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Genetic toxicity: in vivo

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Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 Mar 2017 to 10 Aug 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
24 Mar 2017 to 10 Aug 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
July 29, 2016
Deviations:
no
GLP compliance:
yes
Test type:
traditional method
Limit test:
no
Specific details on test material used for the study:
- Name of the test item (as cited by study report): 4,4’-Diphenylmethane Diisocyanate (MDI)
- Batch No.: P4DB005186
- Purity: 98.89%.
- Retest date: 30 Jun 2017
- Appearance: White solid
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI (Han)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: Approximately 7 weeks
- Weight at study initiation: 168g - 216 g
- Assigned to test groups randomly: yes, the animals judged suitable for assignment to
the study were selected for use in a computerized randomization procedure based on body weight stratification in a block design.
- Housing: Upon arrival, all animals were housed 2 to 3 per cage in clean, solid bottom cages containing ground corncob bedding material (Bed O’Cobs®; The Andersons, Cob Products Division, Maumee, OH). Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.
- Diet: The basal diet used in this study, PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal), is a certified feed with appropriate analyses performed by the manufacturer and provided to Charles River. The basal diet was provided ad libitum throughout the study, except during exposure periods.
- Water: Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system, was provided ad libitum throughout the study, except during exposure periods. Municipal water supplying the facility was analysed for contaminants according to SOPs.
- Acclimation period: All animals were housed for an 14-day acclimation. During acclimation, each animal was observed twice daily for mortality and changes in general appearance or behaviour.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3 – 22.6
- Humidity (%): 37.9 – 46.5
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Remarks:
Filtered
Mass median aerodynamic diameter (MMAD):
>= 1.8 - <= 3.7 µm
Geometric standard deviation (GSD):
>= 2.2 - <= 2.86
Remark on MMAD/GSD:
The MMAD for the 2 mg/m3 group was higher than the protocol-specified target range (1 to 3 microns). While the MMAD was greater than 3, the particle size was still considered acceptable for an acute inhalation study based on OECD 403 guideline expectations. See 'Any other information on materials and methods incl. tables'.
Details on inhalation exposure:
EXPOSURE SYSTEMS
Exposures were conducted using a 2 tier (7.9-L) stainless steel, conventional nose-only exposure systems (CNOS), with grommets in the exposure ports to engage animal holding tubes. Four dedicated exposure systems were used: 1 for the filtered-air control group and 1 for each of the test substance groups. Air supplied to the nose-only systems was provided from the Charles River Inhalation Department breathing quality, in-house compressed air source. All nose-only system exhaust passed through a Solberg canister filter prior to entering the facility exhaust system, which consists of redundant exhaust blowers preceded by activated-charcoal and HEPA-filtration units.

CHAMBER DESCRIPTION
All animals were housed in a normal animal colony room during non-exposure hours. Prior to the exposure, the animals selected for exposure were placed into nose-only restraint tubes in the colony room and transported to the exposure room. Animals were then placed on the nose-only systems and exposed for the requisite duration. After being transported to the exposure room, the animals assigned for exposure were held in restraint tubes for approximately 23 to 26 minutes before the initiation of exposure to allow the animals breathing rates to return to normal baseline values. Food and water were withheld during nose-only restraint tube acclimation and during the exposure period. Oxygen content of the exposure atmospheres was measured during the method development phase of the study using a Dräger PAC III equipped with a calibrated oxygen sensor (Serial No.ERRH-0148, Draeger Safety Inc.; Pittsburgh, PA) and was 20.9% for all groups. Due to the local weather conditions near/at the start of animal exposures, the compressed air used was not as dry as expected. This caused the average humidity for Group 4 to be higher than the protocol-specified target range. The humidity was kept as low as practical to minimize test substance dimer formation.

CONTROL EXPOSURE SYSTEM (EXPOSURE SYSTEM 1)
The control exposure system (Group 1) was operated as follows: dry air was added to the CNOS inlet using a regulator and controlled using a rotameter-type flowmeter.

TEST SUBSTANCE EXPOSURE SYSTEMS (EXPOSURE SYSTEMS 2 TO 4)
A dedicated generation system was used for each test substance exposure system and was operated as follows: A liquid droplet aerosol atmosphere of the test substance was generated using a single-jet Collison nebulizer filled with an appropriate amount of test substance, and heated to approximately 80°C to melt and maintain the test substance in liquid form. Using a regulator, dry, compressed air at a controlled pressure was supplied to the generation port of each nebulizer to affect aerosolization of the test substance. The air entering each nebulizer was also heated. The resulting aerosol from each nebulizer was delivered to a mixing plenum, where it mixed with additional dry, dilutionair. In order to permit reduction of the exposure concentration, a portion of the aerosol output from each nebulizer and/or mixing plenum was directed toward facility exhaust. The remaining aerosol within each mixing plenum was delivered to the “T”-fitting located prior to the inlet of the respective nose-only exposure system, where it was mixed with additional dry air prior to entering each CNOS to permit further reduction of the aerosol concentration.

NOMINAL EXPOSURE CONCENTRATIONS
Nominal exposure concentrations were not calculated for this study due to the nature of each aerosol generation system, where a large portion of the test substance aerosol was removed prior to the final dilution to the target concentration. However, the amount of test substance used during the exposure was calculated by weighing each test substance nebulizer prior to and postexposure.

ACTUAL EXPOSURE CONCENTRATIONS
Aerosol exposure concentrations were measured using standard gravimetric methods. Sample flow was measured using a mini-Buck calibrator. The mass concentration in (mg/m3) was calculated from the filter weight difference divided by the sample volume. Samples were collected at least 4-6 times during each exposure for Exposure Systems 2 to 4 and once for Exposure System 1. Each test substance exposure atmosphere was continuously monitored for aerosol concentration using a light scattering type real time aerosol monitor. These instruments were not intended to define exposure concentration, but were to provide exposure personnel with
an indication of approximate aerosol concentration for guidance in making appropriate system adjustments and achieving the most stable exposure concentration possible.

AEROSOL PARTICLE SIZE MEASUREMENT
Aerosol particle size measurements were conducted using a 7-stage brass cascade impactor. Aerosol particle size measurements were conducted once during the exposure period for each test substance group (Groups 2 to 4). Samples were collected at approximately 1.8 to 1.9 LPM for 360, 150, and 60 minutes for Groups 2,3, and 4, respectively. Following sample collection, substrates were re-weighed and the particle size was calculated based on the impactor stage cut-offs. The particle-size was expressed as the mean aerodynamic diameter (MMAD) in microns and the geometric standard deviation (GSD). See 'Any other information on materials and methods incl. tables' for a summary of the aerosol particle size for each test substance-treated group.

POSITIVE CONTROL SUBSTANCE PREPARATION
The positive control substance formulation was prepared on each day of dose administration (Study Days 0 and 1) as a weight/volume (EMS/0.9% saline) mixture.
The positive control substance, ethyl methanesulfonate (EMS; CAS 62-50-0), was administered via oral gavage to rats in Group 5 at a dose of 200 mg/kg/day on Study Days 0 and 1. The vehicle used in preparation of the positive control substance formulation was 0.9% sodium chloride for injection. The positive control substancewas stored refrigerated (2°C to 8°C), purged with nitrogen,andwas considered stable under these conditions. The route of administration of the positive control substance (oral gavage) was chosen based on previous data in which BioReliance demonstrated that orally administered EMS produced a significant increase in DNA damage (comet response) in the BAL cells, liver, and glandular stomach.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
See 'Any other information on materials and methods incl. tables'
Duration of exposure:
6 h
Concentrations:
- Nominal concentrations: 2, 5, and 11 mg/m3
- Analytical concentrations: 2.5, 4.9, and 12 mg/m3

JUSTIFICATION OF DOSE LEVELS
Doses were selected based on the outcome of the dose-range finding study and other supporting studies (See Acute Toxicity: Inhalation; Pauluhn, 2000; Hotchkiss, 2017) while taking the recommendations in OECD TG 489 to consider cytotoxicity in setting maximum tolerated concentration (MTC). In the range-finding study, Wistar rats were exposed to 3.2, 7.7, and 11.9 mg MDI/m3 for 6 h and biomarkers for local cytotoxicity were analysed 1 h after the exposure and 18 h following completion of exposure. Results demonstrate both concentration- and time-dependent effects of inhaled 4,4’-MDI aerosol on biomarkers of exposure. Compared to control rats, dose dependent increases associated with macrophage activation (β-glucuronidase activity), inflammation (neutrophil infiltration and total protein)) at ≥3.2mg MDI/m3, apoptosis (Annexin V activity; ≥ 7.7 mg MDI/m3), and necrosis (LDH; ≥ 11.9 mg MDI/m3) were noted. This is supported by other supporting studies that demonstrate that acute exposure during 6 h to a concentration of 10-12 mg/m3 resulted in significant increases in biomarkers for inflammation, cytotoxicity/apoptosis, and macrophage activation. Moreover, concentrations > 20 mg/m3 (for 3 hr) were sufficient to induce cytotoxicity induced DNA dam age, but 10 mg/m3 (for 6h) were not (Sutter, 2016). Taken together, the MTC was considered 11.9 mg/m3 on the basis of marked increases in inflammation, apoptosis/necrosis, and cytotoxicity. The remaining 2 concentrations be appropriately spaced as to demonstrate a dose response with the lowest producing little to no toxicity.
No. of animals per sex per dose:
- Test groups (Group 2-4): 12 males
- Vehicle control (Group 1): 12 males
- Positive control (Group 5): 6 males
Control animals:
yes
Remarks:
concurrent vehicle; positive control (comet assay): ethyl methanesulfonate (EMS; CAS 62-50-0)
Details on study design:
SURVIVAL
All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.

CLINICAL OBSERVATIONS
Clinical examinations were performed prior to exposure for animals in the 2, 5, and 11 mg/m3 groups on Study Day 0 for the Group 5 animals prior to dosing and 0 to 1 hour (+0.25 hr) following dose administration. The absence or presence of findings was recorded for individual animals at the scheduled intervals. Detailed physical examinations were conducted on all animals within 4 days of receipt, on the day of randomization, and on Study Day 0. In addition, the social groups were observed at the appropriate intervals for findings that could not be attributed to a single animal; only positive findings were recorded. A separate computer protocol was used to record any observations noted outside of the above-specified intervals and treatments.

BODY WEIGHTS
Individual body weights were recorded within 4 days of receipt, on the day of randomization, and on Study Day 0. Mean body weights and mean body weight changes were calculated for the corresponding intervals.

BRONCHOALVEOLAR LAVAGE
At the scheduled euthanasia, BAL was performed on the lungs of all animals. As the site of contact tissue, bronchoalveolar lavage cells were selected; these primarily consist of alveolar macrophages, which are the primary cells responsible for the removal of inhaled aerosols from the alveoli and are commonly selected in the assessment of pulmonary genotoxicty after inhalation or instillation. The trachea was exposed and a dosing cannula was tied in place in the trachea. The lungs were lavaged 6 times with a lavage solution volume of 25 µL/gram body weight (based on the most recent body weights) of room temperature Hank’s Balanced Salt Solution (sterile) without calcium, magnesium, or phenol red, up to a maximum volume of 4 mL (per lavage). The BALF from the first and second lavages was recovered after remaining in the lung for approximately 1 minute and placed into a 15-mL polypropylene centrifuge tube after the volume was recorded. For the third through sixth lavages, the recovered volumes were recorded and the BALF was collected into a single polypropylene centrifuge tube. Recovered BALF was stored on ice until processed. The following parameters were evaluated from the BAL fluid or cell pellet: Alkaline phosphatease (ALP), Annexin V, ß-glucuronidase, cell differential (cytology), lactate dehydrogrenase (LDH) and total protein.

BRONCHOALVEOLAR LAVAGE FLUID PROCESSING
The BALF was isolated in a refrigerated centrifuge and the supernatant fluid from the first and second lavages was transferred to a sealable vial and stored on an ice bath until used for analysis of BAL clinical chemistry (alkaline phosphatase, lactate dehydrogenase, and total protein) and/or transferred to the internal immunotoxicology department for analysis of beta. The supernatant fluid from the third through sixth lavages was decanted and discarded. The cell pellets obtained from the first and second lavages or third through sixth lavages were pooled separately and retained. The cell pellet from the first and second lavages was resuspended in cold Roswell Park Memorial Institute (RPMI) media with 10% fetal calf serum, and this cell suspension was also used to resuspend the cell pellet from the third through sixth lavages. Total cell counts were obtained using a haemocytometer with cell viability assessed by trypan blue exclusion. A portion of the cell suspensions were transferred into Dulbecco’s Phosphate-Buffered Saline (DPBS; with calcium and magnesium) for future processing and analysis of Annexin V. The remainder of the cell suspension was centrifuged again and a portion of the cell pellet was used for the comet assay (see cross-referenced comet assay, of section 7.6.2)

QUANTIFICATION OF BETA-GLUCURONIDASE BY SANDWICH ELISA
A portion of the supernatant from the first and second lavages was evaluated using a commercially available Rat GUSB/Beta Glucuronidase ELISA kit (Catalog No. LS-F24073, Seattle, WA). Briefly, standard (STD), blank (sample diluent) or diluted BALF samples was added into the appropriate wells of the 96-well ELISA plate. The plate was covered, gently agitated on an orbital shaker, and incubated for approximately 90 minutes at approximately 37 °C. Detection antibody was added into each well, the plate was covered, gently agitated on an orbital shaker and, incubated at approximately 37 °C for approximately 60 minutes. The liquid was decanted and the wells were washed 3 times. After the last wash, remaining buffer was aspirated and HRP Conjugate working solution was added to each well. The plate was covered and incubated for up to approximately 30 minutes at approximately 37 °C. The liquid was decanted and the wells were washed again. After the last wash, remaining buffer was aspirated and TMB substrate solution was added to each well. The plate was covered and incubated for up to 15 minutes at approximately 37 °C and protected from light. The incubation was stopped by adding Stop Solution to each well and the plates were read in a spectrophotometer reader set at endpoint measurement at 450 nm. For study samples, only back-calculated concentrations of diluted samples that fell within the linear range of the standard curve were reported. Values that fell outside the linear range of the standard curve (R) were reported as below the instrument range and were excluded from analysis.

QUANTIFICATION OF ANNEXIN V BY FLOW CYTOMETRY
The Annexin V apoptosis assay quantifies the percentage of cells in the BALF that are in different stages of apoptosis by staining with Annexin V conjugated to FITC and the vital dye propidium iodide (PI). The assay detects cells that are viable (FITC-/PI-), in the early stages of apoptosis (FITC+/PI-),necrotic cells (FITC-/PI+), and cells in the late stages of apoptosis or necrotic cells (FITC+/PI+).

A portion of the combined cell pellet from the first through sixth lavages was evaluated for Annexin V using a BD Biosciences, FITC Annexin V Apoptosis Detection Kit Iand a flow cytometer. Briefly, the DPBS cell suspension was centrifuged in a refrigerated centrifuge set to maintain approximately 4 °C and the supernatant was discarded. The cells were be resuspended with binding buffer and centrifuged again. To the remaining cell pellet, FITC Annexin V and Propidium Iodide were added to each tube and the mixture was incubated at room temperature, protected from light. Following incubation, binding buffer was added to each sample. Samples were maintained on wet ice until sample acquisition on a Beckman Coulter Cytomics FC500 flow cytometer. Flow cytometry analysis completed using FlowJo X Software. Prior to analysis of study samples for Annexin V, BAL cells were collected from stock animals and were used for setting up the controls for flow cytometry. A total of 4 distinctive control groups designated were used. One group was designated as Unstained Control Group, and BAL cells in this group were untreated with Camptothecinand negatively stained for Annexin V FITC or PI. This group was used to detect the level of inherent auto-fluorescence or background staining associated with BAL cells. A second group contained cells that were treated with Camptothecin to induce apoptosis, and subsequently singly stained with either Annexin V FITC or PI. This group was used to create gates for individual fluorochrome, and also to correct for spectral overlap resulting from each dye by applying compensation. A third group contained cells that were untreated with Camptothecin, and stained double positive for both FITC and PI, with the aim of obtaining the basal levels of apoptosis, and late apoptotic/necrotic cells. The fourth group was designated as the positive control group, and contained cells that were treated with Camptothecin, and stained double positive for both Annexin V FITC and PI.

MACROSCOPIC EXAMINATION
All animals were anesthetized by isoflurane inhalation and euthanized by exsanguination. The animals were not fasted overnight prior to the scheduled euthanasia, and a gross necropsy was not performed. Immediately following euthanasia, BAL was performed on all animals and processed. Additionally, samples of the liver and glandular stomach were collected from all animals for comet assay evaluation (see cross-referenced comet assay, of section 7.6.2). At the time of euthanasia, samples of the following tissues were collected and placed in 10% neutral-buffered formalin. The carcasses and remaining tissues were discarded.

HISTOLOGY AND MICROSCOPIC EXAMINATION
After fixation, protocol-specified tissues were trimmed according to the test laboratory SOPs and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned according to the test laboratory SOPs, mounted on glass microscope slides, and stained with haematoxylin and eosin from all animals at the scheduled necropsies.
Statistics:
All statistical tests were performed using WTDMS™. Analyses were conducted using two-tailed tests for minimum significance levels of 1% and 5%, comparing each test substance-exposed group to the vehicle control group. Body weight, body weight change, and BALF 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-exposed groups to the vehicle control group.
Preliminary study:
A preliminary dose range-finding study was conducted to integrate the response of previously observe d non-specific toxicity endpoints and establish a maximum tolerated concentration for a definitive in vivo Comet Assay. Aerosol generated from the test substance (4,4’ Diphenylmethane Diisocyanate) was administered via a single nose-only inhalation exposure for 6 hours to 3 groups of 6 male Wistar Hannover (Crl:WI[Han]) rats at 0, 3.2, 7.7, and 11.9 mg/m3. A bronchoalveolar lavage (BAL) was performed from all animals at the scheduled necropsies, and the BAL fluid (BALF) was assessed for the following endpoints: BAL clinical chemistry (alkaline phosphatase, lactate dehydrogenase, and total protein), cell differential (cytology), and measurement of Annexin V expression and β-glucuronidase activity. Three rats/group were terminated on Study Day 0 within 1 hour postexposure and the remaining 3 rats/group were terminated on Study Day 1 at approximately 18 hours postexposure. All animals survived to the scheduled necropsies and no test substance related clinical observations or body weight effects were observed. Compared to control rats, dose dependent increases associated with macrophage activation (β- glucuronidase activity), inflammation (neutrophil infiltration and total protein)) at ≥3.2 mg MDI/m3, apoptosis (Annexin V activity; ≥ 7.7 mg MDI/m3), and necrosis (LDH; ≥ 11.9 mg MDI/m3) were noted in treated groups. From this range-finding study, inhalation exposure to MDI results in marked local acute toxicity at ≥10-12 mg/m3 for a 6 h exposure as identified by inflammation, apoptosis/necrosis and cytotoxicity supporting results from previous studies. Therefore, using the cellular toxicity parameters identified in the range-finder, the maximum tolerated concentration (MTC) was identified as 11.9 mg/ m3.
Key result
Sex:
male
Dose descriptor:
other: maximal tolerated dose
Effect level:
12 mg/m³ air (analytical)
Based on:
test mat.
Exp. duration:
6 h
Remarks on result:
other: Based on local effects: BALF chemistry parameters (cytology, alkaline phosphatase, lactate dehydrogenase, total protein, β-glucuronidase, and Annexin V) and changes in the liver
Mortality:
All animals survived to the scheduled necropsies.
Clinical signs:
other: There were no test substance-related clinical observations. Observations in the test substance-exposed groups were limited to detailed physical examinations performed prior to exposure.
Body weight:
Body weights were unaffected by test substance exposure. Body weights were collected prior to test substance exposure or positive control substance dose administration. Average body weights in animals designated for inhalation exposure (Groups 1–4) were considered similar prior to the exposure.
Other findings:
BRONCHOALVEOLAR LAVAGE FLUID CHEMISTRY (See 'Any other information on results incl. tables' for a summary of the results on BALF parameters)
BAL fluid clinical chemistry parameters were collected to assess local cellular toxicity and included lactate dehydrogenase (LDH; biomarker of cell death), total protein (TOTPROTEIN; dysfunction of the alveolar/capillary barrier), alkaline phosphatase (ALP; type II cell activation) and β-glucuronidase activity (macrophage activation). Inhalation exposure of aerosolized 4,4’-Diphenylmethane Diisocyanate (MDI) in male Wistar rats resulted in alterations in bronchoalveolar lavage fluid lactate dehydrogenase, total protein, and β-glucuronidase.

MDI-associated differences in BALF chemistry parameters were observed at Study Day 0 (approximately 1-hour post inhalation exposure) and included higher lactate dehydrogenase (LDH), total protein (TOTPROTEIN), and β-glucuronidase concentration.

MDI-related higher bronchoalveolar lavage fluid LDH activity (+34% to +64%) and dose-dependent, higher TOTPROTEIN (+175% to +457%) were evident at the 2, 5, and 11 mg/m3 exposure levels. Compared with the control group mean, test substance-related, higher β-glucuronidase concentrations were observed in all test substance-treated animals in a dose dependent manner, reaching statistically significance at the 5 and 11 mg/m3 exposure levels. From a simple comparison of mean ALP activities, a concentration-related increase was evident, and achieved statistical significance at 11 mg/m3 (+31, +31, and +54% when the 2, 5, and 11 mg/m3 exposure levels were compared to the filtered air control group, respectively). Even if this trend can also be described in the animals euthanized on Study Day 1, the high background in ALP activity in the Study Day 1 filtered air control animals and the high variability of the data in general complicate a conclusive assessment of the biological relevance of this observation.

Approximately 18 hours post exposure to MDI, higher LDH activity (+32.6%) was limited to rats at the 11 mg/m3 exposure level. Dose-dependent, higher total protein concentration was evident at 2, 5, and 11 mg/m3 (+148% to +973%) when compared with the concurrent control group mean value. Furthermore, at the 5 and 11 mg/m3 exposure levels, TOTPROTEIN was increased when compared with respective Study Day 0 group mean values. Compared with the control group mean, higher β-glucuronidase concentrations were observed in all test substance-treated animals in a dose dependent manner, reaching statistically significance at the 11 mg/m3 exposure level. There were no MDI-associated differences in ALP activity at any exposure level at Study Day 1. There were no EMS-associated differences in BALF chemistry parameters when compared with the filtered air group.

BRONCHOALVEOLAR LAVAGE CYTOLOGY (See 'Any other information on results incl. tables' for a summary of the results)
Bronchoalveolar lavage (BAL), conducted approximately 1-hour post exposure to MDI, resulted in higher mean neutrophil absolute counts and percentages at 11 mg/m3. However, the higher mean NEUT count was primarily due to higher neutrophil counts in 2 animals . The higher NEUT count in one animal was considered to be proportional to red blood cells (RBCs) and was not considered MDI-related. Two BALF samples from animals in the 2 mg/m3 exposure level group had low numbers of ciliated columnar epithelial cells representative of minimal upper airway contamination during sample collection. The presence of these cells did not alter sample integrity or interpretation.

Approximately 18 hours post exposure to MDI, bronchoalveolar lavage fluid mean neutrophil counts and percentages were higher in the 11 mg/m3 exposure group animals when compared with the filtered air group mean.

All other differences in BAL cytology were minor and interpreted as normal biological variation.

BRONCHOALVEOLAR LAVAGE CELL VIABILITY (See 'Any other information on results incl. tables' for a summary of the results on BAL cell parameters)
Annexin V assay was conducted to detect the percentage of BAL cells, if any, in different stages of apoptosis or necrosis following MDI inhalation exposure.

Compared to the control group, there were statistically significant higher numbers in early apoptotic cells (5 and 11 mg/m3 exposure levels), late apoptotic or necrotic cells (5 and 11 mg/m3 exposure levels), and necrotic cells (2, 5, and 11 mg/m3 exposure levels), along with a concomitant decrease in viable cells at all exposure levels.

After approximately 18 hours post exposure to MDI, a statistically significant increase in the early apoptotic cells along with a corresponding decrease in the viable cells was observed at the 11 mg/m3 exposure level.

HISTOLOGICAL CHANGES
Test substance-related microscopic findings were noted in the liver of the 2, 5, and 11 mg/m3 groups on Study Day 0. Test substance-related minimal to mild increased mitotic figures were observed in hepatocytes throughout the parenchyma. Similar findings were not observed on Study Day 1 (approximately 18 hours post-exposure), although increased mitoses were observed in the EMS (positive control group). Additionally, MDI gave a negative (non-DNA damaging) response in the Comet assay for the liver at both the 1 hour (Study Day 0) and 18 hour post exposure (Study Day 1) time points, as noted by the lack of an increase in % Tail DNA relative to the concurrent filtered air control group.

In absence of histological changes indicative of toxicity, the transient increase in hepatocyte cell division that is resolved after a recovery period as short as 18 hours, bears no toxicological relevance. Further, increases in mitotic figures in the liver have been shown to be physiological response to internal and external stresses. Therefore, the increase in mitotic figures (cell proliferation) observed at Study Day 0 were considered to be an adaptive response to administration of test substance.

There were no other test substance-related histologic changes in the liver or stomach. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Table: Exposure-related Differences in Bronchoalveolar Lavage Fluid Chemistry Parameters

Parameter

Exposure (mg/m3)

0

2.5

4.9

12

200 mg/kg EMS (a)

β-glucuronidase(pg/mL)

 

 

 

 

 

Study Day 0

138.05

271.44 (+96.6%)

644.75 (+367%)

846.99 (+513%)

NA

Study Day 1

103.76

424.62 (+309%)

761.68 (+634%)

2344.69** (+2160%)

336.94 (+225%)

LDH (IU)

 

 

 

 

 

Study Day 0

44

59 (+34.1%)

72* (+63.6%)

69* (+56.8%)

NA

Study Day 1

46

44 (-4.3%)

53 (+15.2%)

61 (+32.6%)

39 (-15.2%)

ALP (IU)

 

 

 

 

 

Study Day 0

52

68 (+30.8%)

68 (+30.8%)

80* (+53.8%)

NA

Study Day 1

74

54* (-27.0%)

78 (+5.4%)

80 (+8.1%)

52* (-29.7%)

TPROT (mg/dL)

 

 

 

 

 

Study Day 0

5.1

14.0* (+174.5%)

24.1** (+372.5%)

28.4** (+456.9%)

NA

Study Day 1

4.8

11.9 (+147.9%)

27.9** (+481.2%)

51.5** (+972.9%)

6.8 (+41.7%)

Table: Bronchoalveolar Lavage Fluid Cytology Values

Parameter

Exposure (mg/m3)

0

2.5

4.9

12

200 mg/kg EMS (a)

Alveolar Macrophages (%)

 

 

 

 

 

Study Day 0

98.00

97.50 (-0.5%)

98.25 (+0.3%)

96.00 (-2.0%)

NA

Study Day 1

96.33

95.92 (-0.4%)

97.92 (+1.7%)

94.08 (-2.3%)

96.25 (-0.1%)

Neutrophils (%)

 

 

 

 

 

Study Day 0

0.58

1.25 (+115.5%)

1.33 (+129.3%)

3.33* (+474.1%)

NA

Study Day 1

0.17

2.17 (+1176.5%)

1.17 (+588.2%)

4.17** (+2352.9%)

1.42 (+735.3%)

Table: Exposure-related Differences in Bronchoalveolar Lavage Cell Parameters

Parameter

Exposure (mg/m3)

0

2.5

4.9

12

200 mg/kg EMS (a)

FITC-/PI+

 

 

 

 

 

Study Day 0

0.5

1.2** (140.0%)

1.9** (280.0%)

1.7** (240.0%)

NA

Study Day 1

0.6

0.6 (0.0%)

0.7 (16.7%)

0.8 (33.3%)

0.7 (16.7%)

FITC+/PI+

 

 

 

 

 

Study Day 0

8.2

14.4 (75.6%)

23.0** (180.5%)

17.4* (112.2%)

NA

Study Day 1

5.7

6.0 (5.3%)

5.4 (-5.3%)

7.3 (28.1%)

6.5 (14.0%)

FITC-/PI-

 

 

 

 

 

Study Day 0

88.2

78.9 (-10.5%)

67.5** (-23.5%)

73.3** (-16.9%)

NA

Study Day 1

88.0

7.9 (-0.1%) 

87.5 (-0.6%)

69.4** (-21.1%)

87.6 (-0.5%)

FITC+/PI-

 

 

 

 

 

Study Day 0

3.1

5.4 (74.2%)

7.6** (145.2%)

7.7** (148.4%)

NA

Study Day 1

5.7

5.5 (-3.5%)

6.4 (12.3%)

22.6** (296.5%)

5.2 (-8.8%)

NA = Not applicable

Values in parenthesis (x) represent percent difference relative to the negative control (filtered air) group.

* Significantly different from the negative control group at 0.05 using Dunnett’s test.

** Significantly different from the negative control group at 0.01 using Dunnett’s test.

(a) Positive control.

Bold = Values considered to be test substance-related

FITC-/PI- = indicator of viable cells

FITC+/PI- = indicator of cells in early apoptosis

FITC-/PI+ = indicator of necrotic cells

FITC+/PI+ = indicator of cells in the late stages of apoptosis or necrotic cells

Interpretation of results:
study cannot be used for classification
Conclusions:
Local cellular toxicity at the portal of entry (BAL fluid and cells), characterised by an increased concentration of total protein and the macrophage activation marker β-glucuronidase, an increase in apoptosis/necrosis, were observed in animals exposed to ≥ 2 mg 4,4’-MDI/m3. With the exception of β-glucuronidase and total protein, all other parameter returned to control levels by 18 h post-expo sure at the low- and mid-dose groups. The influx of neutrophils (a hallmark of an acute inflammatory response) was induced at 11 mg 4,4’-MDI/m3 at both D0 and D1 confirming MDI induces a persistent inflammatory response at this dose level. No clinical signs were observed (e.g. laboured breathing, increase breathing rate). Based on the magnitude of the differences noted in the BALF endpoints support, 11 mg/m3 was considered to be the maximum tolerated concentration (MTC) for an in vivo comet assay.
Executive summary:

The acute inhalation toxicity of the test substance to male rats was investigated in a combined in vivo genotoxicity/acute inhalation toxicity study according to OECD TG 489 (genotoxicity assessed by Comet assay) under GLP conditions (Bruce, 2017). Groups of 12 Wistar rats were exposed to an actual concentration of 2.5, 4.9 or 12 mg/m3(corresponding nominal concentrations: 2, 5, 11 mg/m3)of an aerosol-generated form of the test substance administered via a single nose-only inhalation exposure for 6 hours. A concurrent control group received filtered air on a comparable regimen. Bronchoalveolar lavage (BAL) was performed in all animals at the scheduled necropsies, and the BAL fluid (BALF) and cells (BALC) was assessed for biomarkers of cytotoxicity and inflammation.The endpoints alkaline phosphatase (type II alveolar epithelial cell cytotoxicity), lactate dehydrogenase (tissue damage/cytotoxicity), Annexin V + flow cytometry (apoptosis and necrosis), and total protein (cytotoxicity, blood/air barrier dysfunction) were determined to assess the cytotoxicity. β-glucuronidase (indicator for macrophage activation: activated macrophages secrete various inflammatory mediators such as cytokines/chemokines) and cell differential (with particular focus on the % neutrophils, the influx of which is the hallmark of the typical acute inflammatory response in the rat lung) were determined to assess the inflammatory potential of the test substance. Six rats/group were sacrificed approximately 1 hour post-exposure (ca. 1 hour after termination of the 6 hour exposure) and the other six/group approximately 18 hours post-exposure. Samples of the BALC, liver, and glandular stomach were collected from all animals and processed for comet assay evaluation (see Comet assay in section 7.6.2 and Genetic Toxicity Endpoint Summary). All animals survived to the scheduled euthanasia and there were no test substance-related clinical observations or effects on body weight.

Results included:

- Dose-dependent induction of β-glucuronidase was observed 1 h post-exposure at ≥ 4.9 mg/m3 and ≥ 2.5 mg/m3 at 18 h post-exposure, reaching statistical significance for the high dose-group.

- Dose-dependent increases in LDH at ≥ 2.5 mg/m3 at 1h timepoint. At the 18h timepoint, LDH was only increased in BALF at 11 mg/m3.

- Clear dose-dependent increases in total protein was observed ≥ 2.5 mg/m3 at both time points.

- At both 1 and 18 h after cessation of exposure, the % neutrophils in BALF was increased in in the 4,4’-MDI-exposed groups at 12 mg/m3 indicating an acute inflammatory response.

- An increased of late apoptosis and necrotic cells was observed 1 h post-exposure at ≥ 2.5 mg/m3as identified by Annexin V expression which returned to baseline by 18 h. Similarly, the number of early apoptotic cells increased 1 h after exposure at ≥ 4.9 mg/m3. However, by 18 h, an increase was only observed at 12 mg/m3

In summary, local cellular toxicity, characterised by an increased concentration of total protein and the macrophage activation marker β-glucuronidase in BALF, an increase in apoptosis/necrosis, were observed in animals exposed to ≥ 2.5 mg 4,4’-MDI/m3. With the exception of β-glucuronidase and total protein, all other parameter returned to control levels by 18 h post-exposure at the low- and mid-dose groups. The influx of neutrophils (a hallmark of an acute inflammatory response) was induced at 12 mg 4,4’-MDI/m3 at both D0 and D1 confirming MDI induces a persistent inflammatory response at this dose level. No clinical signs were observed (e.g. laboured breathing, increase breathing rate). Based on the magnitude of the differences noted in the BALF endpoints support, 12 mg/m3 was considered to be the maximum tolerated concentration (MTC) for an in vivo comet assay.

 

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
July 29, 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
- Name of the test item (as cited by study report): 4,4’-Diphenylmethane Diisocyanate (MDI)
- Batch No.: P4DB005186
- Purity: 98.89%.
- Retest date: 30 Jun 2017
- Appearance: White solid

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl:WI (Han)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: Approximately 7 weeks
- Weight at study initiation: 168g - 216 g
- Assigned to test groups randomly: yes, the animals judged suitable for assignment to the study were selected for use in a computerized randomization procedure based on body weight stratification in a block design.
- Housing: Upon arrival, all animals were housed 2 to 3 per cage in clean, solid bottom cages containing ground corncob bedding material (Bed O’Cobs®; The Andersons, Cob Products Division, Maumee, OH). Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.
- Diet: The basal diet used in this study, PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal), is a certified feed with appropriate analyses performed by the manufacturer and provided to Charles River. The basal diet was provided ad libitum throughout the study, except during exposure periods.
- Water: Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system, was provided ad libitum throughout the study, except during exposure periods. Municipal water supplying the facility was analysed for contaminants according to SOPs.
- Acclimation period: All animals were housed for an 14-day acclimation. During acclimation, each animal was observed twice daily for mortality and changes in general appearance or behaviour.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3 – 22.6
- Humidity (%): 37.9 – 46.5
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: aerosol
Vehicle:
Filtered air
Details on exposure:
TYPE OF INHALATION EXPOSURE: nose only

EXPOSURE SYSTEMS
Exposures were conducted using a 2 tier (7.9-L) stainless steel, conventional nose-only exposure systems (CNOS), with grommets in the exposure ports to engage animal holding tubes. Four dedicated exposure systems were used: 1 for the filtered-air control group and 1 for each of the test substance groups. Air supplied to the nose-only systems was provided from the Charles River Inhalation Department breathing quality, in-house compressed air source. All nose-only system exhaust passed through a Solberg canister filter prior to entering the facility exhaust system, which consists of redundant exhaust blowers preceded by activated-charcoal and HEPA-filtration units.

CHAMBER DESCRIPTION
All animals were housed in a normal animal colony room during non-exposure hours. Prior to the exposure, the animals selected for exposure were placed into nose-only restraint tubes in the colony room and transported to the exposure room. Animals were then placed on the nose-only systems and exposed for the requisite duration. After being transported to the exposure room, the animals assigned for exposure were held in restraint tubes for approximately 23 to 26 minutes before the initiation of exposure to allow the animals breathing rates to return to normal baseline values. Food and water were withheld during nose-only restraint tube acclimation and during the exposure period. Oxygen content of the exposure atmospheres was measured during the method development phase of the study using a Dräger PAC III equipped with a calibrated oxygen sensor (Serial No.ERRH-0148, Draeger Safety Inc.; Pittsburgh, PA) and was 20.9% for all groups. Due to the local weather conditions near/at the start of animal exposures, the compressed air used was not as dry as expected. This caused the average humidity for Group 4 to be higher than the protocol-specified target range. The humidity was kept as low as practical to minimize test substance dimer formation.

CONTROL EXPOSURE SYSTEM (EXPOSURE SYSTEM 1)
The control exposure system (Group 1) was operated as follows: dry air was added to the CNOS inlet using a regulator and controlled using a rotameter-type flowmeter.

TEST SUBSTANCE EXPOSURE SYSTEMS (EXPOSURE SYSTEMS 2 TO 4)
A dedicated generation system was used for each test substance exposure system and was operated as follows: A liquid droplet aerosol atmosphere of the test substance was generated using a single-jet Collison nebulizer filled with an appropriate amount of test substance, and heated to approximately 80°C to melt and maintain the test substance in liquid form. Using a regulator, dry, compressed air at a controlled pressure was supplied to the generation port of each nebulizer to affect aerosolization of the test substance. The air entering each nebulizer was also heated. The resulting aerosol from each nebulizer was delivered to a mixing plenum, where it mixed with additional dry, dilutionair. In order to permit reduction of the exposure concentration, a portion of the aerosol output from each nebulizer and/or mixing plenum was directed toward facility exhaust. The remaining aerosol within each mixing plenum was delivered to the “T”-fitting located prior to the inlet of the respective nose-only exposure system, where it was mixed with additional dry air prior to entering each CNOS to permit further reduction of the aerosol concentration.

NOMINAL EXPOSURE CONCENTRATIONS
Nominal exposure concentrations were not calculated for this study due to the nature of each aerosol generation system, where a large portion of the test substance aerosol was removed prior to the final dilution to the target concentration. However, the amount of test substance used during the exposure was calculated by weighing each test substance nebulizer prior to and postexposure.

ACTUAL EXPOSURE CONCENTRATIONS
Aerosol exposure concentrations were measured using standard gravimetric methods. Sample flow was measured using a mini-Buck calibrator. The mass concentration in (mg/m3) was calculated from the filter weight difference divided by the sample volume. Samples were collected at least 4-6 times during each exposure for Exposure Systems 2 to 4 and once for Exposure System 1. Each test substance exposure atmosphere was continuously monitored for aerosol concentration using a light scattering type real time aerosol monitor. These instruments were not intended to define exposure concentration, but were to provide exposure personnel with
an indication of approximate aerosol concentration for guidance in making appropriate system adjustments and achieving the most stable exposure concentration possible.

AEROSOL PARTICLE SIZE MEASUREMENT
Aerosol particle size measurements were conducted using a 7-stage brass cascade impactor. Aerosol particle size measurements were conducted once during the exposure period for each test substance group (Groups 2 to 4). Samples were collected at approximately 1.8 to 1.9 LPM for 360, 150, and 60 minutes for Groups 2,3, and 4, respectively. Following sample collection, substrates were re-weighed and the particle size was calculated based on the impactor stage cut-offs. The particle-size was expressed as the mean aerodynamic diameter (MMAD) in microns and the geometric standard deviation (GSD). See 'Any other information on materials and methods incl. tables' for a summary of the aerosol particle size for each test substance-treated group.

POSITIVE CONTROL SUBSTANCE PREPARATION
The positive control substance formulation was prepared on each day of dose administration (Study Days 0 and 1) as a weight/volume (EMS/0.9% saline) mixture.

JUSTIFICATION OF DOSE LEVELS
Doses were selected based on the outcome of the dose-range finding study (see cross-referenced acute inhalation toxicity record, see section 7.2.2) and other supporting studies (See Acute Toxicity: Inhalation; Pauluhn, 2000; Hotchkiss, 2017) while taking the recommendations in OECD TG 489 to consider cytotoxicity in setting maximum tolerated concentration (MTC). In the range-finding study, Wistar rats were exposed to 3.2, 7.7, and 11.9 mg MDI/m3 for 6 h and biomarkers for local cytotoxicity were analysed 1 h after the exposure and 18 h following completion of exposure. Results demonstrate both concentration- and time-dependent effects of inhaled 4,4’-MDI aerosol on biomarkers of exposure. Compared to control rats, dose dependent increases associated with macrophage activation (β-glucuronidase activity), inflammation (neutrophil infiltration and total protein)) at ≥3.2 mg MDI/m3, apoptosis (Annexin V activity; ≥ 7.7 mg MDI/m3), and necrosis (LDH; ≥ 11.9 mg MDI/ m3) were noted. This is supported by other supporting studies that demonstrate that acute exposure during 6 h to a concentration of 10-12 mg/m3 resulted in significant increases in biomarkers for inflammation, cytotoxicity/apoptosis, and macrophage activation (See Acute Toxicity: Inhalation; Pauluhn, 2000; Hotchkiss, 2017). Moreover, concentrations > 20 mg/m3 (for 3 hr) were sufficient to induce cytotoxicity induced DNA damage, but 10 mg/m3 (for 6h) were not (Sutter, 2016). Taken together, the MTC was considered 11.9 mg/m3 on the basis of marked increases in inflammation, apoptosis/necrosis, and cytotoxicity. The remaining 2 concentrations be appropriately spaced as to demonstrate a dose response with the lowest producing little to no toxicity. In the range-finding study, concentrations of 3.2 and 7.7 mg/m3 resulted in mild to moderate concentration-dependent changes as measured by inflammation and macrophage activity (β-glucuronidase). As the midconcentration, 5 mg/m3 is selected as it is anticipated to induce mild to moderate cytotoxic effects. As the low-concentration, 2 mg/m3 is selected as it is expected to result in little to no cellular toxicity. The selection of concentrations is in agreement with the recommendations described in OECD TG 489.
Duration of treatment / exposure:
6 hours
Frequency of treatment:
One exposure period
Post exposure period:
Six rats/group terminated on Study Day 0 within approximately 1 hour postexposure (Groups 1–4), on Study Day 1 at approximately 18 hours postexposure (Groups 1–4), or on Study Day 1 between 2 and 4 hours after the second dose of EMS (Group 5); bronchoalveolar lavage fluid (BALF), liver, and glandular stomach collected and processed for in vivo comet assay evaluation from 6 animals/sex/group at each time point; bronchoalveolar lavage and fluid analysis performed on all animals; carcasses and remaining tissues discarded.
Doses / concentrationsopen allclose all
Dose / conc.:
2 mg/m³ air (nominal)
Remarks:
Analytical concentration: 2.5 mg/m³
Dose / conc.:
5 mg/m³ air (nominal)
Remarks:
Analytical concentration: 4.9 mg/m³
Dose / conc.:
11 mg/m³ air (nominal)
Remarks:
Analytical concentration: 12 mg/m³
No. of animals per sex per dose:
-Test groups (Group 2-4): 12 males
- Vehicle control (Group 1): 12 males
- Positive control (Group 5): 6 males
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control substance, ethyl methanesulfonate (EMS; CAS 62-50-0), was administered via oral gavage to rats in Group 5 at a dose of 200 mg/kg/day on Study Days 0 and 1. The vehicle used in preparation of the positive control substance formulation was 0.9% sodium chloride for injection. The positive control substancewas stored refrigerated (2°C to 8°C), purged with nitrogen,andwas considered stable under these conditions. The route of administration of the positive control substance (oral gavage) was chosen based on previous data in which BioReliance demonstrated that orally administered EMS produced a significant increase in DNA damage (comet response) in the BAL cells, liver, and glandular stomach.

Examinations

Tissues and cell types examined:
Samples of the BALF, liver and stomach were collected from all animals and processed for the comet assay evaluation.

BAL cells have been used have been regularly used in the assessment of pulmonary genotoxicity after inhalation or instillation of various substances. Pulmonary macrophages are predominant in the BAL fluid, usually one of the first cell types to come into contact with aerosols and thus represent the site of contact in the lung; this is especially relevant for very reactive substances such as 4,4’-MDI. The use of BAL cells, versus using minced lung tissue, reduces the dilution of effects from unexposed cells that occurs when for instance alveolar epithelial cells are isolated. It is technically difficult to harvest and sort different cell types from minced lung tissue. Harsh treatment often results in high background damage and a major share of cells in minced lung tissue consist of haematopoetic cells. This means that only a relatively large induction of DNA damage would be identified by the sensitive Comet Assay. Importantly, during development of the historical control database in the performing laboratory in a preliminary study, it was observed that the positive control substance EMS rapidly distributes from the lymph system to the BAL to induce DNA damage. This was confirmed during the main study and further demonstrates the value of BAL cells as an appropriate tissue to be examined. Liver was included since it is the site of primary metabolism; and it was included to investigate systemic (as opposed to local) genotoxicity. Glandular stomach was included due to possible exposure after clearance of 4,4’-MDI via the mucociliary escalator.
Details of tissue and slide preparation:
MACROSCOPIC EXAMINATION
All animals were anesthetized by isoflurane inhalation and euthanized by exsanguination. The animals were not fasted overnight prior to the scheduled euthanasia, and a gross necropsy was not performed. Immediately following euthanasia, BAL was performed on all animals and processed. At the time of euthanasia, samples of the following tissues were collected and placed in 10% neutral-buffered formalin. The carcasses and remaining tissues were discarded.

HISTOLOGY AND MICROSCOPIC EXAMINATION
After fixation, protocol-specified tissues were trimmed according to the test laboratory SOPs and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned according to the test laboratory SOPs, mounted on glass microscope slides, and stained with hematoxylin and eosin from all animals at the scheduled necropsies.

BRONCHOALVEOLAR LAVAGE
At the scheduled euthanasia, BAL was performed on the lungs of all animals. The trachea was exposed and a dosing cannula was tied in place in the trachea. The lungs were lavaged 6 times with a lavage solution volume of 25 µL/gram body weight (based on the most recent body weights) of room temperature Hank’s Balanced Salt Solution (sterile) without calcium, magnesium, or phenol red, up to a maximum volume of 4 mL (per lavage). The BALF from the first and second lavages was recovered after remaining in the lung for approximately 1 minute and placed into a 15-mL polypropylene centrifuge tube after the volume was recorded. For the third through sixth lavages, the recovered volumes were recorded and the BALF was collected into a single polypropylene centrifuge tube. Recovered BALF was stored on ice until processed. The following parameters were evaluated from the BAL fluid or cell pellet: Alkaline phosphatease (ALP), Annexin V, ß-glucuronidase, cell differential (cytology), lactate dehydrogrenase (LDH) and total protein (see cross-referenced acute inhalation study, section 7.2.2).

BRONCHOALVEOLAR LAVAGE FLUID PROCESSING
The BALF was isolated in a refrigerated centrifuge and the supernatant fluid from the first and second lavages was transferred to a sealable vial and stored on an ice bath until used for analysis of BAL clinical chemistry (alkaline phosphatase, lactate dehydrogenase, and total protein) and/or transferred to the internal immunotoxicology department for analysis of beta. The supernatant fluid from the third through sixth lavages was decanted and discarded. The cell pellets obtained from the first and second lavages or third through sixth lavages were pooled separately and retained. The cell pellet from the first and second lavages was resuspended in cold Roswell Park Memorial Institute (RPMI) media with 10% fetal calf serum, and this cell suspension was also used to resuspend the cell pellet from the third through sixth lavages. Total cell counts were obtained using a hemocytometer with cell viability assessed by trypan blue exclusion. A portion of the cell suspensions were transferred into Dulbecco’s Phosphate-Buffered Saline (DPBS; with calcium and magnesium) for future processing and analysis of Annexin V (see cross-referenced acute inhalation study, section 7.2.2). The remainder of the cell suspension was centrifuged again and a portion of the cell pellet was used for the comet assay.

TISSUE SELECTION FOR COMET ASSAY
A section of the liver was placed in 3 mL of chilled mincing solution (Hanks’ balanced salt solution with EDTA and DMSO), then minced with fine scissors to release the cells. A section of the glandular stomach was placed in 1 mL of chilled mincing solution then scraped using a plastic spatula to release the cells. The cell suspensions were strained into a pre-labeled conical polypropylene tube through a Cell Strainer and were kept on wet ice during preparation of the slides. The remaining cell pellet from the bronchoalveolar lavage was mixed with chilled mincing solution. The mixture was kept on wet ice during the preparation of the slides.

PREPARATION OF COMET SLIDES
- Preparation of slides: From each liver, stomach and BAL cell suspension, an aliquot of 2.5 µL, 7.5 µL and 7.5 µL, respectively, were mixed with 75 µL (0.5%) of low melting agarose. The cell/agarose suspension was applied to microscope slides commercially available pre-treated multi-well slides. Commercially purchased multi-well slides were used and these slides have 20 individual circular areas, referred to as wells in the text below. The slides were kept refrigerated for at least 15 minutes to allow the gel to solidify. At least two Trevigen, Inc. 20-well slides were prepared per animal per tissue. Slides were identified with a random code that reflects the study number, group, animal number, and organ/tissue. Three wells were used in scoring and the remaining wells were designated as a backup. The backup slides/wells may be used in additional scoring, if deemed necessary. Following solidification of agarose, the slides were placed in jars containing lysis solution.
- Lysis: Following solidification of agarose, the slides were submerged in a commercially available lysis solution supplemented with 10% DMSO on the day of use. The slides were kept in this solution at least overnight at 2-8 °C.
- Unwinding: After cell lysis, slides/wells were washed with neutralization buffer (0.4 M tris hydroxymethyl aminomethane in purified water, pH ~7.5) and placed in the electrophoresis chamber. The chamber reservoirs were slowly filled with alkaline buffer composed of 300 mM sodium hydroxide and 1 mM EDTA (disodium) in purified water. The pH was > 13. All slides remained in the buffer for 20 minutes at 2-10 °C and protected from light, allowing DNA to unwind.
- Electrophoresis: Using the same buffer, electrophoresis was conducted for 30 minutes at 0.7 V/cm, at 2-10 °C and protected from light. The electrophoresis time was constant for all slides.
- Neutralization: After completion of electrophoresis, the slides were removed from the electrophoresis chamber and washed with neutralization buffer for at least 10 minutes. The slides (gels) were then dehydrated with 200-proof ethanol for at least 5 minutes, then air dried for at least 2 hours and stored at room temperature with desiccant.
- Staining: Slides were stained with a DNA stain (i.e., Sybr-gold™) prior to scoring. The stain solution was prepared by diluting 1 µL of Sybr-gold™ stain in 15 mL of 1xTBE (tris-boric acid EDTA buffer solution).

METHOD OF ANALYSIS
Three wells per organ/animal/treatment were used for the first five animals in each group/time point. The wells from the 6th animal in each group/time point will only be scored due to loss of any animals or rejection of the samples (on quality grounds) after consultation between the sponsor and study director. Fifty randomly selected, non-overlapping cells per slide/well were scored resulting in a total of 150 cells evaluated per animal for DNA damage using the fully validated automated scoring system Comet Assay IV from Perceptive Instruments Ltd. (UK).
The following endpoints of DNA damage were assessed and measured:
- Comet Tail Migration; defined as the distance from the perimeter of the Comet head to the last visible point in the tail.
- % Tail DNA; (also known as % tail intensity or % DNA in tail); defined as the percentage of DNA fragments present in the tail.
- Tail Moment (also known as Olive Tail moment); defined as the product of the amount of DNA in the tail and the tail length [(% Tail DNA x Tail Length)/100.
Each slide/well was also examined for indications of cytotoxicity. The rough estimate of the percentage of “clouds” was determined by scanning 150 cells per animal, when possible (percentage of “clouds” was calculated by adding the total number of clouds for all slides scored, dividing by the total number of cells scored and multiplying by 100). The “clouds”, also known as “hedgehogs”, are a morphological indication of highly damaged cells often associated with severe genotoxicity, necrosis or apoptosis. A “cloud” is produced when almost the entire cell DNA is in the tail of the comet and the head is reduced in size, almost nonexistent. “Clouds” with visible gaps between the nuclei and the comet tail were excluded from comet image analysis.
Evaluation criteria:
CRITERIA FOR DETERMINATION OF A VALID TEST
- The DNA damage data (% tail DNA) in the filtered air control group is expected to be within the historical vehicle control range.
- The mean number of clouds for a tissue from a filtered air control group animal should not exceed 30%.
- The positive control group must be significantly increased relative to the concurrent filtered air control group (p ≤ 0.05).

EVALUATION OF THE TEST RESULTS
- The test substance will be considered to induce a positive response in a particular tissue if the mean % tail DNA (or other parameters of DNA damage) in one or more test substance groups (doses) is significantly elevated relative to the concurrent filtered air control group.
- The test substance will be judged negative for induction of DNA damage if no statistically significant increase in the mean % DNA damage (or other parameters) in the test substance groups relative to the concurrent filtered air control group is observed.
- The historical vehicle control data; a statistically significant increase in the mean % DNA (or other parameters) may not be considered biologically relevant if the values do not exceed the range of historical vehicle control.
- Any statistically significant increase in DNA damage occurring at a cytotoxic dose may not be considered as a positive finding.
- If a dose-response is evident with no statistically significant increase, additional testing, including histopathology evaluation of the tissue, may be considered.
- If criteria for either a positive or negative response are not met, the results may be judged as equivocal.
Statistics:
In order to quantify the test substance effects on DNA damage, the following statistical analysis was performed:
- The use of parametric or non-parametric statistical methods in evaluation of data was based on the variation between groups. The group variances for % tail DNA generated for the vehicle and test substance groups were compared using Levene’s test (significant level of p ≤ 0.05). If the differences and variations between groups were found not to be significant, a parametric one-way ANOVA followed by a Dunnett’s post-hoc test was performed (significant level of p < 0.05).
- A linear regression analysis was conducted to assess dose responsiveness in the test substance treated groups (p < 0.01).
- A pair-wise comparison (Student’s T-test, p ≤ 0.05) was used to compare the positive control group against the concurrent vehicle control group.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
see "Additional information on results"
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
GENOTOXICITY
- No significant differences in group variance was noted for all tissues at 1 hour and 18 hours after exposure. Therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.
- No statistically significant response in the % Tail DNA (DNA damage) was observed in the test substance groups relative to the concurrent filtered air control groups for all tissues at 1 hour and 18 hours after exposure.
- No dose-dependent increase in the % Tail DNA was observed across three test substance doses for all tissues at 1 hour and 18 hours after exposure.
- In the filtered air control groups, % Tail DNA was within the historical vehicle control range for all groups except for the BAL cells and stomach after 18 hours exposure.
- The positive control induced a statistically significant increase in the % Tail DNA in all tissues as compared to the filtered air control groups
- The presence of ‘clouds’ in the test substance groups was not consistent between test substance groups (either higher than, lower than or comparable to the % of clouds in the filtered air control group).
See 'Any other information on results incl. tables' for % tail DNA data.

CYTOTOXICITY
Local toxic effects were noted as described in the cross-referenced acute inhalation toxicity record, see section 7.2.2. At exposure concentrations of 2, 5, and 11 mg/m3, test substance-associated differences in BALF endpoints (cytology, alkaline phosphatase, lactate dehydrogenase, total protein, β-glucuronidase, and Annexin V) and microscopic changes in the liver were observed. Based on the magnitude of the differences noted in the BALF endpoints, 11 mg/m3 was considered to be the maximum tolerated dose (MTD) for local effects.

Any other information on results incl. tables

Table: % Tail DNA in BAL Cells Following Administrations of MDI (1 hour after exposure)

Treatment

Number of Animals

Group Mean % of Clouds

Tail DNA (%)

Mean

S.D. (±)

Negative Control: Filtered air

5

5.8

0.02

0.02

2.5 mg/m3 MDI

5

21

0.03

0.01

4.9 mg/m3 MDI

5

24.8

0.08

0.11

12 mg/m3 MDI

5

20

0.04

0.05

Table: % Tail DNA in BAL Cells Following Administrations of MDI (18 hours after exposure)

Treatment

Number of Animals

Group Mean % of Clouds

Tail DNA (%)

Mean

S.D. (±)

Negative Control: Filtered air

5

19

2.22

3.33

2.5 mg/m3 MDI

5

21.4

0.09

0.08

4.9 mg/m3 MDI

5

32.4

0.43

0.36

12 mg/m3 MDI

5

23.4

0.07

0.06

Positive Control: EMS 200 mg/kg

5

41.8

28.82

4.96*

Table: % Tail DNA in Liver Cells Following Administrations of MDI (1 hour after exposure)

Treatment

Number of Animals

Group Mean % of Clouds

Tail DNA (%)

Mean

S.D. (±)

Negative Control: Filtered air

5

3

0.15

0.09

2.5 mg/m3 MDI

5

2

0.19

0.13

4.9 mg/m3 MDI

5

4

0.27

0.37

12 mg/m3 MDI

5

3.6

0.22

0.13

Table: % Tail DNA in Liver Cells Following Administrations of MDI (18 hours after exposure)

Treatment

Number of Animals

Group Mean % of Clouds

Tail DNA (%)

Mean

S.D. (±)

Negative Control: Filtered air

5

1

0.05

0.03

2.5 mg/m3 MDI

5

2.8

0.08

0.08

4.9 mg/m3 MDI

5

2

0.03

0.02

12 mg/m3 MDI

5

2

0.16

0.14

Positive Control: EMS 200 mg/kg

5

6.2

37.82

4.61*

Table: % Tail DNA in Stomach Cells Following Administrations of MDI (1 hour after exposure)

Treatment

Number of Animals

Group Mean % of Clouds

Tail DNA (%)

Mean

S.D. (±)

Negative Control: Filtered air

5

11.6

10.35

4.39

2.5 mg/m3 MDI

5

5.2

4.75

3.45

4.9 mg/m3 MDI

5

3.6

5.27

4.29

12 mg/m3 MDI

5

7

8.91

3.77

Table: % Tail DNA in Stomach Cells Following Administrations of MDI (1 hour after exposure)

Treatment

Number of Animals

Group Mean % of Clouds

Tail DNA (%)

Mean

S.D. (±)

Negative Control: Filtered air

5

15.8

20.18

5.04

2.5 mg/m3 MDI

5

16.6

18.7

5.55

4.9 mg/m3 MDI

5

4.4

16.22

8.71

12 mg/m3 MDI

5

25

20.42

12.77

Positive Control: EMS 200 mg/kg

5

55.6

51.35

4.72*

S.D. = Standard Deviation          *p ≤ 0.05 (Student's t-test); Statistically significant increase relative to the vehicle control

Applicant's summary and conclusion

Conclusions:
With the exception of the 18 h timepoint for BAL and stomach, the test material fulfilled all requirements described above in the in vivo Comet Assay in all three tissues (BAL cells, liver and stomach) and at both timepoints. For the 18 h timepoint, the % Tail DNA measured for the filtered air control group was outside the existing historical vehicle control range for the lung and stomach. For the lung, the mean % Tail DNA in the control group was 2.22% +/- 3.33 (SD), while the historical control data are in the range 0.01-0.11%. For the stomach, the mean % Tail DNA in the control group was 20.18 +/- 5.04 (SD), the historical control data are in the range 0.94-16.88%. While the 18 h BAL and stomach results were not all within the historical range established for these tissues, it was not deemed to impact on validity of the study outcome or study interpretation since A) there were no statistically significant differences between exposed animals and controls, B) there is no trend towards a dose dependent effect, C) direct evidence of toxicity was demonstrated at the site of contact, and D) there was a statistically significant difference between the filtered air control group and the EMS-exposed positive control group. It is clear from these data that under the conditions of this study, that the administration of 4,4’- Diphenylmethane Diisocyanate (4,4’-MDI) at doses up to and including the maximum tolerated concentration of 12 mg/m3 (actual concentration) did not cause significant increases in % Tail DNA in BAL cells, liver and stomach relative to the concurrent vehicle control. Therefore, 4,4’-MDI is concluded to be negative in the in vivo Comet Assay.
Executive summary:

The genotoxic potential of the test substance to male rats was investigatedin an in vivo mammalian alkaline Comet Assay/acute inhalation toxicity study according to OECD TG 489 (genotoxicity assessed by Comet Assay) under GLP conditions. This study was performed to assess if 4,4’-MDI is a genotoxic substance at the site of contact. As the site of contact tissue, bronchoalveolar lavage cells were selected; these primarily consist of alveolar macrophages, which are the primary cells responsible for the removal of inhaled aerosols from the alveoli and are commonly selected in the assessment of pulmonary genotoxicity after inhalation or instillation. In addition, the liver was included since it is the site of primary metabolism; also, it was included to investigate systemic (as opposed to local) genotoxicity. Finally, the glandular stomach was included due to possible exposure after clearance of 4,4’-MDI via the mucociliary escalator. Groups of 12 Wistar rats were exposed to actual concentrations of 2.5, 4.9 and 12mg/m3 (corresponding nominal concentrations: 2, 5, 11 mg/m3) to an aerosol-generated form of the test substance via a single nose-only inhalation exposure for 6 hours. The top concentration was defined as the maximum tolerated concentration (MTC) from a preliminary range-finding study and other supporting acute sub-lethal inhalation studies. The MTC was selected based on marked local acute toxicity as identified by biomarkers forinflammation, apoptosis/necrosis and cytotoxicity at concentrations ≥ 11.9 mg/m3. A concurrent control group received filtered air on a comparable regimen. A positive control group received ethyl methanesulfonate (EMS) via oral gavage (200 mg/kg/day) for 2 days. In preliminary studies at the performing laboratory, gavage administration of EMS resulted in a strong positive signal in all tissues examined as was determined appropriate for identifying direct acting genotoxicity. Bronchoalveolar lavage (BAL) wasperformed in all animalsat the scheduled necropsies, and the BAL fluid (BALF) was assessed for the following endpoints: clinical chemistry (alkaline phosphatase, lactate dehydrogenase, and total protein), cell differential (cytology), and measurement of Annexin V expression and β-glucuronidase activity.The alkaline phosphatase, lactate dehydrogenase, Annexin V expression + flow cytometry, and total protein were determined to assess the cytotoxicity of the test substance. The β-glucuronidase and cell differential (in particular % polymorphonuclear leukocytes) were determined to assess the inflammatory potential of the test substance.Six rats/groupwere sacrificed approximately1 hour post-exposure and the other six/group approximately 18 hours post-exposure, or 2 to 4 hours after the second dose for the positive control group.At the high concentration of 12 mg/m3, test substance-associated differences in BALF endpoints (neutrophil influx, total protein, β-glucuronidase, and Annexin V) were observed. Therefore, 12 mg/m3 was confirmed to be the MTC.The test substance gave a negative (non-DNA damaging) response in thisassay in the BAL cells, liver and stomach for both the 1 hour and 18 hour time points for males in % Tail DNA. It was therefore concluded that the test substance scored negative in the in vivo Comet Assay up to the maximum tolerated concentration.