Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2015
Reliability:
1 (reliable without restriction)

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
GLP compliance:
yes (incl. QA statement)
Test type:
traditional method

Test material

Constituent 1
Reference substance name:
4,4'-Methylenediphenyl diisocyanate, oligomeric reaction products with butane-1,3-diol, 2,4'-diisocyanatodiphenylmethane, [(methylethylene)bis(oxy)]dipropanol and propane-1,2-diol
EC Number:
500-312-1
EC Name:
4,4'-Methylenediphenyl diisocyanate, oligomeric reaction products with butane-1,3-diol, 2,4'-diisocyanatodiphenylmethane, [(methylethylene)bis(oxy)]dipropanol and propane-1,2-diol
Cas Number:
123714-19-2
Test material form:
liquid: viscous
Details on test material:
Batch # 290832

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Species and species justification: The study was carried out in rats, a rodent species recommended in the test guidelines.

The study was in compliance with the Directive 201 0/63/EU, the Guideline of the Council dated September 22, 2010 on the Reconciliation of Legal and Administrative Regulations of the Member Countries for the Protection of Animals used for Studies and other Scientific Purposes, the Journal of the European Community, Legal Specifications L276, pp. 33-76 as well as the German Tierschutzgesetz and the appendant executive order law in the actual version. The general scope of study has been reviewed by Animal Management (Bayer Pharma AG, Research Center Aprath, 42096 Wuppertal, Germany) and the responsible German Authority. Healthy young adult SPF bred Wistar rats, strain Cri:(Wi)WU BR (SPF), from the experimental animal breeder Charles River, Sulzfeld, Germany were used. Animals of this strain have been used at Bayer Pharma AG in toxicological studies for years. Historical data on their physiology, diseases and spontaneous alterations are available. The state of health of the strain is randomly checked regularly at the instance of the Laboratory Animal Services, Bayer Pharma AG, for the most
important specific infectious pathogens. The results of these examinations are archived.

Acclimatization: The animals were acclimatized to the animal room conditions for at least 5 days before use. During this period, rats were also acclimatized to the restraining tubes.

Identification: Animals were identified by both individual color-marking and cage labels. All animals from this study were located on one cage-rack.

Randomization: Before the start of the study the health status of each animal was assessed. Animals were subsequently assigned to exposure groups at random (randomization procedure is described in section 6.17).

Health status: Only healthy rats free of signs were used for this study. The animals were not vaccinated or treated with anti-infective agents either before their arrival or during the acclimatization or study periods. The females were nulliparous and not pregnant.

Age and weight: At the study start the variation of individual weights did not exceed ± 10 per cent of the mean for each sex. Animals of the weight class used are approximately 2 months old and hence fulfill the criterion for young adults.

Animal housing: During the acclimatization and study periods the animals were housed singly in conventional Makrolon® Type IIIH cages with gnawing sticks(specification is documented in the raw data). All cages and gnawing sticks are changed twice a week while unconsumed feed and water bottles are changed once per week. The legal requirements for housing experimental animals (Directive 201 0/63/EU) are followed.

Bedding: Bedding consisted of low-dust wood granulate type Ssniff BF1 low-dust wood granulate from Brandenburg Holzfaserstoffe, Goldenstedt, Germany.

Animal rooms: All animals were housed in a single room. For reasons of space availability rats from other acute toxicity studies were housed in the same room, however mistakes in animal assignments were excluded by adequate spatial separation, clear cage labeling, and appropriate organization of all work procedures. The housing of several studies in one animal room is not considered to be a deviation from current GLP-requirements since many acute studies comprise of 10 animals only (as required to perform a limit test).

Environmental Conditions in the Animal Room
The animal room environment was as follows:
Room temperature: 22 ± 3 oc
Relative humidity: 40-60%
Dark/light cycle: 12 h/12 h; artificial light from 6.00 a.m. to 6.00 p.m. Central
European Tim
Light intensity: approximately 150-200 Lux
Ventilation: approximately 10 air changes per hour


The room humidity and temperature were continuously monitored and documented using a calibrated thermohygrograph. Occasional deviations from these conditions occurred, e.g. as a result of animal room cleaning, but these had no detectable influence on the outcome of this study.

Cleaning, disinfection, and pest control: The animal room was regularly cleaned and disinfected once a week with aqueous solution of TEGO® 2000. Contamination of the feed and contact with the test system were excluded. Pest control measures using pesticides were not taken in the animal room.

Feeding: Ration consisted of a standard fixed-formula diet (ssniff® R/M-H pellets maintenance diet for rats and mice; ssniff Spezialdiaten GmbH, http://www.ssniff.de) and tap water (drinking bottles). Both food and water were available ad libitum. The pelletized feed was contained in a rack in the stainless-steel wire cage cover. The nutritive composition and contaminant content of the standard diet was checkedregularly by random sampling by the Laboratory Animal Services, Bayer Pharma AG, Wuppertal. Details concerning general feed specification are provided in the
Appendix.

Water: Drinking quality municipality tap-water (current versions of the Drinking Water Decree (TrinkwV)) was provided ad libitum in polycarbonate bottles containing approximately 300 ml (based on A. Spiegel and R. Gonnert, Zschr. Versuchstierkunde, 1. 38 (1961) and G. Meister, Zschr. Versuchstierkunde, z. 144-153 (1965)). The results of feed and water analyses are retained by Bayer Pharma AG, Wuppertal. The available data provided no evidence of an impact on the study objective.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose/head only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 2.02 - <= 2.15 µm
Geometric standard deviation (GSD):
>= 1.55 - <= 1.57
Duration of exposure:
4 h
Concentrations:
349, 548 and 711 mg/m3 (based on gravemetric determination)
No. of animals per sex per dose:
5
Statistics:
Body weights: Means and single standard deviations of body weights are
calculated. Mean body weights are also depicted graphically as a function of time
(see result section). Since in acute studies individual group means may differ prior to
commencement of the first exposure, the body weight gain was statistically evaluated
for each group. For these evaluations a one-way ANOVA (vide infra) is used.
Particle size analysis: The statistical methods used in the evaluation of the particlesize distribution were described in Section 6.8.
Physiological data: Data of rectal temperature measurements are statistically
evaluated using the ANOVA procedure (vide infra).
Randomization: A computerized list of random numbers served the purpose to
assign animals at random to the treatment groups.
Calculation of the LCso: It is performed by computer (PC) according to the method
of Rosiello et al. (1977) as modified by Pauluhn (1983). This method is based on the
maximum-likelihood method of Bliss (1 938). If only 2 pairs of values with greater than
0% lethality and less than 1 00% are available then the first linear approximation is
based on these values and a x2-homogeneity test is not performed. In this case the
interpolated concentration at 50% lethality is designated the approximate LCso.
Analysis of variance (ANOVA): This parametric method checks for normal
distribution of data by comparing the median and mean. The groups are compared at
a confidence level of (1-a) = 95 % (p = 0.05). The test for the between-group
homogeneity of the variance employed Box's test if more than 2 study groups were
compared with each other. If the above F-test shows that the intra-group variability is
greater than the inter-group variability, this is shown in the Appendix as "no statistical
difference between the groups". If a difference is found then a pairwise post-hoc
comparison is conducted (1- and 2-sided) using the Games and Howell modification
of the Tukey-Kramer signifi

Results and discussion

Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
518 mg/m³ air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
Mortality occurred at 349, 548 and 711 mg/m3 in a dose dependent manner. The
mortality patterns were typically of a lung edema (e.g.: nose: white foamy discharge;
trachea: white foamy content, lung: less collapsed, cyanosis, labored breathing,
dyspnea, breathing sounds, stridor). These findings are reflected as indicator for
pulmonary irritation. Furthermore necropsy lung findings as e.g. dark-red marbled
and/or brown-red marbled lungs as well as many hepatoid areas and/or many firm
areas in the lungs are considered as atelectasis due to the sticky physic-chemical
properties of the test item.
Body weight:
Significantly decreased body temperatures and incremental body weight gain found
at 349 mg/m3 and above, are reflected as indicators of a deteriorating health
condition. .
Gross pathology:
Necropsy findings were unremarkable in surviving rats whereas in rats that
succumbed in the course of study the following findings of toxicological importance at
349 mg/m3 and above were observed (e.g. nose: red encrusted, white foamy
discharge, firm yellow deposits; trachea: white foamy content; lung: less collapsed,
dark-red marbled, brown-red marbled, many hepatoid areas, many firm areas; Gltract: bloated; glandular stomach: mucosa reddened; intestines: yellowish mucous content, bloated; small intestine: mucosa reddened; liver: many light-colored areas;
spleen: light colored, reduced in size; heart: hardened, reduced in size, ventricles
exsanguinous, ventricle walls thickened; abdominal aorta: lumen dilated, kidneys:
renal pelvis bilaterally reddened, thymus: dark-red areas).
Other findings:
Rectal temperature
4,4'-MDI/1 ,3-BD/TPG/PG
Results of the evaluation of the rectal temperature are summarized in the Appendix
and Figure 5. Mean values are shown in Figure 5. Statistical comparisons between
the control and the exposure groups revealed significant changes in body
temperature at 349 mg/m3 test item and above.

Any other information on results incl. tables












































































Group/sexTarget Conc.Toxicological resultOnset and duration of signsOnset and duration of mortalitymean rectal temperture

1/m


00/0/5----37.5
2/m4001/5/50d -8d1d*31.7
3/m5503/5/50d-14d

0d (5h*, 8hr*)


1d (<24hr)


29.3
4/m7004/5/50d-10dd (7h*, 8h*)
1d*, 2d*
29.9
1/f00/0/5----37.9
2/f4001/5/50d -8d1d*31.6
3/f5503/5/50d-14d

0d (6h*, 7hr*)


30.0
4/f7003/5/50d-14d0d (7h*, 7h*)
1d*, (<24h)
29.6

N = group assignment, m = males, f = females, Od = day of exposure
* = animals moribund euthanized according to OECD TG 403
Values given in the 'Toxicological results' column are as follows:
1st number = number of dead animals
2nd number = number of animals with signs after exposure cessation
3rd number = number of animals exposed

Applicant's summary and conclusion

Interpretation of results:
other: expert judgment with acute tox 4 H332 EU GHS 1272/2008 CLP classification
Conclusions:
n summary, there is evidence of portal-of-entry toxicity in rats after exposure (4h) of
aerosolized test item. The calculated LC5o for males & females combined is
518 mg/m3 test item. There was no gender specific susceptibility observed.
Executive summary:

The respirable aerosol of test substance had a moderate acute inhalation toxicity to rats. The signs observed demonstrated marked respiratory tract irritation with mortality associated with lower respiratory tract irritation (alveolar edema). Such lower respiratory tract effects are dependent on a highly respirable aerosol not encountered in the workplace, which needs to be taken into account for classification.


Using the strict GHS LC50 cut-off for classification, the LC50 values obtained for the test substance would trigger a Category 3. However, classification for these substances according to GHS legal text allows for the application of scientific judgement. It must be considered that the LC50 cut-off is well above the saturated vapor concentration for the test material.


Furthermore, the aerosols were generated using sophisticated techniques in the laboratory, whereby extremely small particles are generated in order to meet international guidelines for testing. This size and concentration of aerosol is not generated in the workplace even under foreseeable worst-case conditions (Ehnes et al., 2019). The particle size distribution of aerosols formed during actual spraying applications has virtually no overlap with that of the highly respirable aerosol generated in inhalation studies (see EC (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 based on 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. Due to a very low vapor pressure (<0.01 Pa) MDI substances are not inherently toxic by inhalation since the saturated vapor concentration would be orders of magnitude below toxic concentration. It is only with modification and input (in terms of heat, cooling and size screening) that MDI substances become toxic after inhalation. The European Chemical Industry Council have discussed and given guidance for these situations, and on the classification of respective aerosols. Classification of MDI as “Harmful” is consistent with this guidance.  


The acute inhalation data of the worst-case substances in the MDI category (pMDI and 4,4’-MDI) were considered by EU experts, and their conclusion that MDI be classified as “Harmful” and  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 both MDI substances remain 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 (EC, 2008). With the enforcement of the CLP regulation (Regulation (EC) No 1272/2008) in 2009, the Dangerous Substance/Preparation Directive (DSD) was repealed and harmonized classifications were formally transferred to the CLP regulation; MDI is classified with Acute Tox. 4 H332 (Annex VI Regulation (EC) No 1272/2008 (CLP regulation).  According the MDI Category hypothesis described in detail in the Category Justification Document, MDI/1,3-BD/TPG/PG is also classified as a Acute Tox. 4 H332.  

Categories Display