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extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study planned
Justification for type of information:

- Name of the substance on which testing is proposed to be carried out: 4,4'-MDI/DPG/HMWP

- Available GLP studies: There are no GLP-compliant fertility toxicity studies available for MDI (members for MDI/ of the MDI group approach ), neither screening for reproductive/developmental toxicity (OECD 421 or 422) Annex VIII 8.7.1. or Extended One-Generation Reproductive Toxicity Study (B.56 of the Commission Regulation on test methods as specified in Article 13(3) or OECD 443) Annex X 8.7.3. An additional screening study (OECD 422) will be performed on the test material to define the study design and as a basis for bridging to the other substances within the MDI category.
- Available non-GLP studies: There are no non-GLP-compliant fertility toxicity studies available on the substance.
- Historical human: There are no appropriate historical human data that address the fertility toxicity endpoint on the substance.
- (Q)SAR: There are no QSAR models available for this higher tier human heath endpoint that are sufficiently validated and acceptable.
- In vitro methods: There are no validated tests that use in vitro methodologies that could be used to meet the standard requirement of the REACH regulation for fertility toxicity.
- Weight of evidence: The available repeated dose toxicity study, addressing effects on reproductive organs were found not sufficient to meet REACH standard requirement for fertility because fertility toxicity is not sufficiently examined in the sub-chronic and chronic repeated dose toxicity studies available for MDI.
- Grouping and read-across: The substance is part of a grouping category that uses a 2-generation reproductive study an analogue substance read-across. However as no test is available on a substance within the group, this test will support lack of reproductive effects within the category.
- Substance-tailored exposure driven testing [if applicable] – Not applicable
- Approaches in addition to above [if applicable] Not applicable
- Other reasons [if applicable] – Not applicable
- none of the adaptation possibilities in REACH Annex VIII to X is adequate to generate the necessary information or waive an extended one-generation reproductive toxicity study. Relevant human exposure cannot be excluded as the substance is used in an industrial setting under non-closed conditions.

The substance is part of a category read-across based on the hypothesized MoA that predicts local effects in the lungs and no significant systemic exposure to unreacted NCO since it reacts with biological nucleophiles before being absorbed as GHS/protein adducts. With no systemic exposure to unreacted isocyanate or toxic metabolite, no effects on fertility are predicted. This is justified by weight of evidence is based on three independent sources of information including a) Absence of effects on reproduction and fertility in a guideline two-generation reproductive toxicity study on an analogue aromatic diisocyanate TDI, b) Evidence for a lack of systemic availability of toxicologically active parent or metabolite, and c) A lack of systemic toxicity and effects on reproductive organs in repeated dose toxicity of representative MDI substances and TDI. As all substances of the MDI category as well as the analogous substance TDI contain significant quantities of bioaccessible NCO groups required for the hypothesized MoA.
However, as no fertility data from the required test guideline for Annex X is available for any category test substance, the current study is proposed on the boundary substances (4,4’-MDI and 4,4’-MDI/DPG/HMWP) to support the common mechanism. Additional screening studies (OECD 422) will be performed on selected category members from each sub-group to act as bridging studies. These additional studies will confirm that hypothesis that additional structural features to not contribute to the systemic and reproductive toxicity and that 4,4’-MDI would still be considered the worst-case substance. If these additional studies suggest additional toxic potential, additional OECD 443 studies will be proposed.

Data source

Materials and methods

Test guideline
according to guideline
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Justification for study design:

- Premating exposure duration for parental (P0) animals:
The toxicity of MDI substances is driven by the bio-accessible NCO value which is responsible for chemical and physiological reactivity and subsequent toxicological profile. The hypothesized MoA of toxicity is the reaction of the electrophilic NCO group with extracellular biological nucleophiles such as cellular proteins and is limited to point of contact effects without subsequent systemic toxicity. Reliable inhalation data is available for 4,4’-MDI, pMDI and 4,4’-MDI/DPG/HMWP). Results from these studies are consistent with the hypothesized MoA and assertion that MDI substance toxicity after repeated inhalation exposure in animals is limited to local effects caused by inflammation and irritation to the respiratory tract without subsequent systemic toxicity. In regard to these local effects after inhalation, the lungs are the primary organ affected since olfactory lesions were only observed at higher concentrations/doses. As an MDI substance enters the lung, NCO groups react with biological nucleophiles at the MDI/lung fluid interface to form MDI-conjugates. Formation of these MDI-adducts depletes protective nucleophiles in the lung and results in pulmonary irritation and inflammatory cell influx. MDI only enters the systemic circulation in the form of MDI-GSH or protein adducts. Consequently, there is no systemic exposure to the toxic NCO functional group which is consistent with the lack of distal toxicity in any study conducted.
- Basis for dose level selection: An OECD 422 study will be performed prior to main study to serve as a dose-range finder and basis for comparing bridging studies for multiple category members. In addition, study data from existing sub-chronic and chronic studies on other category members will be used for dose selection (Hoymann et al. 1994 for 4,4’-MDI, Reuzel et al. 1994 for pMDI). These supporting studies demonstrate that even with a chronic/lifetime exposure duration with group sizes equivalent to or greater than guideline reproductive studies, effects from pMDI and 4,4’-MDI aerosols are confined to the lungs. Effects on systemic organs including reproductive organs were not observed at exposure concentrations associated with marked respiratory tract toxicity.
- Inclusion/exclusion of extension of Cohort 1B: not planned, based on reliable repeated dose toxicity study data there is no indication of distal toxicity induced by MDI, pMDI or 4,4'-MDI/DPG/HMWP see above. However, if preliminary OECD 422 studies indicate concern, additional cohorts will be added to the definitive OECD 443 study.
- Termination time for F2: not applicable, the available data indicate no specific concern regard to neuronic see above
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: not applicable, the available data indicate no specific concern regard to neurotoxicity in adult animals or to developmental neurotoxicity, see above. However, if preliminary OECD 422 studies indicate concern, additional cohorts will be added to the definitive OECD 443 study.

- Inclusion/exclusion of developmental immunotoxicity Cohort 3: not applicable, the available data indicate no specific concern with regard to immunotoxicity in adult animals or developmental immunotoxicity. However, if preliminary OECD 422 studies indicate concern, additional cohorts will be added to the definitive OECD 443 study.
- Route of administration: inhalation route
A need for an oral hazard assessment for MDI category substances is not indicated by their use or by toxicokinetic profile of exposure routes. Furthermore, oral toxicity data for MDI category substances cannot be extrapolated for risk assessment of inhaled aerosols as the relevant route of exposure for human risk assessment. Therefore, there is no apparent benefit from any oral toxicity data for MDI category substances. Toxicokinetic data for the inhalation route of exposure is sufficient, and the performance of an additional oral animal toxicity study would not create data that would influence the risk management measures and therefore would be in conflict with the principles of animal use and welfare. These differences are described below for 4,4'-MDI and also apply to 4,4'-MDI/DPG/HMWP (for details see Category Justification Document in section 13):

Route of exposure specific differences in MDI metabolism

4,4’-MDI (MDI) contains two highly reactive NCO-groups, which are responsible for the distinct portal of entry toxicity described by the available toxicological data. The NCO-group reacts readily with nucleophilic biomolecules, and depending on the chemical and physico-chemical composition of the interphase at the site of primary contact, distinct differences in primary reaction products can be described. Therefore significant differences in bioavailability and metabolic fate can be described for the oral, dermal and inhalation route of exposure (see end point summary on toxicokinetics\):
• in the pH neutral medium of the lung inhaled respirable MDI aerosols react with the proteins and peptides (mainly glutathione) of the bronchioalveolar fluid, partly representing bioavailable adducts,
• direct intubation of large MDI doses into the stomach is an artificial exposure route and can only simulate accidental swallowing. In the acidic pH of the stomach MDI polymerizes with the stomach content and forms solid and inert polyureas. Information from analogous diisocyanates and US reports on accidental ingestion of MDI based glues in domestic animals describe formation of high molecular primary reaction products with CO2 liberation, without apparent systemic chemical toxicity. Polymeric reaction products are of low bioavailability.
• Following oral swallowing of traces of MDI, reactions will commence at once with biological macromolecules in the buccal region and will continue along the oesophagus prior to reaching the stomach. Reaction products will be a variety of polyureas and macromolecular conjugates with for example mucus, proteins and cell components.
• at the interface of the skin, reactions with nucleophilic groups of the skin matrix and polymerization to a solid polyurea crust occurs, significantly reducing bioavailability. (Based on this knowledge it should only be speculated on the effects of an agglutination of the reactive MDI with blood proteins following i.v. application.)

In conclusion the toxicokinetic behaviour of MDI needs to be considered with respect to the specific physico-chemical and chemical exposure conditions at the site of first contact. For MDI, significant differences in primary reaction products and by this in the subsequent bioavailability and metabolic fate imply a high level of uncertainty for route to route extrapolation of toxicological data. In accordance to REACh Annex I (0.3.), the chemical safety assessment of a substance shall be based on a “…known or reasonably foreseeable exposure…”, accidental exposures are not considered. Therefore, and in accordance to ECHA Guidance Chapter R.7a and the REACh Annexes on information requirements the potential hazard of MDI should be determined on the most relevant route of exposure for risk assessment which is inhalation.

Relevance of oral route of exposure for risk and hazard assessment:
ECHA Guidance Chapter R.14 (Occupational exposure estimation, R.14.2 Types and routes of exposure) indicates that, exposure through ingestion is “…generally not considered further in the assessment of workplace exposure“. For proof of concept, working processes for professional uses of MDI include handling and spray application of foams in concentrations up to max 30 % pbw. Other uses include handling and application of coatings, adhesives and paints in which MDI is not contained as such but as pre-reacted high viscous polymer with low monomer content. For spray applications in which aerosols are generated respiratory protection by full mask, and for all application’s effective dermal protection by full body protection and gloves is prescribed. These risk reduction measures effectively prevent from any oral exposure, e.g. via contaminated skin or clothing or inhalation cross-contamination. Cured PU applications, e.g. spray foam, do not contain residual unreacted MDI (see Topic 6 below). In conclusion, no significant uncertainties regarding a potential oral exposure can be anticipated in the holistic exposure assessment for professionals.

ECHA Guidance Chapter R.15 (Consumer exposure estimation, R.15.2.2 Reasonable worst-case situations) indicates that, “…the consumer exposure estimation should normally address the intended uses of the products that contain the substances under investigation. However, since consumers may not accurately follow instructions for use of products, an estimation of other reasonably foreseeable uses should be made. Consideration of deliberate abuse is not part of the exposure estimation process.” Consumer uses include one component rigid foam available in cans as well as coatings, adhesives & sealants, and paintings. These uses contain pre-reacted and polymerized MDI derivatives (prepolymers and higher oligomers originating form polymeric MDI (PMDI)) with low amounts of residual monomer. Reaction is readily with humidity in the air, resulting in entirely cured product, free of residual MDI.

Consumer uses of MDI are covered by national regulations, which e.g. restrictively prescribe selling in correspondingly equipped DIY stores, if the consumer agrees to information provided by the staff addressing the risk. In addition, the appropriate storage and handling is explicitly prescribed, e.g. by the recommendation to use gloves which are delivered with every can as part of an existing restriction under REACH Annex XVII as follow up of the 2005 Risk Assessment. Therefore, there is a very low possibility for oral exposure by the intended use, since e.g. skin contact should be prevented by the use of gloves, and by this dermal to oral cross-contamination is minimized. Since cured PU applications, e.g. spray foam, do not contain residual unreacted MDI (see Appendix 1 on Toxicological information, DNEL justification provided as a separate document) migration from articles through sucking, chewing or licking can likewise be excluded. In addition, no foreseeable misuse other than abuse has to reasonably be anticipated due to specific regulations of the consumer application.

- Other considerations, e.g. on choice of species, strain, vehicle and number of animals [if applicable]: Wistar rats will be used as the species as they have been extensively used in MDI substances and provide an appropriate basis for comparison between studies.

Test material

Constituent 1
Chemical structure
Reference substance name:
1,1'-Methylenebis(4-isocyanatobenzene) and oligomeric reaction products of 1,1'-methylenebis(4-isocyanatobenzene) and oxydipropanol and oligomerization reaction products of oxydipropanol
EC Number:
Molecular formula:
C8H6NO [C7H5NO [C3H6O]n C8H7NO ]m C7H4NO with 2<=n<20 and 0<=m<=3
1,1'-Methylenebis(4-isocyanatobenzene) and oligomeric reaction products of 1,1'-methylenebis(4-isocyanatobenzene) and oxydipropanol and oligomerization reaction products of oxydipropanol

Results and discussion

Applicant's summary and conclusion