Isocyanic acid, polymethylenepolyphenylene ester, 2-ethyl-1-hexanol-blocked

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item  Desmodur MT was initially investigated using the Salmonella/microsome plate incorporation test for point mutagenic effects in doses up to and including 5000 µg per plate on five Salmonella typhimurium mutants. these comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102. The independent repeat was performed as preincubation for 20 minutes at 37°C. Doses up to and including 50 µg per plate did not cause any bacterio-toxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a stain-specific bacterio-toxic effect. Substance precipitation occurred at the dose of 500 µg per plate and above. Due to these effects this range could only partly be used up to 5000 µg per plate for assessment purpose. Evidence of mutagenic activity of Desmodur MT was not seen. No biologically relevant increase in the mutant count, in  comparison with the negative control, was observed. The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C cumene hydroperoxide and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant in mutant colonies compared to the  corresponding negative control.

Therefore, Desmodur MT was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well in the preincubation modification of the Salmonella/microsome test.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 471 "Bacterial Reverse Mutation Test" adopted 21st July 1997

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch number: LL6-077

Species / strain / cell type:

S. typhimurium, other: TA 98, TA 100, TA 102, TA 1535, TA 1537

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male rat liver S9 mix

Test concentrations with justification for top dose:

First test: 0, 50, 158, 500, 1581, 5000 µg/plate (all TA strains, without and with S9 mix)
Repeat test: 0 - 5000 µg/tube (different concentrations for the 5 TA strains, without and with S9 mix)

Vehicle / solvent:

Solvents used: ethylene glycol dimethylether (EGDE) dried with a molecular sieve 0.3nm (test substance), deionized water (mitomycin C), DMSO (sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide, 2-aminoanthracene)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (only TA 1535), nitrofurantoin (only TA 100), 4-nitro-1,2-phenylene diamine (TA 1537 and TA 98), mitomycin C (only TA 102 in plate incorporation assay), cumene hydroperoxide (only TA 102 in preincubation assay), 2-aminoanthracene.

Remarks:

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C and cumene hydroperoxide were only used without S9 mix; the positive control 2-aminoanthracene was only used with S9 mix.

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Statistics:

Not specified.

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

precipitation at 500 µg/plate and higher

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxic effects at doses up to and including 158 µg/plate and higher precipitation at 500 µg/plate and higher

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxic effects at doses up to and including 5000 µg/plate precipitation at 500 µg/plate and higher

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxic effects at doses up to and including 158 µg/plate and higher precipitation at 500 µg/plate and higher

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1581 µg/plate and higher precipitation at 500 µg/plate and higher

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Ames test:
- Signs of toxicity: yes
- Individual plate counts: yes
- Mean number of revertant colonies per plate and standard deviation: yes

Table 1: Summary of results from the Salmonella mutagenicity assay (first test) with Desmodur MT (mean values of revertants per plate)

Dose (µg per plate)	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (EGDE)	14	183	7	28	178
50	12	143	6	27	189
158	14	159	6	23	183
500	11	64	6	10	191
1581	9	45	5	8	185
5000	---	57	---	8	175
Positive control	579	415	76	126	511
Dose (µg per plate )	With metabolic activation (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (EGDE)	8	187	6	21	321
50	9	191	7	18	314
158	8	219	8	28	332
500	8	139	7	22	234
1581	7	138	7	24	213
5000	---	149	---	19	211
Positive control	130	1000	126	1114	440

--- = no value available

Table 2: Summary of results from the Salmonella mutagenicity assay (repeat test) with Desmodur MT (mean values of revertants per tube)

Dose (µg per tube)	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (EGDE)	22	151	7	33	274
5	28	134	n.t.	30	n.t.
16	29	148	7	33	n.t.
50	28	136	6	33	263
158	25	115	7	18	294
500	17	100	5	14	268
1581	n.t.	n.t.	6	n.t.	235
5000	n.t.	n.t.	n.t.	n.t.	246
Positive control	617	432	109	156	506
Dose (µg per tube )	With metabolic activation (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (EGDE)	15	210	8	34	252
16	11	213	7	n.t.	n.t.
50	11	193	8	40	269
158	10	200	10	43	316
500	10	183	8	47	348
1581	12	163	8	45	340
5000	n.t.	n.t.	n.t.	38	327
Positive control	140	1372	135	885	467

n.t. = not tested

Doses up to and including 50 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a strain-specific bacteriotoxic effect. Substance precipitation occurred at the dose 500 µg per plate and above. Due to these effects this range could only partly be used up to 5000 µg per plate for assessment purposes.

Evidence of mutagenic activity of Desmodur MT was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C, cumene hydroperoxide and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Conclusions:

negative

Executive summary:

In an Ames test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535, and TA 1537 Desmodur MT revealed no mutagenic activity in the absence and in the presence of a metabolic activation system (OECD TG 471).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

There are no in vitro cytogenetic study data for MDI MT available. A read across to valid in vivo data of the source substance 4,4’-MDI was performed. For the endpoint cytogenetic reliable in vivo micronucleus assays are available. In an OECD guideline study (OECD 474) Pauluhn et al. (2001) conducted a micronucleus study under GLP. Rats were exposed via both whole body and nose only to 4,4’-MDI for 1 hour per week for 3 weeks, with bone marrow examinations at one and two days post-exposure at concentrations up to 118 mg/m³. Although toxic effects at the portal of entry (e.g. respiratory distress, increased lung weights) were observed, there was no evidence of an MDI-induced increase in the frequency of micronucleated polychromatic erythrocytes (MN-PCE) at any of the time points selected. MN-PCEs were significantly increased in rats treated with the positive control when compared to both the negative control and MDI-exposure groups.

In a supporting study Lindberg et al. (2011) investigated the genotoxicity of inhaled 4,4’-MDI in male C57Bl/6J mice by examining micronucleated polychromatic erythrocytes (PCE) in bone marrow and peripheral blood. Mice were exposed head-only to 4,4’-MDI aerosols (mean concentrations 10.7, 20.9 and 23.3 mg/m³), 1 h/day for 5 consecutive days. Bone marrow and peripheral blood were collected 24 hours after the last exposure. Haemoglobin adducts detected in the exposed mice resulted from direct binding to globin of MDI and adducts originating from the diamine (MDA) were not observed. No significant increase in the frequency of micronucleated PCEs was detected in the bone marrow or peripheral blood of the mice exposed to MDI. The authors concluded that inhalation of MDI (1 h/day for 5 days), at levels that induced toxic effects (decreased respiratory frequency, decreased body weights and an influx of inflammatory cells into the lung were observed) and formation of MDI-specific adducts in haemoglobin, did not have detectable systemic genotoxic effects in mice, as investigated by the micronucleus assay.

An older mouse micronucleus study has been reported in summary from JETOC (1982) also gave comparable results. In summary, 4,4’-MDI was dissolved in dry DMSO, mixed with corn oil and administered to mice by intra-peritoneal injection at doses of 32, 80 or 200 mg/kg). The mice were killed 24 hours following final treatment and incidence of polychromatic and normochromatic erythrocytes with micronuclei evaluated. There was no difference in incidence of micronuclei between animals treated with MDI and the untreated control group. It was concluded that 4,4’-MDI did not cause micronuclei, and therefore did not induce in vivo genotoxic effects.

In contrast, a bone marrow micronucleus study by Zhong and Siegel (2000) exposed Brown Norway rats (males, n = 6) were exposed via inhalation (whole body, 2 at a time) to either 7 or 113 mg/m³ 4,4’-MDI for 1 hour, once a week for 3 weeks with sacrifice 1 week later. A dose-dependent increase in the frequency of micronucleated polychromatic erythrocytes (MN-PCEs) was noted: 1.5- and 4.5-fold increases of micronuclei were reported for the two exposure groups over control (a control group of 4 rats). No difference was found in the ratio of PCEs and NCEs between exposure and control groups, suggesting the absence of bone marrow cytotoxicity. However, this study was deemed to be low reliability (Klimisch 3) since the in vivo protocol used was not standard for a micronucleus test (evaluation at 7-days post-exposure but not before, absence of positive controls) and the findings are not consistent with the existing genotoxicity profile of MDI (or MDA).

Based on a weight-of-evidence, inhalation of 4,4’-MDI by rats does not induce MN formation in systemic target organs despite circulating levels of MDI metabolites and detection of radioactivity in the bone following inhalation of radiolabelled 4,4’-MDI in ADME studies (i.e. Gledhill (2003b; 2005).  Taken together it can be assumed that the bone marrow has been exposed to the 4,4’-MDI via metabolites and conjugates. Given the reactivity of 4,4’-MDI and the toxicokinetic profile described above that 4,4’-MDI is available exclusively to MDI metabolites and conjugates (and not free mMDI), the absence of systemic genotoxicity after an inhalation exposure is not unexpected and in line with the hypothesized MoA.

There are no in vitro mammalian cell mutagenicity study data available for MDI MT. A read across to valid in vivo data of the source substance 4,4’-MDI was performed. Randazzo (2017)  conducted a guidance study (OECD TG 489) under GLP conditions with 4,4’-MDI. As the site of contact tissue, bronchoalveolar lavage (BAL) cells were selected to be analysed. In addition to the BAL cells, the liver and glandular stomach was included in the analysis. The liver was analysed since it is the site of primary metabolism (and potential systemic genotoxicity), while the glandular stomach was included due to possible secondary exposure after clearance of 4,4’-MDI via the mucociliary escalator (i.e. local effects at a secondary site of contact). Groups of 12 Wistar rats were exposed to actual concentrations of 2.5, 4.9 and 12 mg/m3 for 6 hours with the maximum dose selected from pilot range-finding study and previous studies as a concentration that will induce significant local cellular damage (Hotchkiss et al., 2017).  Bronchoalveolar lavage (BAL) was performed in all animals at the scheduled necropsies, and the BAL fluid (BALF) was assessed for cytotoxicity and inflammation to determine non-specific or direct toxicity at the sites of contact following acute exposure as recommended by the test guideline. Animals were sacrificed approximately one-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 (MNU via gavage). An inflammatory response was observed in the lung in the high-concentration group, characterized by a significant increased influx of neutrophils. Also, time and dose-dependent apoptosis/necrosis was induced in the lung in all treatment groups, as shown by Annexin V staining and LDH activity. However, these effects were only persistent to the 18 hour time point at 12 mg/m³.  In the stomach and liver, there were no indications for 4,4’-MDI-induced toxicity as indicated by histopathology. Overall, these results are consistent with local cellular toxicity of the lung as the critical mode of action of MDI toxicity.  Based on the magnitude of the differences noted in the BAL endpoints, 12 mg/m³ (measured concentration) was considered to be the maximum tolerated concentration (MTC) to avoid confounding secondary DNA damage resulting from local cytotoxic effects. DNA damage was investigated using the Comet Assay with results clearly negative for all three investigated tissues (BAL cells; liver; stomach) indicating that 4,4’-MDI is not genotoxic at the portal of entry at exposures of up to the maximum tolerated concentration, as indicated by local cytotoxicity.

In a former non-guideline comet assay (Suter, 2016) adopted to explore the sequence of irritation-related events on pulmonary cells harvested by bronchoalveolar lavage (BAL) from rats exposed by inhalation to aerosolized 4,4’-MDI. Groups of rats were nose-only exposure to air and concentration x time (C x t) intensities in the range of 50 and 500 mg/m³xh (10, 20, 100, or 180 mg 4,4’-MDI/m³ for 3 or 6 hours). BAL-fluid/-cells were collected 3 hours (day 0), 1 and 3 days post-exposure to establish a C x t and postexposure duration-related matrix of acute inflammation (lung weights, LDH, total protein, and cytodifferentiation of BAL-cells), restoratively increased annexin V activity, increased caspase 3/7 activity correlated with increased %Tail intensity of BAL-cells. The dose levels, inflammation endpoints, and sampling points were selected based on earlier inhalation toxicity studies in rats (Pauluhn, 2011a).  Aerosolized paraffin at about 50 mg/m³xh 21 served as non-irritant/ genotoxic reference for the phagocytosis of waxy aerosols by alveolar macrophages (AM) and the dose level was selected to match one of the 4,4'-MDI dose levels (but a different time point was examined for that dose).

Results from this study indicated that inhalation exposure to 4,4'-MDI led to a dose dependent mild positive response in the Comet assay observed at day 0 and 1 stating with an exposure of 20 mg/m³ for 3 hours (54.3 mg/m³xh). Increase in tail length (approximately 2.3-fold increase compared to approximately 11 to 22-fold increase in the positive control (MNU administrated orally)) correlated with markers of cytotoxicity, apoptosis and inflammation. A no observed adverse effect concentration (NOAEC) of 10 mg/m³ for 6 hours (61.8 mg/m³xh) was identified for the Comet response but yet still induced a clear acute toxic effect in the BAL. At day three after exposure there was no significant effect on tail length even at a very high C x t of 518.4 mg/m³xh (100 mg/m³ for 6 hours). Effects on markers of alveolar toxicity at 20 mg/m³ 4,4'-MDI for 3 hour (54.3 mg/m3xh) and sampled at day 0 correlated with results obtained for the inert organic particle solid paraffin at 25 mg/m³ at day one. For example, increase in alveolar macrophages were not identified, but increase in markers for apoptosis (caspase 3/7 or annexin V) was observed together with a mild comet response. The increased apoptosis observed for at least one of the two apoptosis parameters after inhalation of 4,4'-MDI, but not after exposure to the positive control for direct genotoxicity (MNU), put the positive Comet assays result into perspective of primary versus secondary effects. There is understood to be a link between macrophage activation and reactive oxygen species generation (as seen in the current cytodifferentiation results), leading to an inflammatory response including oxidative burst, finally resulting in oxidative DNA damage. Accordingly, these results do not point to primary, test substance-induced suggest that DNA damage caused the 4,4’-MDI at concentrations ≥ 20 mg/m³ is the result of excessive toxicity (cytotoxicity, apoptosis and/or inflammation) rather than direct genotoxicity.

In summary, as mentioned above, the in vivo comet assay conducted by Randazzo (2017) also included analysis of the liver to assess the potential for genetic damage at a distal tissue (e.g. site of first pass metabolic clearance) and the stomach for secondary exposure following mucociliary clearance. No DNA-damage was noted in either tissue at either 1 or 18 hours after exposure at the maximum tolerated dose for respiratory toxicity (12 mg/m³) further indicating that 4,4’-MDI is not systemically genotoxic, which is in line with the hypothesized MoA.

As the source substance 4,4'-MDI and the target substance MDI MT contain sufficient monomeric MDI, the driver of toxicity, similarities in reaction is predicted. In addition, as the higher molecular weight non-monomeric content of the UVCB substance MDI MT do not contains reactive centres and is consequently inert and thus do not contribute to the expected toxicity, it is reasonable to assume that using read across to the source substance 4,4'-MDI is warranted. 

Therefore, the target substance MDI MT is not classified for genotoxicity according to Regulation (EC) No.1272/2008 (CLP Regulation).

Link to relevant study records

Reference

Reference 1

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

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

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

Details on species / strain selection:

Crl:WI (Han)

Sex:

male

Details on test animals or test system 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

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.

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 substance was stored refrigerated (2°C to 8°C), purged with nitrogen, and was 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.

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.

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.

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

Conclusions:

The current study is a source study used (Randazzo 2017) for read-across to the target substance MDI MT. The study data did not indicate a genotoxic potential. The genotoxic potential of the test substance to male rats was investigated in 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 for inflammation, 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) was performed in all animals at 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/group were 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 this assay 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.

Executive summary:

Randazzo (2017)  conducted a guidance study (OECD TG 489) under GLP conditions with 4,4’-MDI. As the site of contact tissue, bronchoalveolar lavage (BAL) cells were selected to be analyzed. In addition to the BAL cells, the liver and glandular stomach was included in the analysis. The liver was analyzed since it is the site of primary metabolism (and potential systemic genotoxicity), while the glandular stomach was included due to possible secondary exposure after clearance of 4,4’-MDI via the mucociliary escalator (i.e. local effects at a secondary site of contact). Groups of 12 Wistar rats were exposed to actual concentrations of 2.5, 4.9 and 12 mg/m³ for 6 hours with the maximum dose selected from pilot range-finding study and previous studies as a concentration that will induce significant local cellular damage (Hotchkiss et al., 2017).  No DNA damage was noted for all three investigated tissues (BAL cells; liver; stomach) indicating that 4,4’-MDI is not genotoxic at the portal of entry at exposures of up to the maximum tolerated concentration, as indicated by local cytotoxicity. 

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

The study (exposure technology) was performed according to OPPTS guideline 870.1300 as far as applicable to this particular study. The OPPTS guideline 870.5.395 was used for the evaluation in the bone marrow micronucleus assay. It meets also OECD Guideline 474.

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

rat

Strain:

Brown Norway

Sex:

male

Details on test animals or test system and environmental conditions:

Age at study initiation: animals were approx. 2 months old
Weight: the variation of individual weights did not exceed +/- 10% of the mean.

Route of administration:

inhalation

Vehicle:

none

Details on exposure:

MODE OF EXPOSURE: inhalation; were either exposed whole-body to conditioned air (negative control) or respirable MDI-aerosol at actual breathing zone concentrations of 9.2+/- 1.5 and 118 +/-8.6 mg/m3. One additional group of rats was exposed to 110 +/- 14.4 mg/m3 using a directed-flow nose-only mode of exposure utilizing a more refined aerosol generation technology.

AEROSOL GENERATION: In whole-body exposed rats the principles of aerosol generation used by Siegel (1999) were duplicated (MDI condensation aerosol formed by bubbling air through an impinger containing MDI monomer at 125°C) whilst in the nose-only exposed group a combined dispersion-condensation aerosol principle was devised (dispersion aerosol from heated MDI @80°C and evaporation/re-condensation under controlled conditions). In whole body exposed rats the mean mass median aerodynamic diameter (MMAD) was in the range of 2.4 - 3.1 gm (GSD 1.6) whereas in nose-only exposed rats it was 1.2 gm (GSD 1.5). In addition to gravimetric analyses, the nitro-reagent and the modified Marcali methods were used to characterize exposure atmospheres. Though both methods showed somewhat lower concentrations when compared to the gravimetric methods, it does neither appear that the principle of aerosol generation nor that the different humidity (whole-body: ab. 40%, nose-only: ab. 5%) appeared to have any impact on the chemical stability of airborne MDI.

SAMPLING TIME: Bone marrow smears were prepared from rats sacrificed 1, 2 and 7 days after cessation of exposure.

Duration of treatment / exposure:

1 hr/day, 1 exposure per week, 3 consecutive weeks

Frequency of treatment:

one hour/day, one exposure per week on three consecutive weeks

Post exposure period:

IN-LIFE OBSERVATIONS: Body weights were recorded prior to exposure weekly, clinical signs before and after each exposure. Clinical signs on exposure-free days: once/day. Rectal temperature measurements: before, directly after, and approx. 2-hrs after cessation of each exposure.

Dose / conc.:

7 mg/m³ air (nominal)

Dose / conc.:

113 mg/m³ air (nominal)

No. of animals per sex per dose:

4 groups of young adult male Brown-Norway rats (Strain: BN/RijHsd); 6 per serial sacrifice 1, 2 and 7 days after last treatment/exposure in all but in the negative control group (day 1 only)

Positive control(s):

a) for clastogenic effects; cyclophosphamide (20 mg/kg, gavage) b) for spindle Poison effects - Colcemid (4 mg/kg, i.p.) Negative control; Conditioned, dry filtered air (whole-body exposure)

Details of tissue and slide preparation:

SACRIFICE: 24, 48 hours and one week after last exposure (pentobarbital anesthesia).

TISSUE PREPARATION: Bone marrow from femur by aspiration with fetal bovine serum (FBS-solution); 5 replicate slides/animal were prepared and allowed to dry overnight. The slides were fixed with absolute methanol for 5 minutes. After completion of the in-live phase of study the slides were shipped to Dr. Bhaskar Gollapudi for staining and evaluation.

CELL STAINING: Acridine Orange and Wright-Giemsa (from duplicate slides) SCORING: 200 erythrocytes for PCE:NCE per animal and 2000 PCE per animal (coded and blind), Mast cells: 1000 nucleated cells per animal for determining the mast cell incidence.

FIXATION AND WEIGHT OF LUNGS: Lungs were fixed by instillation of formaldehyde fixative and stored in the same fixative for possible future histopathological examination. Prior to fixation, after exsanguination of rats the lung weights were determined.

Statistics:

One-way ANOVA with Tukey post-hoc test following arcsine-square root transformation, if necessary to achieve normal distribution of data

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Effects related to respiratory irritation were noted. No systemic effects were observed.

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

- All rats exposed to 0 (air) and 9.2 mg MDI/m3 tolerated the whole-body exposure without specific signs whilst the 118 and 110 mg MDI/m3-exposed rats experienced labored and irregular breathing patterns, bradypnea and a serous nasal discharge. Mortality did not occur in any group. Apart from the 'irregular breathing pattern' both the incidence and intensity of clinical findings were almost identical in whole-body and nose-only exposed rats. The signs observed were related to respiratory tract irritation.

- Comparisons of control and MDI-exposed rats did not reveal any statistically significant effect on body weights, though there was a tendency of slightly decreased body weight gains in nose-only exposed rats.

- Statistical analysis of measurements of rectal temperatures revealed a significant hypothermia in the nose-only exposed rats.

- in nose-only exposed rats, lung weights were increased to a greater extent than in the in whole-body exposed rats. These findings are taken as indirect evidence that the nose-only mode of exposure provides a more rigorous means the expose rats to MDI. In whole-body exposed rats measures were taken to prevent their huddling together or burying their noses into the fur of neighboring rats in order to minimize their exposure to irritant particulates. Nonetheless, the irritant-related, reflexively induced hypothermia, associated with elevated lung weights , was only discernible in nose-only exposed rats.

- At no time point was there evidence of any effect on the frequency of mast cells. The frequency of mast cells was low enough not to interfere with the outcome of test. Rats treated with the positive control substances displayed increased MN-PCE on sampling days 1 and 2, but not on day 7. At no time point there was any evidence of an effect on the frequency of MN-PCE in MDI-exposed animals. -No differences in outcome existed following staining with the DANN-specific Acridine -Orange or Wright-Giemsa. These results indicate that, under the experimental conditions used, aerosolized MDI did not induce cytogenetic damage in vivo.

Conclusions:

The current study is used (Pauluhn and Gollapudi 2001) as source record for read-across to the target substance MDI MT. The study data did not indicate a mutagenic potential. The guideline inhalation rat micronucleus study conducted indicate that aerosolised, inhaled 4,4’-MDI at concentrations as high as 118 mg/m³ air, (a concentration high enough to produce portal-of-entry specific toxic effects, including statistically significantly increased lung weights), failed to induce formation of micronuclei and cytogenetic damage in vivo.

Executive summary:

Pauluhn et al. (2001) conducted a micronucleus study under GLP. Rats were exposed via both whole body and nose only to 4,4’-MDI for 1 hour per week for 3 weeks, with bone marrow examinations at one and two days post-exposure at concentrations up to 118 mg/m³. Although toxic effects at the portal of entry (e.g. respiratory distress, increased lung weights) were observed, there was no evidence of an MDI-induced increase in the frequency of MN-PCE at any of the time points selected. MN-PCEs were significantly increased in rats treated with the positive control when compared to both the negative control and MDI-exposure groups.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

The source substances 4,4’-MDI and target substance MDI MT contain the reactive NCO group  and therefore a similar toxicokinetic response is indicated. The available genotoxic study data and the hypothesized underlying mechanism clearly demonstrate that the source substances and target substance are not expected to be mutagenic which is supported by reliable Ames testing data which give negative responses. Taken together, the available data indicate that MDI substances lack genotoxic potential both at the site of contact and systemically and is consistent with the overall hypothesis that effects are driven by the toxicokinetic activities at the site of contact. Specifically for mutagenicity, this mechanism is reflected by a) the reactive NCO groups on the MDI substances is unavailable in the cell cytosol and MDI-adducts have not been found to be reactive with DNA either with cells at the site of contact (BALC) or systemically (bone marrow and blood cells), and b) absorption and metabolism of MDI substances occurs without detectable formation of the mutagenic diamine (MDA). As reactive NCO groups are a common feature of source substances and target substance, it is predicted that these have a similar reactivity profile with GSH and proteins and not available to react with DNA.  Therefore, the target substance MDI MT is not classified for genotoxicity according to Regulation (EC) No.1272/2008 (CLP Regulation).

Justification for classification or non-classification

According to Regulation (EC) No.1272/2008 (CLP Regulation) the non-classification of 4,4'-MDI (CAS No.101-68-8) is considered for the non-classification of MDI MT (CAS No.147993-65-5): 4,4'-MDI was not classified as a germ cell mutagen by Regulation (EC) No. 1272/2008 (CLP Regulation) (there is conclusive data but it is not sufficient for classification). MDI MT was not mutagenic in the Ames test.