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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
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. certificate)
Type of assay:
transgenic rodent mutagenicity assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: clear liquid
Details on test material:
Purity 99.7%
Certicicate of Ananlysis attached.
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.

Test animals

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 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.

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Test results
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.

Any other information on results incl. tables

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).

Applicant's summary and conclusion

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.