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EC number: 701-173-1
CAS number: -
The test substance is covered by the category approach of
methylenediphenyl diisocyanates (MDI).
Hence, data of the category substances can be used to cover this
endpoint. The read-across category justification document is attached in
IUCLID section 13. It is important to note that the MDI category
approach for read-across of environmental and human hazards between the
MDI substances belonging to the MDI category is work in progress under
REACH. Therefore the document should be considered a draft.
The MDI is harmful by inhalation according to EU (H332; R20) and GHS
(Cat. 4) classification. The MDI is non toxic after single oral and
animal studies demonstrated that MDI has low acute oral toxicity.
In the available acute oral toxicity key study (OECD Guideline 425)
(Mallory, 2009), one female Sprague Dawley rat was dosed at 175 mg/kg.
No mortality was observed at 175 mg/kg and dosing continued in another
female at 550 mg/kg. Dosing continued in an additional female at 2000
mg/kg as per protocol guidelines and then in one female at 5000 mg/kg.
Based on these results two additional females were dosed at 5000 mg/kg.
A total of 6 females were dosed. Mortality checks were made once daily.
Clinical observations were recorded prior to dosing, as well at 30
minutes, 4 hours post-dose, and daily thereafter through Day 15. Body
weights were recorded on the day of dosing (Day 1), and on Days 8 and
15. All rats were euthanized by CO2 asphyxiation and necropsied on Day
1. For doses of 175, 550, 2000 or 5000 mg/kg, no mortality was observed.
All animals appeared normal throughout the study at 175, 550, 2000 and
5000 mg/kg. No biologically significant effect was seen on body weights
on Days 8 and 15. Terminal necropsy revealed no visible lesions in any
of the animals at 175, 550, 2000 and 5000 mg/kg. Based on these
LD50>5000 mg/kg was determined.
supporting study describing the acute oral toxicity of MDI conducted as
a limit test according to EU guideline (84/449/EEC) with GLP did not
find any mortality up to the maximum dose tested (Bomhard, 1990). The
acute LD50 was found to be > 2000 mg/kg bw. Another supporting study
describing the acute oral toxicity of polymeric MDI (pMDI) conducted
similar to OECD 401 guideline (Reliability 2) also did not find any
mortality up to the maximum dose tested, hence the LD50 is > 10000 mg/kg
bw (Wazeter, 1964). Other studies on MDI are consistent with this.
to acute oral toxicity data, the available animal studies demonstrated
that the MDI has low acute dermal toxicity. The key study describing the
acute dermal toxicity of pMDI in rabbits did not find lethality up to
the maximum dose tested and the LD50 was > 9400 mg/kg bw (Wazeter et
al., 1964). Other studies on pMDI or 4,4’-MDI are consistent with this.
inhalation toxicity: Mortality studies
key guideline acute inhalation study (OECD 403) was performed in male
and female rats at five exposure concentrations of 4,4-MDI (Pauluhn,
2008). The animals were nose-only exposed for 4 hours to liquid aerosol
in concentrations of 0, 300, 354, 399, 500 and 554 mg/m3 (analysed
conc.), with a post exposure observation period of two weeks. Mass
median aerodynamic diameter ranged between 2.1 and 3.5 µm across all
groups. Male rats were apparently more susceptible than females. All MDI
exposure groups exhibited clinical signs consistent with respiratory
irritation, so the NO(A)EC was <300 mg/m3. Mortality occurred in a
concentration related manner at 354 mg/m3 and above. The LC50 for male
rats was calculated as 368 mg/m3 (95% confidence interval 296-458 mg/m3)
and for females approximately 559 mg/m3.For both sexes combined, the
LC50 was 431 mg/m3.
acute inhalation study was performed in rats at only one concentration
level of 2.24 mg/L/1h (Pauluhn 2003). This study was specifically
designed to comply with NFPA 704, and also complied with the limit test
of the OECD guideline 403 with deviations (only 1 hr exposure,
concentration lower than limit test concentration) and is therefore
reliable with restrictions. Exposure of 4,4’-MDI for 1 hr resulted in
mortality shorty after exposure of one out of ten rats. Clinical signs
were characterised by typical signs of respiratory tract irritation.
Necropsy findings were unremarkable in surviving rats, whilst the rat
that succumbed displayed signs of lung oedema which was considered to be
the cause of death. The LC50 >2.24 mg/L/1h (analytical) in both males
and females was determined.
inhalation toxicity: Sub-lethal Mode of Action studies
a study by Pauluhn (2000), Wistar rats were acutely (6 hours) exposed
nose-only to pMDI (0, 0.7, 2.4, 8 or 20 mg/m3) and markers for lung
injury were evaluated in bronchoalveolar lavage fluid (BALF) or cells
(BALC) at various times up to 7-days post-exposure. The data provide
evidence for an immediate (0 hours post-exposure) loss in barrier
function and a change in surfactant homeostasis at all concentrations
tested (≥0.7 mg/m3); these effects returned to control levels by 3 days
post-exposure. In contrast, evidence of cytotoxicity (LDH) and oxidative
stress (GSH) were seen at exposure concentrations ≥8 mg/m3.
acute inhalation toxicity of the 4,4-MDI to male rats was investigated
to characterize the mechanism of local effects in the lung following an
acute inhalation exposure (Hotchkiss, et al. 2017). Groups of 12 Wistar
rats were exposed to 4, 12 and 27 mg/m3 (analytical concentrations,
determined by gravimetry) to an aerosol-generated form of the test
substance via a single nose-only inhalation exposure for 6 hours. A
concurrent control group received filtered air on a comparable regimen.
Half of the rats in each exposure group were euthanized immediately
after exposure with the remaining rats in each group euthanized
approximately 18 hours later. At necropsy bronchoalveolar lavage was
performed on all experimental animals. The lavage fluid supernatant was
analysed for biomarkers of injury and inflammation (total protein, LDH,
alkaline phosphatase, β-glucuronidase) and oxidative stress (GSH and
GSSG levels). The lavage cells were analysed for total and differential
cell counts, targeted gene expression (including signals for
inflammation, macrophage activation, apoptosis, and oxidative stress),
and markers of apoptosis (Annexin V and Caspase-3). All animals survived
to the scheduled euthanasia and there were no test substance-related
clinical observations or effects on body weight.
increases in total protein and β-glucuronidase was noted at the end of
the exposure and 18 hours post-exposure was noted at ≥ 4 mg/m3 which was
statistically significant in the high exposure group.
increases in LDH activity were detected at the end of exposure and 18
hours post-exposure (statistically significant in 4 and 27 mg/m3
ratio of GSH/GSSG was reduced (indicative of oxidative stress) compared
to controls at the end of exposure at ≥4 mg/m3. GSH/GSSG ratio remained
lower than control rats in 12 and 27 mg/m3 exposed rats at 18h but was
slightly elevated in rats exposed to 4 mg/m3 due to a significant
increase in GSH levels
inflammation was noted at > 12 and 27 mg/m3 immediately and was still
evident at 18hr post-exposure.
concentration-dependent increase in % Annexin (+) / PI (-) cells in
nucleated BAL cellswas
observed in 4,4’-MDI exposed animals immediately after exposure and 18
summary, a single 6 hour exposure to respirable aerosols of 4,4’-MDI to
4, 12, and 27 mg/m3 resulted in concentration- and time dependent
increases in oxidative stress, inflammation, and markers of apoptosis.
There was some evidence of inflammation, oxidative stress, and/or
apoptosis at every dose, although the clearest effects were observed in
the 12 and 27 mg/m3 exposures and especially by 18 hours post exposure.
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 (Randazzo, 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.
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.
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.
increases in LDH at ≥ 2.5 mg/m3 at 1h timepoint. At the 18h timepoint,
LDH was only increased in BALF at 12 mg/m3.
dose-dependent increases in total protein was observed ≥ 2.5 mg/m3 at
both time points.
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.
increased of late apoptosis and necrotic cells was observed 1 h
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
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.
inhalation toxicity: discussion
Mode of Action studies:
together, data from non-lethal acute inhalation studies indicate that a
disruption of surfactant homeostasis is an early event in the pulmonary
response to the deposition of MDI and occur at relatively lower MDI
concentrations (NOAEC 0.7 mg/m3). Aerosols of MDI deposited in the
alveolar epithelial lining fluids (surfactant) react with macromolecular
nucleophiles (e.g. GSH, proteins, peptides), and when exposure
concentration and/or duration is sufficient, the nucleophilic capacity
of this layer becomes overwhelmed and deterioration of cell membranes
and cytotoxicity occurs (e.g. total protein, LDH, apoptosis). In
addition to the disruption of the surfactant homeostasis, reaction
products of the MDI with alveolar macromolecules are phagocytised by
activated macrophages. When activated, these macrophages release
pro-inflammatory cytokines which recruit neutrophils (with a possible
subsequent oxidative burst and reactive oxidative species production).
The available data demonstrate early indications for toxic lung effects
such as inflammation / cytotoxicity (e.g. lactate dehydrogenase (LDH),
Annexin V expression) and oxidative stress (e.g. BALF glutathione (GSH)
levels) at concentrations ≥8mg/m3and with consistent
moderate toxicity at ≥12mg/m3. This suggests that acute exposures to MDI
concentrations ≥ 12mg/m3 results in significant portal of entry cellular
should be noted that in an analysis of acute and chronic inhalation
studies with MDI, Pauluhn (2011) concurred with the report by Feron et
al. (2001) that the pulmonary effects seen in chronic investigations are
but rely on both the concentration used and the time of exposure, i.e. C
x T (day) rather than being concentration dependent. Using C x T (day)
as a metric, Pauluhn (2011) showed that pulmonary effects thresholds
from acute and short term inhalation studies are more predictive of
effects in the chronic investigations than concentration per se without
any evidence of any cumulative dose effects in the chronic studies.
classification, there were two well conducted, guideline studies, with
reliably generated aerosol atmospheres and analysed concentrations taken
into account (Appelman and de Jong , 1982a&b, Pauluhn, 2008). It was
preferred to use these studies with reliable valid data rather than
taking a mean of data reported in databases which do not critically
assess the experimental set-up and analysis of the test-atmosphere. The
GHS guidance has cut off values for Category 1 of 0.5 mg/L (500 mg/m3)
for vapours and 0.05 mg/L for aerosols. GHS guidance section 22.214.171.124
note (d) states that where vapour and aerosol exist together the cut of
value for Category 1 should be 100 ppmV. The result of Appelman and de
Jong (1982a, b) is an LC50 of 490 mg/m3 (0.49 mg/L), which falls just
under the cut off for GHS Category 1 for vapours and mixed
vapours/aerosols, but would be Category 2 for aerosols. Similarly the
Pauluhn (2003,2004) study result of LC50>2.24 mg/L and Pauluhn (2008)
study result of 431 mg/m3 would also result in Category 2 (aerosol).
of chemicals allows for the application of scientific judgement. It must
be taken into account that the LC50 cut-off of 500 mg/m3 (approximately
50 ppm for pMDI), is over 2500-fold above the saturated vapour
concentration for pMDI. At the saturated vapor concentration MDI has no
effect on animals. Furthermore the aerosols were generated using
sophisticated techniques in thelaboratory
(often involving heating), were ofextremely
small particle size only in order to meet international guidelines for
testing of aerosols, and this sort and concentration of aerosol is not
generated in the workplace (ISOPA, 2015). In spraying applications
aerosols are formed where the particle size distribution has virtually
no overlap with that of the highly respirable aerosol generated in
inhalation studies (see EU Risk Assessment Report on methylene
diphenyldiisocyanate, EINECS-No. 247-714-0, 2005). In addition the EU
legislation for classification and labelling of chemicals, the
67/548/EEC Substances Directive in Article 1(d) makes it clear that the
object of classification is to approximate the laws of the Member States
in relation to substances dangerous to man or the environment. In
Article 4 in points 1 and 2 it is clearly stated that substances shall
be classified on the basis of their intrinsic properties according to
the categories of danger as detailed in Article 2(2) and that the
general principles of classification shall be applied as in Annex VI.
Intrinsic properties are those inherent in the substance. MDI is not
inherently toxic by inhalation, as evidenced by its lack of any effect
at the saturated vapour concentration. It is only with modification and
input (in terms of heat, cooling and size screening) that MDI becomes
toxic after inhalation. The European Chemical Industry Council have
discussed and given guidance for situations like that, and on the
classification of respective aerosols (attached in 7.2.2 'Acute Toxicity
Endpoint Summary Attached Documents'). Classification of MDI as
“Harmful” is consistent with this guidance.
Appelman and de Jong (1982a&b) data were considered by EU experts and
their conclusion that MDI be classified as “Harmful” (Xn, R20) is
reported in the 25th Adaptation to Technical Progress (ATP) to the
Dangerous Substances Directive (67/548/EEC). This was endorsed in the
28th ATP and MDI remains as “Harmful” in the 30th ATP ( adopted by
Member States on 16 February 2007 and published 15th September 2008).
The original decision was upheld in the EU Risk Assessment of MDI
(Directive 793/93/EEC, 3rd Priority List) published in 2005, noting that
considering “the exposure assessment, it is reasonable to consider MDI
as harmful only and to apply the risk management phrase ‘harmful by
inhalation’. This classification was also endorsed by the Scientific
Committee on Toxicity, Ecotoxicity and the Environment (CSTEE, now
SCHER) in giving their opinion on the Risk Assessment. This
classification is equvialant to H332 (Harmful if inhaled) under CLP
principle is considered to apply equally to the Appelman and de Jong
(1982a&b) study and the Pauluhn (2008) results.
classification as “Harmful”, is equivalent to GHS Category 4. For these
reasons, the GHS proposal follows the EU Regulatory lead accepting that
the animal data are inappropriate and classified pMDI as GHS acute
toxicity category 4 (ISOPA 2007).
of the available acute toxicity data indicates that inhalation exposure
to the aerosols of MDI results in toxicity confined predominantly to the
respiratory tract. In terms of hazard characterization, MDI is harmful
by inhalation according to EU (H332)
and GHS (Cat. 4) classification. MDI is non-toxic after single oral and
EU classification according to CLP: H332
GHS classification (GHS UN rev.2, 2007):
Inhalation route (vapour): Acute Category 4.
Not toxic by the dermal or oral routes.
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