lutea, implantations, live fetuses, resorptions and dead fetuses, pre- and post-implantation losses, and dominant lethal mutation rate were examined.
GLP compliance:
no
Type of assay:
rodent dominant lethal assay
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Orient Bio Co. Ltd. (Gyeonggi-Do, Korea).
- Total No. of mice: 66 males and 720 females.
- Age at study initiation: Males 7 weeks, females 4 to 7 weeks old.
- Assigned to test groups randomly: yes.
- Housing: Animals were housed in a stainless-steel wire-mesh cage during the acclimation and mating periods. Mated females were housed in clear polycarbonate cages with stainless steel wire lids.
- Diet: Pelleted foods was gamma-ray irradiated and provided ad libitum.
- Water: UV-irradiated municipal tap water was given ad libitum.
- Quarantine and acclimation period: 1 week.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3 ºC.
- Humidity (%): 50 ± 10%.
- Air changes (per hr): 13 to 18 charges per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle.
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: Corn oil.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: An appropriate amount of the test material was suspended in corn oil and was freshly prepared daily before the treatment.
Duration of treatment / exposure:
Males only were treated for ten consecutive days before mating.
Frequency of treatment:
Daily.
Post exposure period:
Post exposure animals were mated for 6 weeks. Details of the mating procedure can be seen in the field 'Any other information on materials and methods incl. tables'.
Remarks:
Doses / Concentrations:
300 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
600 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
Twenty males per dose. An additional twelve mice were used for determination of serum testosterone levels and histopathological changes.
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: Cyclophosphamide monohydrate dissolved in a sterilized physiological saline.
- Route of administration: Fifteen males were dosed intraperitoneally.
- Doses / concentrations: 40 mg/kg for five consecutive days before mating.
Tissues and cell types examined:
Cells collected for analysis: blood from the vena cava and testes, serum and sperm.
Tissue collected for analysis: testes.
Details of tissue and slide preparation:
TREATMENT, SAMPLING TIMES AND PREPARATION:
Serum testosterone levels and histopathological examination: Six mice were orally treated with the test material at the dose of 600 mg/kg for 10 consecutive days, and the remnants were administered with corn oil. All animals were sacrificed on the next day after the final treatment. Blood was collected from the cauda vena cava and the testes were fixed in Bouin’s solution. Serum samples were stored at -80 °C until assayed for testosterone. The testes were embedded in paraffin, cut and stained with hematoxylin and eosin, and examined microscopically.
Sperm analysis: To evaluate sperm quality at 5 weeks after the final treatment, 12 male mice aged 8 weeks were used. Six mice were orally treated at a dose level of 600 mg/kg for 10 consecutive days and the remnants were administered with corn oil. At 5 weeks after the final treatment, all mice were sacrificed. The left cauda epididymis were retrieved, and then sperms were suspended by incision of the tissue in CO2 independent medium supplemented with 0.5% bovine serum albumin.

SPERM ANALYSIS:
Major motion parameters such as motility, straight line velocity (VSL), curvilinear velocity (VCL), path velocity (VAP), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) were analyzed with HTM-TOX IVOS sperm analyzer at 5–10 min after preparation of the sperm suspension.
Statistics:
Continuous data such as body weights were analyzed by using Bartlett’s test for variance homogeneity. When the Bartlett’s test indicated no significant deviations from variance homogeneity, the ANOVA multiple comparison test was conducted to determine which pairs of group comparison were significantly different (α = 0.05). When significance was found by ANOVA, Dunnett’s t-test was used to determine the difference between the control and treatment groups. In the case that significant deviations from variance homogeneity were observed, a non-parametric comparison test (Kruskal-Wallis (H) Test) was conducted. When a significant difference was observed in the Kruskal–Wallis (H) test, the Dunn’s Rank Sum test was conducted to quantify the specific pairs of group comparison, which are significantly different. The significance of a difference between one treatment group and control group such as serum testosterone levels, parameters of sperm motions was evaluated statistically by analysis of Student’s t-test. The level of significance was taken as P < 0.05 or 0.01. Litter data such as numbers of the corpora lutea, resorptions and dead fetuses were statistically evaluated using the statistical unit as a litter. Statistical analyses were performed by comparing the different dose groups with the vehicle control group using the statistical software (Path/Tox System Version 4.2.2 or SAS/STAT Version 8).
Sex:
male
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
- Clinical signs of toxicity in test animals: No treatment-related changes in clinical signs and body weights were observed in any of the males during the treatment and non-treatment periods. No notable changes in behavior, clinical signs or abortion were observed in any of the females of all groups.

- Mating performance and pregnancy status (see Table 1): The mating index in the vehicle control group ranged from 95% to 100% through the different mating intervals. However, a decreasing trend was observed in the first week (87.5%) of the 300 mg/kg dose group, the second week (85.0%) of the 600 mg/kg dose group, and the first (83.3%), third (83.3%), fifth (86.7%) and sixth (80.3%) weeks of the positive control group as compared to the vehicle control group (95–100%). However, these were considered to be of no toxicological significance since all males of each group could impregnate females, and there was no consistency among the mating intervals. In addition, the gestation index of all treatment groups including the positive control group was not statistically significantly different from that of the vehicle control group.

- Caesarean section findings (see Table 2): The test material did not affect the numbers of implantations, live fetuses, dead fetuses, and fetal deaths. However, a decrease in the numbers of implantations was found during the first 2 weeks of the positive control group as compared to the vehicle control group. In addition, a decrease in the number of live fetuses and an increase in the number of resorptions and fetal deaths (resorptions + dead fetuses) were observed during the first 3 weeks in the positive control group as compared to the vehicle control group. There was no change in the number of dead fetuses in any of the groups.

- Pre- and post-implantation loss and dominant mutation rates (see Table 3): No significant change in these parameters was observed in the 300 mg/kg dose group as compared to the vehicle control group. A significant increase in pre-implantation loss was observed during the fifth week of the 600 mg/kg dose group as compared to the vehicle control group. On the contrary, post-implantation loss was significantly decreased during the same mating period of the 600 mg/kg dose group. There was no change in dominant lethal mutation rate in the 600 mg/kg group. In the positive control group, a significant increase in pre- and post-implantation loss was observed during the first 2 weeks, and post-implantation loss was also increased during the third week as compared to the vehicle control group. Additionally, dominant lethal mutation rate was markedly increased during the first 3 weeks in the positive control group.

- Serum testosterone: Levels were 0.96 ± 1.13 (mean ± S.D.) ng/mL and 1.32 ± 1.61 ng/mL for the treatment group and the vehicle control group, respectively. There was no statistically significant change in serum testosterone levels. However, degeneration of pachytene spermatocytes was increased in males treated with the test material compared to the controls.

- Sperm parameters (see Table 4): In the results of examination for motion parameters of sperms in the cauda epididymis at 5 weeks after treatment, motility was numerically decreased in the treatment groups (70.3 ± 16.7%) as compared to the vehicle controls (85.7 ± 7.5), although there was no statistical significance. No treatment related changes were observed in any other parameters.

Table 1. Mating and Pregnancy Status

Group

Test Week

No. of Females Paired

Mating Index (%)

Gestation Index (%)

 

Vehicle Control

1

40

95.0

94.7

 

2

40

100

97.5

 

3

40

97.5

100

 

4

40

95.0

94.7

 

5

40

100

90.0

 

6

40

97.5

94.9

 

Test Material, 300 mg/kg

1

40

87.5

88.6

 

2

40

92.5

100

 

3

40

92.5

97.3

 

4

40

92.5

97.3

 

5

40

92.5

83.8

 

6

40

95.0

97.4

 

Test Material, 600 mg/kg

1

40

97.5

94.9

 

2

40

85.0

100

 

3

40

95.0

100

 

4

40

100

97.5

 

5

40

92.5

100

 

6

40

100

92.5

 

Positive Control

1

30

83.3

92.0

 

2

30

90.0

100

3

30

83.3

96.0

4

30

90.0

96.3

5

30

86.7

92.3

6

30

80.3

95.8

 

Table 2. Corpora Lutea, Live and Dead Implantations

Group

Test Week

No. of Corpora Lutea (± SD)

No. of Implantations (± SD)

No. of Live Fetuses (± SD)

No. of Resoptions (± SD)

No. of Dead Fetuses (± SD)

Fetal Deaths (± SD)*

Vehicle Control

1

15.2 (2.8)

14.1 (2.7)

13.1 (2.5)

1.1 (1.3)

0.1 (0.4)

1.3 (1.2)

2

15.2 (2.2)

14.3 (2.0)

13.5 (2.1)

0.5 (0.9)

0.2 (0.4)

0.7 (1.0)

3

13.7 (2.0)

13.5 (2.0)

12.6 (2.0)

0.7 (1.0)

0.1 (0.4)

0.8 (1.0)

4

15.6 (1.9)

15.1 (2.1)

14.3 (2.2)

0.6 (0.8)

0.2 (0.5)

0.8 (0.9)

5

14.1 (1.9)

13.6 (2.4)

12.9 (2.8)

0.7 (1.0)

0.1 (0.2)

0.7 (1.1)

6

15.9 (2.4)

14.8 (2.4)

14.2 (2.6)

0.7 (2.2)

0.1 (0.3)

0.8 (2.1)

Test Material, 300 mg/kg

1

14.8 (1.9)

13.8 (1.7)

12.8 (1.3)

0.8 (0.9)

0.1 (0.4)

1.0 (1.1)

2

15.2 (2.0)

14.4 (2.2)

13.3 (2.5)

0.8 (1.2)

0.3 (0.6)

1.1 (1.1)

3

15.0 (3.2)

14.4 (2.8)

13.4 (2.7)

0.9 (1.1)

0.2 (0.5)

1.1 (1.2)

4

15.6 (2.2)

14.5 (1.9)

13.3 (2.2)

1.0 (1.5)

0.3 (0.5)

1.3 (1.6)

5

14.7 (2.1)

13.8 (2.9)

12.7 (3.3)

1.0 (1.7)

0.1 (0.3)

1.1 (1.8)

6

15.6 (2.4)

14.5 (2.8)

13.4 (3.1)

0.8 (1.0)

0.1 (0.2)

0.8 (1.0)

Test Material, 600 mg/kg

1

15.9 (2.4)

14.6 (2.5)

13.6 (2.3)

0.9 (1.3)

0.1 (0.3)

1.0 (1.4)

2

14.8 (1.8)

14.0 (1.7)

13.1 (1.6)

0.6 (0.9)

0.3 (0.7)

0.9 (1.2)

3

15.0 (2.9)

14.5 (2.8)

13.9 (2.8)

0.5 (0.8)

0.1 (0.4)

0.6 (0.8)

4

15.3 (2.1)

14.8 (2.5)

14.3 (2.6)

0.4 (0.7)

0.2 (0.4)

0.5 (0.9)

5

15.8 (2.0)

13.8 (3.2)

13.7 (3.2)

0.4 (0.7)

0.2 (0.5)

0.6 (0.9)

6

15.1 (2.1)

13.9 (2.4)

13.3 (2.7)

0.5 (0.7)

0.2 (0.4)

0.7 (0.7)

Positive Control

1

14.0 (2.4)

11.8 (2.5)**

6.0 (2.6)**

5.5 (2.3)**

0.1 (0.3)

5.6 (2.3)**

2

12.7 (2.4)**

10.7 (2.3)**

5.4 (2.6)**

5.3 (2.8)**

0.0 (0.2)

5.3 (2.8)**

3

14.0 (2.3)

13.1 (2.3)

10.3 (2.7)**

2.8 (1.6)**

0.0 (0.2)

2.8 (1.6)**

4

15.0 (2.4)

13.9 (2.4)

12.9 (3.2)

0.7 (1.2)

0.2 (0.4)

0.9 (1.2)

5

14.8 (2.6)

13.8 (3.3)

13.1 (3.2)

0.6 (0.9)

0.2 (0.5)

0.8 (1.0)

6

15.7 (1.9)

13.7 (2.6)

13.1 (2.4)

0.5 (0.8)

0.1 (0.3)

0.7 (0.8)

* Fetal deaths = no. of resorptions + no. dead fetuses.

**Significant difference from control group (p<0.01)

Table 3. Pre- and Post- Implantation Losses and Dominant Lethal Mutation rate

Group

Test Week

Pre-implantation loss % (±SD)

Post-implantation loss % (±SD)

Dominant Lethal Mutation Rate % (±SD)

Vehicle Control

1

7.1 (9.7)

6.6 (7.7)

 

2

5.8 (8.8)

5.0 (7.3)

 

3

1.6 (3.6)

6.3 (7.6)

 

4

3.7 (6.6)

5.4 (6.0)

 

5

3.9 (9.1)

5.7 (9.0)

 

6

6.5 (9.3)

4.4 (8.0)

 

Test Material, 300 mg/kg

1

6.3 (8.5)

6.6 (7.1)

0.17

2

5.8 (8.2)

7.8 (8.3)

2.17

3

3.2 (6.7)

7.1 (8.0)

0.3

4

6.0 (10.3)

8.8 (9.7)

3.14

5

6.7 (15.3)

7.7 (13.7)

2.98

6

9.6 (18.8)

6.6 (8.7)

3.68

Test Material, 600 mg/kg

1

8.1 (9.7)

6.4 (8.5)

-0.26

2

5.1 (6.7)

6.0 (7.6)

0.88

3

2.9 (6.9)

4.1 (5.3)

-2.71

4

3.4 (8.0)

3.6 (5.7)

-2.03

5

12.9 (16.9)*

0.8 (2.7)**

-4.66

6

7.4 (12.5)

5.3 (7.2)

0.27

Positive Control

1

15.3 (12.9)**

48.8 (19.2)**

45.27

2

14.9 (13.4)**

49.1 (19.9)**

46.54

3

5.9 (9.1)

21.7 (13.1)**

15.76

4

7.6 (15.1)

6.9 (9.8)

2

5

8.2 (15.7)

4.6 (6.2)

0.08

6

12.1 (13.8)

3.9 (4.7)

0.34

* Significant differences from control group (p<0.05).

** Significant differences from control group (p<0.01).

Table 4. Major Motion Parameters of Sperm at 5 Weeks

Group

Mortality % (± SD)

Path Velocity µm/s (±SD)

Straight Line Velocity µm/s (±SD)

Curvilinear velocity µm/s (±SD)

Lateral Head Displacement µm (±SD)

Beat Cross Frequency Hz (±SD)

Vehicle Control

85.7 (7.5)

198.9 (19.9)

178.8 (18.7)

278.6 (23.3)

10.8 (1.1)

30.1 (4.4)

Test Material, 600 mg/kg

70.3 (16.7)

188.9 (21.3)

165.9 (22.3)

277.6 (19.4)

11.7 (0.9)

29.8 (2.4)

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of the test, the test material did not induce dominant lethality in mice. There were no treatment related changes in clinical signs, gross findings, mating index, gestation index, number of corpora lutea and implantations, pre-implantation loss, live fetuses, resorptions, dead fetuses, post-implantation loss at any of the dose levels. However there was an increase in pre-implantation loss at the fifth week following treatment, this was considered to be due to low sperm quality.
Executive summary:

Paternally transmitted genetic defects were investigated in mice using a dominant lethal assay. The test material was administered once daily by gavage to male mice for 10 consecutive days before mating. Positive and vehicle controls were run concurrently for comparison. After completion of the dosing period males were mated with untreated females during six sequential mating periods of a week each. Males were sacrificed at the end of mating and so were the pregnant females on days 15–17 of gestation. Clinical signs, gross findings, mating index, gestation index, the numbers of corpora lutea, implantations, live fetuses, resorptions and dead fetuses, pre- and post-implantation losses, and dominant lethal mutation rate were examined.

Under the conditions of the test, the test material did not induce dominant lethality in mice. There were no treatment related changes in clinical signs, gross findings, mating index, gestation index, number of corpora lutea and implantations, pre-implantation loss, live fetuses, resorptions, dead fetuses, post-implantation loss at any of the dose levels. However there was an increase in pre-implantation loss at the fifth week following treatment, this was considered to be due to low sperm quality.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17th November 1994 - 10th May 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
Only a single dose (not the limit dose) was evaluated in the main test.
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Iffa Crédo, L'Arbresle, France.
- Age at study initiation: ca. 7 weeks.
- Weight at study initiation: NDA
- Assigned to test groups randomly: yes, by sex.
- Fasting period before study: NDA
- Housing: Polycarbonate cages each containing 5 animals.
- Diet (e.g. ad libitum): Animals had free access to A04 C pelleted sustenance diet.
- Water (e.g. ad libitum): Animals had free access to tap water.
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 ºC
- Humidity (%): 50 ± 20 %
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12 (light period from 07:00 - 19:00)


FIRST STUDY IN-LIFE DATES: From: 09/02/1995 To: 14/02/1995
SECOND STUDY IN-LIFE DATES: From: 16/03/1995 To: 22//03/1995
THIRD STUDY IN-LIFE DATES: From: 04/05/1995 To: 09/05/1995
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil
- Justification for choice of solvent/vehicle: NDA
- Concentration of test material in vehicle: from 10 - 80 mg/mL depending on dosage requirements.
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Type and concentration of dispersant aid (if powder): N/A
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was suspended in the vehicle in order to achieve the required concentrations and then homogenised with a magnetic stirrer. The preparations were made immediately before use.
Duration of treatment / exposure:
48 hours from 1st dose to necropsy
Frequency of treatment:
Twice, once at 0 hours and then again at 24 hours.
Post exposure period:
24 hours from final dose to necropsy.
Remarks:
Doses / Concentrations:
100, 400 and 80 mg/kg/day
Basis:
other: actual administrated concentration. First study, males and females.
Remarks:
Doses / Concentrations:
800 mg/kg/day
Basis:
other: actual administrated concentration. Second study, males and females.
Remarks:
Doses / Concentrations:
600 mg/kg/day
Basis:
other: actual administrated concentration. Third study, males only.
No. of animals per sex per dose:
First study. 5 animals per sex per dose. 3 supplementary animals per sex at 800 mg/kg/day dose.
Second study: 5 animals per sex per dose. 3 supplementary animals per sex at 800 mg/kg/day dose.
Third study: 5 male animals per dose. 3 supplementary male animals at 600 mg/kg/day dose.
Control animals:
yes, concurrent vehicle
Positive control(s):
CPA cyclophosphamide
- Justification for choice of positive control(s): NDA
- Route of administration: oral
- Doses / concentrations: 50 mg/kg/day
Tissues and cell types examined:
Bone marrow from the femur of the mouse was examined.

For each animal, the micronucleated polychromatic erythocytes were counted in 2000 polychromatic erythocytes; the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).
Details of tissue and slide preparation:
The femurs of the mice were removed and the bone marrow eluted out using fetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were suspended by shaking. A drop of this cell suspension was placed and spread on a slide. The sides were air-dried and stained with Giemsa. All the slides were coded for scoring.
Evaluation criteria:
Biological relevance of the results was considered first. In addition, the following criteria may be used as an aid for determining a positive response:
• a statistically significant increase in the frequency of MPE when compared to the vehicle group
• this increase should double the frequency of MPE of available historical data.
Statistics:
The mean number of micronucleated polychromatic erythrocytes (MPE) and the PE/NE ratio from the treated groups were compared to the simultaneous vehicle groups. The intergroup comparison was performed using:
• for MPE, the χ^2 test,
• for the PE/NE ratio, the Student's t test,
in which ρ = 0.05 was used as the lowest level of significance.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
lowest dose observed was 800 mg/L
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Sex:
female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
dosed up to 800 mg/L
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 250, 500, 750, 1000, 150 and 2000 mg/kg/day
- Solubility: NDA
- Clinical signs of toxicity in test animals: In males, prostration (3/4 animals) and mortality (1/4 animals) observed at 1000 mg/kg, prostration (1/3 animals), mortality (3/3 animals) and sedation (2/3 animals) at 1500 mg/kg and prostration (3/3 animals) and mortality (3/3 animals) at 2000 mg/kg. In females, sedation (1/3 animals) and mortality (1/3 animals) observed at 1500 mg/kg and hypoactivity (1/3 animals) and mortality (1/3 animals) oberserved at 2000 mg/kg.


RESULTS OF FIRST DEFINITIVE STUDY
No clinical signs and no mortality were observed after treatment with the test substance at 100 or 400 mg/kg/day. After treatment at 800 mg/kg/day, mortality was observed in 5/8 males.
Since the PE/NE ration in the vehicle control group was lower than obtained typically, the results of the first study have not been taken into account. The biological significance of such reults obtained in the conditions of an insufficient erythropiesis is difficult to assess.

RESULTS OF SECOND AND THIRD DEFINITIVE STUDIES
Therefore, a second cytogenetic study was performed with one dose: 800 mg/kg/day. Piloerection was noted in all treated mice 2 hours after the first administration. Thereafter no clinical signs were noted in females, but 6 of the 8 males were found dead 24 hours after the initial treatment.
Therefore a third study was conducted with males only at 600 mg/kg/day. Piloerection was observed in 5/8 males at 2 hours after the second administration.
In the vehicle control group, the mean value of nucleated polychromatic erythrocytes (MPE) was within the range of available historical data.

POSITIVE CONTROL
Cyclophosphamide induced a highly significant increase (ρ < 0.001) in the number of MPE, indicating the sensitivity of the test system. In addition, the PE/NE ratio decreased significantly (ρ<0.01) showing the toxic effect of this substance to bone marrow cells.)


In the groups treated with 1-bromopropane, the mean values of MPE were considered to be equivalent to those of their respective vehicle groups, and within the range of available historical data. The PE/NE ratio was not significantly different from that of the respective vehicle control groups.

Table 1: Summary of Second Study (females only)

Group

Doses (1)

MPE/1000PE

PE/NE ratio

Time of sacrifice after last administration

(mg/kg/day)

Mean

SD

Mean

SD

Vehicle

-

1.1

0.7

0.9

0.2

24 h

Test substance

800

1.9

1.5

0.8

0.2

24 h

Cyclophosphamide

50

31.7

7.2

0.3

0.1

24 h

 

Doses (1) frequency – vehicle and test substance : 2 administratins at 24 h intervals

                                    – cyclophosphamide : 1 administration (oral route)

Table 2: Summary of Third Study (males only)

Group

Doses (1)

MPE/1000PE

PE/NE ratio

Time of sacrifice after last administration

(mg/kg/day)

Mean

SD

Mean

SD

Vehicle

-

1.3

0.8

0.7

0.2

24 h

Test substance

600

2.0

1.2

0.8

0.1

24 h

Cyclophosphamide

50

43.8

13.3

0.7

0.2

24 h

 

Doses (1) frequency – vehicle and test substance : 2 administratins at 24 h intervals

                                    – cyclophosphamide : 1 administration (oral route)

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of the test, 1-bromopropane did not induce damage to the chromosomes or the mitotic apparatus in the bone marrow cells of mice treated by the intraperitoneal route at 600 mg/kg/day for the males or 800 mg/kg/day for the females in the micronucleus test.
Executive summary:

In an in vivo micronucleus study (12122 MAS), 7 week old male and female Swiss OFI/ICO:OFI (IOPS CAW) mice were administered with 1-bromopropane via the intraperitoneal route twice in 48 hours with 24 hour intervals. The study was perfomed according to the OECD 474 guideline to GLP standard. The doses used in the first definitive study were 100, 400 and 800 mg/kg/day. This study was discounted due to lower than typically achieved PE/NE ratio. A second study was therefore performed with a single dose of 800 mg/kg. Since 6 out of the 8 males died, a further male only study was performed at 600 mg/kg/day.

Under the conditions of the test, 1-bromopropabe did not induce damage to the chromosomes or the mitotic apparatus in the bone marrow cells of mice treated by the intraperitoneal route at 600 mg/kg/day for the males or 800 mg/kg/day for the females in the micronucleus test.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: 5 to 6 weeks
- Weight at study initiation: 23.4 - 23.7 g (male) and 19.7 - 20.4 g (female)
- Fasting period before study: NDA
- Housing: Individually in stainless steel, wire bottom (Lab Products, Inc., Seaford, DE) cages; changed weekly, rotated weekly. Untreated paper cage pan liner (Sheperd Specialty Papers, Kalamazoo, MI); changed daily.
- Diet (e.g. ad libitum): NTP-2000 irradiated pelleted diet (Zeigler Brothers, Inc., Gardners, PA); available ad libitum, except during exposure periods; changed weekly.
- Water (e.g. ad libitum): Tap water (Richland, WA, municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI); available ad libitum.
- Acclimation period: 12 days
Before the studies began, five male and five female mice were randomly selected for parasite evaluation and gross observation for evidence of disease. Serological analyses were performed on five male and five female sentinel mice during week 1 and five male and five female chamber control mice at the end of the studies.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Temperature: 72° ± 3° F
- Humidity (%): 50% ± 15%
- Air changes (per hr): 15 ± 2/hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light/day

IN-LIFE DATES: From: 11 November 2002 To: 13 February (males) or 14 February (females) 2003
Route of administration:
inhalation: vapour
Vehicle:
Air
Details on exposure:
VAPOR GENERATION AND EXPOSURE SYSTEM
A preheater was necessary for the 3-month study. 1-Bromopropane was pumped through a preheater and into a heated glass column filled with glass beads that increased the surface area for vaporization. Heated nitrogen entered the column from below and assisted in vaporizing the chemical while conveying it into a short distribution manifold. Concentration in the manifold was determined by the chemical pump rate and nitrogen flow rate. The pressure in the distribution manifold was kept fixed to ensure constant flow through the manifold and into all chambers as the flow of vapor to each chamber was adjusted.
Metering valves at the manifold controlled flow to each chamber through individual Teflon® delivery lines that carried the vapor from the manifold to three-way exposure valves at the chamber inlets. The exposure valves diverted vapor delivery to exposure chamber exhaust until the generation system was stable and exposures were ready to proceed. To initiate exposure, the chamber exposure valves were rotated to allow the 1-bromopropane vapor to flow to each exposure chamber inlet duct where it was further diluted with filtered, conditioned air to achieve the desired exposure concentration.
The study laboratory designed the inhalation exposure chamber (Harford Systems Division of Lab Products, Inc., Aberdeen, MD) so that uniform vapor concentrations could be maintained throughout the chamber with the catch pans in place. The total active mixing volume of each chamber was 1.7 m3. A small particle detector was used with and without animals in the exposure chambers to ensure that 1-bromopropane vapor, and not aerosol, was produced. No particle counts above the minimum resolvable level (approximately 200 particles/cm3) were detected.
Duration of treatment / exposure:
Animals were exposed for 6 hours and 10 minutes, the 10 minute period at the beginning, being the time for the vapour concentration to reach at least 90% of the target concentration.
Frequency of treatment:
6 hours per day, 5 days per week for 14 weeks.
Post exposure period:
Not reported
Remarks:
Doses / Concentrations:
62.5 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
125 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
250 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
500 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
5 male, 5 female
Control animals:
yes, concurrent vehicle
Positive control(s):
Not reported
Tissues and cell types examined:
A detailed discussion of this assay is presented by MacGregor et al. (1990)*. At the end of the 3-month toxicity study, peripheral blood samples were obtained from male and female mice.
*MacGregor, J.T., Wehr, C.M., Henika, P.R., and Shelby, M.D. (1990). The in vivo erythrocyte micronucleus test: Measurement at steady state increases assay efficiency and permits integration with toxicity studies. Fundam. Appl. Toxicol. 14, 513-522.
Details of tissue and slide preparation:
Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs; immature erythrocytes) per animal. In addition, the percentage of polychromatic erythrocytes (PCEs or reticulocytes) in a population of 1,000 erythrocytes was determined as a measure of bone marrow toxicity.
Evaluation criteria:
The results were presented as the mean of the pooled results from all animals within an exposure group plus or minus the standard error of the mean. The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over exposure groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposed group and the chamber control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single exposed group is less than or equal to 0.025 divided by the number of exposed groups. A final call of positive for micronucleus induction is preferably based on reproducibly positive trials (as noted above). Results of the 3-month study were accepted without repeat tests, because additional test data could not be obtained. Ultimately, the final call is determined by the scientific staff after considering the results of statistical analyses, the reproducibility of any effects observed, and the magnitudes of those effects.
In this assay, a positive response is defined as a reproducible, dose-related response. An equivocal response is defined as a response that is not dose related, is not reproducible, or is not of sufficient magnitude to support a determination of mutagenicity. A negative response is obtained when no response is observed following chemical treatment. There is no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive, although positive calls are typically reserved for responses that are at least twofold over background.
Statistics:
These are the basic guidelines for arriving at an overall assay result for assays performed by the National Toxicology Program. Statistical as well as biological factors are considered. For an individual assay, the statistical procedures for data analysis have been described in the preceding protocols. There have been instances, however, in which multiple aliquots of a chemical were tested in the same assay, and different results were obtained among aliquots and/or among laboratories. Results from more than one aliquot or from more than one laboratory are not simply combined into an overall result. Rather, all the data are critically evaluated, particularly with regard to pertinent protocol variations, in determining the weight of evidence for an overall conclusion of chemical activity in an assay. In addition to multiple aliquots, the in vitro assays have another variable that must be considered in arriving at an overall test result. In vitro assays are conducted with and without exogenous metabolic activation. Results obtained in the absence of activation are not combined with results obtained in the presence of activation; each testing condition is evaluated separately. The results presented represent a scientific judgment of the overall evidence for activity of the chemical in an assay.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not examined

Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice Following Treatment with 1-Bromopropane by Inhalation for 3 Monthsa

 

Compound

Exposure Concentration (ppm)

Number of Micewith Erythrocytes Scored

Micronucleated NCEs / 1000 NCEsb

P valuec

PCEs (%)b

Male

 

 

 

 

 

 

 

 

 

 

 

Aird

0

5

2.00 ± 0.61

 

2.52 ± 0.30

 

 

 

 

 

 

1-Bromopropane

62.5

5

3.10 ± 0.81

0.0615

2.16 ± 0.31

 

125

5

2.70 ± 0.64

0.1533

2.96 ± 0.11

 

250

5

1.30 ± 0.41

0.8887

2.80 ± 0.18

 

500

5

2.30 ± 0.46

0.3235

2.80 ± 0.44

 

 

 

 

 

 

 

 

 

P=0.757e

 

 

 

 

 

 

 

 

Female

 

 

 

 

 

 

 

 

 

 

 

Aird

0

5

1.80 ± 0.25

 

2.24 ± 0.52

 

 

 

 

 

 

1-Bromopropane

62.5

5

1.70 ± 0.25

0.5672

2.94 ± 0.23

 

125

5

1.60 ± 0.19

0.6343

2.74 ± 0.52

 

250

5

1.40 ± 0.33

0.7604

2.42 ± 0.27

 

500

5

1.80 ± 0.20

0.5000

2.48 ± 0.29

 

 

 

 

 

 

 

 

 

P=0.500

 

 

 

a Study was performed at ILS, Inc. The detailed protocol is presented by MacGregor et al.(1990).*

PCE=polychromatic erythrocyte; NCE=normochromatic erythrocyte.

b Mean ± standard error

c Pairwise comparison with the chamber control group; significant at P≤0.008

d Chamber control

e Significance of micronucleated NCEs/1,000 NCEs tested by the one-tailed trend test; significant at P≤0.025

 

*MacGregor, J.T., Wehr, C.M., Henika, P.R., and Shelby, M.D. (1990). The in vivo erythrocyte micronucleus test: Measurement at steady state increases assay efficiency and permits integration with toxicity studies. Fundam. Appl. Toxicol. 14, 513-522.

Conclusions:
Interpretation of results (migrated information): negative
No increases in the frequencies of micronucleated normochromatic erythrocytes were seen in peripheral blood of male or female B6C3F1 mice exposed for 3 months to 62.5 to 500 ppm 1-bromopropane via inhalation. The percentage of reticulocytes (polychromatic erythrocytes) in the peripheral blood of male and female mice was unaltered by 1-bromopropane exposure, suggesting a lack of chemical-associated bone marrow toxicity.
Executive summary:

No increases in the frequencies of micronucleated normochromatic erythrocytes were seen in male or female B6C3F1 mice exposed for 3 months to 62.5 to 500 ppm 1-bromopropane via inhalation. The percentage of reticulocytes (polychromatic erythrocytes) in the peripheral blood of male and female mice was unaltered by 1-bromopropane exposure, suggesting a lack of chemical-associated bone marrow toxicity.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Dominant lethal studies were conducted in SD male rats with five halogenated 3-carbon compounds that are structurally similar to a known mutagen 1,2-dibromo-3-chloropropane (DBCP).
GLP compliance:
no
Type of assay:
rodent dominant lethal assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles RIver Japan Inc.
- Age at study initiation: 10 weeks
- Weight at study initiation: NDA
- Assigned to test groups randomly: NDA
- Fasting period before study: NDA
- Housing: NDA
- Diet (e.g. ad libitum): NDA
- Water (e.g. ad libitum): NDA
- Acclimation period: At least 7 days

Route of administration:
oral: gavage
Vehicle:
Olive Oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The chemicals were dissolved in olive oil before use.
Duration of treatment / exposure:
5 successive days of gastric intubation.
Frequency of treatment:
Once a day
Post exposure period:
N/A
Remarks:
Doses / Concentrations:

Basis:
actual ingested
400 mg/kg/day
No. of animals per sex per dose:
15 males used for the dosage group and for vehicle and positive controls. Each male mated with 1 female.
Control animals:
yes, concurrent vehicle
Positive control(s):
1,2-Dibromo-3-chloropropane
- Justification for choice of positive control(s): NDA
- Route of administration: Oral (gavage)
- Doses / concentrations: 50 mg/kg/day
Tissues and cell types examined:
Numbers of corpora lutea, implants, live embroys and early and late embronic deaths were counted.
Details of tissue and slide preparation:
N/A
Evaluation criteria:
The frequency of induced dominant lethal mutations was calculated according to the method of Röhrborn (1970).
Statistics:
Fisher's exact method and Mann-Whitney U test were used for the statistical evaluation of results.
Sex:
female
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
At week 8, the frequency of dead implants in the test group was higher than in the control group, but there was no increase in the dominant mutation index.

DBCP, used as the positive contol, induced dominant lethals, particularly in the early spermatid stage (weeks 4 and 5), and also caused testicular degenrtation in 5 out of 15 males.

Table 1: Dominant Lethal Mutation in Sprague-Dawley rats

Time of mating after treatment (weeks)

Chemical

Dose (mg/kg/day)

Number of females with implants

Number of corpora lutea per femalea

Number of implants per femalea

Number of live embryos per femalea

Mean dead implantsb (%)

Dominant lethal mutation indexc

1

Control

-

14

17.4 ± 3.4

15.1 ± 2.1

13.9 ± 2.7

7.7

 

DBCP

50

15

15.9 ± 0.7

15.6 ± 0.7

12.9 ± 1.8

17.6**

7.6

1-BP

400

14

16.0 ± 1.6

14.9 ± 2.0

14.2 ± 2.1

4.2

-2.1

2

Control

-

13

18.5 ± 3.9

16.5 ± 1.1

15.6 ± 1.3

5.2

 

DBCP

50

15

16.6 ± 1.3

15.9 ± 1.1

13.8 ± 2.4*

13.1*

11.6

1-BP

400

15

16.8 ± 1.7

16.0 ± 1.9

15.3 ± 2.0

4.2

1.8

3

Control

-

15

17.7 ± 2.8

16.4 ± 1.6

15.3 ±1.9

6.7

 

DBCP

50

15

16.7 ± 2.8

13.1 ± 5.6

11.7 ± 5.9

9.2

23.1

1-BP

400

15

17.5 ± 2.2

16.2 ± 2.4

15.2 ± 3.0

6.8

0.4

4

Control

-

12

17.3 ± 2.1

16.9 ± 1.8

15.7 ± 1.7

7.1

 

DBCP

50

11

17.3 ± 2.6

15.0 ± 3.3

7.8 ± 5.0**

50.8**

50.1

1-BP

400

13

17.2 ± 1.6

16.3 ± 1.9

15.5 ± 1.8

38.7**

1.3

5

Control

-

15

17.4 ± 1.8

15.9 ± 2.9

15.1 ± 2.7

48

 

DBCP

50

12

16.8 ± 2.0

13.7 ± 3.2

6.1 ± 4.9**

59.5**

59.6

1-BP

400

7**

16.8 ± 1.6

15.8 ± 2.2

14.6 ± 2.0

7.5

3.3

6

Control

-

14

16.3 ± 1.6

15.9 ± 1.5

15.2 ± 1.6

4.1

 

DBCP

50

12

16.9 ± 1.6

16.1 ± 1.2

14.9± 1.8

7.3

2.0

1-BP

400

14

15.6 ± 1.2

14.6 ± 3.6

14.0 ± 2.0

5.5

8.0

7

Control

-

15

16.4 ± 2.0

15.6 ± 1.5

15.0 ± 1.5

3.8

 

DBCP

50

13

15.1 ± 3.6

14.3 ± 4.2

13.2 ± 4.2

14.2

11.8

1-BP

400

15

16.1 ± 1.3

15.5 ± 1.5

14.9 ± 2.1

4.5

0.9

8

Control

-

14

16.5 ± 1.1

15.9 ± 1.0

15.3 ± 2.3

4.4

 

DBCP

50

12

16.7 ± 2.2

15.7 ± 2.5

14.2 ± 2.5*

9.2*

7.3

1-BP

400

15

17.8 ± 2.7

15.5 ± 2.9

13.9 ± 3.6

11.2**

9.3

a values represent the mean ± S.D.

b values are given as mean per pregnancy

c (1 - (live embryos per test female / live embryos per control female)) x 100

*,** significantly different from control at ρ<0.05, ρ<0.01

Conclusions:
Interpretation of results (migrated information): negative
1-Bromopropane was found to have no effect on dominant lethal mutations in rats.
Executive summary:

A dominant lethal study was performed on Sprague-Dawley rats. Dosed male rats were mated with 1 female per week over an 8 week period. Pregnant females were killed 13 or 14 days after copulation and the number of corpora lutea, implants, live embryos and early and late embryonic death were counted. The frequency of induced dominant lethal mutations was then calculated.

1-Bromopropane was found to have no effect on dominant lethal mutations in rats.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The animals used in this test were those that had already been dosed as part of a repeated dose inhalation toxicity test. The animals, after finishing exposure to testing material, were suffocated using dry ice. The blood of each animal was extracted from the abdomen main artery, and then the thighbone was cut and marrow was extracted from the thighbone using fetal bovine serum (Gibco) into a centrifugal tube to centrifuge at 1,000 rpm, for 5 minutes. Then the upper clear solution layer was eliminated and the precipitate was suspended with small amount of fresh serum. The suspended solution was smeared on a glass slide and then dried at room temperature, then settled with methanol for 5 minutes. To the settled specimen, a drop of acridine orange solution (40 µg/ml, Sigma Lot 11H3650) was added and covered with cover-glass then fluorescence stained. Blind method was adopted to the specimen observation using fluorescence microscope (NIKON OPTIPHOT-2) with magnification of 400 or more.
The erythrocyte cells having micronuclei among 1,000 of polychromatic erythrocytes (PCE) in the area of good smeared state on the slide were counted. A cell with a red fluorescent color without a nucleus was classified as a PEC. A micronucleus was identified in the size range from as small as distinguishable up to 112 of RBC size. Mainly circular shapes, but also doughnut or semicircle shapes were accepted for a micronucleus. Green fluorescence color, which is same as nucleus color of normal cell, is accepted as micronucleus color. The appearance frequencies of micronuclei in the testing material dosed groups were compared with that of the control group, the difference significance was checked using the method of Kastenbaum, et al.
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Korea Animal Experiment Centre.
- Age at study initiation: 10 weeks old
- Weight at study initiation: Males 267.20 ± 6.16 g. Females 199.29 ± 3.80 g.
- Assigned to test groups randomly: Rats separated into testing groups depending on weight differences. Each group was formed with least variation compared to other groups.
- Fasting period before study: NDA
- Housing: The rats of the 1 control and 3 test groups were accomadated in 4 sets of inhalation chambers with 5 connected metal cages inside each chamber.
- Diet (e.g. ad libitum): Pasteurized animal feed.
- Water (e.g. ad libitum): Sterlilized potable water always available to the rats.
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2 ºC
- Humidity (%): 40 - 70 %
- Air changes (per hr): 11 -15
- Photoperiod (hrs dark / hrs light): 12 hours. Light from 9 am - 9 pm. Light intensity of 15 - 300 lux

IN-LIFE DATES: From: To: NDA
Route of administration:
inhalation: vapour
Vehicle:
Not used
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Incubation chamber with a capacity of 1 cubic meter using SIS-20RG, SHIBATA Co., LTD, Japan.
- Method of holding animals in test chamber: Barrier system.
- System of generating particulates/aerosols: Gas generator
- Temperature, humidity, pressure in air chamber: 23 ± 2 ºC, 40 - 70 % humidity, 1 - 15 mmAq pressure.
- Air change rate: 11 - 15 air changes per hour.
- Method of particle size determination: NDA
- Treatment of exhaust air: NDA

TEST ATMOSPHERE
- Brief description of analytical method used: GC was used to measure the concentration of test compound in the chamber. The concentration was measured every 15 minutes. An automatic sampling device is attached to the vessel.
- Samples taken from breathing zone: NDA
Duration of treatment / exposure:
6 hours a day
Frequency of treatment:
5 days a week for 8 weeks
Post exposure period:
N/A
Remarks:
Doses / Concentrations:
50, 300 and 1800 ppm.
Basis:
nominal conc.
No. of animals per sex per dose:
10 animals per sex per dose.
Control animals:
yes
Positive control(s):
Not used
Tissues and cell types examined:
Red blood cells from bone marrow of rats.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on preliminary tests

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The samples were taken after 8 days inhalation exposure.

DETAILS OF SLIDE PREPARATION:
The blood of each animal was extracted from the abdomen main artery. The thighbone was cut and marrow was extracted using fetal bovine serum into a centrifuge at 1000 rpm for 5 minutes. The upper clear solution layer was eliminated and the precipitate was suspended with a small amount of fresh serum. The suspended solution was smeared on a glass slide and dried at room temperature, the settles on methanol for 5 minutes. A drop acridine orange was added and covered with cover glass, then fluorescence stained.

METHOD OF ANALYSIS:
The Blind method was used to observe the slides using a fluorescence microscope with magnification of 400x or greater.

OTHER: N/A
Evaluation criteria:
The erythrocyte cells having micronuclei among 1000 of polychromatic erythrocytes (PCE) in the well smeared area of the slide were counted. A micronucleous was identified in the size range from as small as distinguishable, to half the size of a red blood cell (RBC). They are mainly circular, but also doughnut or semi-circular in shape. To be accepted as a micronucelus, they must be fluorescent green, the same colour as a normal cell nucleus.
Statistics:
The frequency of micronuclei in the test groups were compared to the control. The significance in difference was checked using the Kastenbaum et al, method.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Additional information on results:
The appearance frequency rate of red blood cells with micronuclei among polychromatic erythrocytes of the contol rats was 0.26 ± 0.13, 0.19 ± 0.08 for the 50 ppm groupm, 0.23 ± 0.16 for the 30 ppm group and 0.26 ± 0.15 for the 1800 ppm group.

Table 1: Mean Appearance Frequencies of Micronuclei Induced by 1 -bromopropane

Sex

Group 0 (control)

Group 1 (50 ppm)

Group 2 (300 ppm)

Group 3 (1800 ppm)

Male

0.28 ± 0.13

0.21 ± 0.10

0.41 ± 0.16

0.35 ± 0.12

Female

0.23 ± 0.12

0.18 ± 0.06

0.23 ± 0.11

0.16 ± 0.11

Total

0.26 ± 0.13

0.19 ± 0.08

0.32 ± 0.16

0.25 ± 0.15

Conclusions:
Interpretation of results (migrated information): negative
The micronucelus frequency in marrow of rats exposed to 1 -bromopropane was found to not to have increased with respect to the control group and the result is therefore considered negative.
Executive summary:

Using marrow samples extracted form 10 week old male and female Sprague-Dawley rats, males weighing 267.20 ± 6.16 g, females weighing 199.29 ± 3.80 g, a micronucleus assay was performed. The rats had previously been exposed to 1-bromopropane via inhalation in a repeat dose study for 8 weeks, with 6 hours exposure per day for 5 days each week. The appearance frequency rate of red blood cells with micronuclei among polychromatic erythrocytes of the contol rats was 0.26 ± 0.13, 0.19 ± 0.08 for the 50 ppm groupm, 0.23 ± 0.16 for the 30 ppm group and 0.26 ± 0.15 for the 1800 ppm group.

The micronucleus frequency in marrow of rats exposed to 1-bromopropane was found to not to have increased with respect to the control group and the result is therefore considered negative.

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

Additional information

All Ames studies on n-propyl bromide gave negative genotoxicity results, except for Barber et. al. (1981), which gave a positive result for two of the Salmonella strains tested both with and without metabolic activation. However, this test was a non-GLP study conducted using non-standard test methods in a closed system to control volatility of the test substance.

An in vitro mouse lymphoma study on n-propyl bromide gave a positive result. This was a well conducted, reliable study using standard test guidelines, however only limited details regarding the methodology were available, and hence this study was assigned a Klimisch score of 2.

All available in vivo studies on n-propyl bromide (three Mouse Micronucleus studies and two Dominant Lethal Assay) give negative results.


Short description of key information:
Six in vitro Ames studies are available for this substance:
Barber E.D., Donish W.H. and Meuller K.R. (1981): Positive for S. typhimurium TA 1535 and TA 100
Lehrer. S (1981): Negative for all strains tested
Décharioux. H (1993): Negative for all strains tested
Moon. Y.H. et al (1998): Negative for all strains tested (including E.coli)
USA National Institute of Health (2011): Negative for all strains tested
USA National Institute of Health (2011): Negative for all strains tested (including E.coli)

An in vitro mouse lymphoma study is available for this substance:
Molinier. B (1996): Positive

Three in vivo Mouse Micronucleus studies are available for this substance:
Molinier. B (1995): 2 intraperitoneal doses - Negative
Moon. Y.H. et al (1998): 13-week inhalation exposure - Negative
USA National Institutes of Health (2011): 3-month inhalation exposure - Negative

Two in vivo Dominant Lethal Assay are available for this substance:
Saito-Suzuki R., Teramoto S. and Shirasu Y. (1982): Negative
Yu W. J. et al (2008): Negative

One in vivo Transgenic Rodent Somatic Cell Gene Mutation Assay in female mice is available for this substance:
Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In in vitro genotoxicity screening studies, where the potential to detect any genotoxicity potential (including mutagenicity, clastogenicity, cytogenetic effects, aneugeneicity, polyploidy) was maximised, no genotoxicity by 1-bromopropane was detected. In three in vivo mouse micronucleus assays and two Dominant-lethal assays, no genotoxicity was detected, and in an occupational exposure setting there was no reliable evidence for a genotoxic effect. Therefore 1-bromopropane does not require classification as a mutagen in accordance with Directive 67/548/EEC and the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.