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Key value for chemical safety assessment

Effects on fertility

Description of key information

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.

MDI is not classified as a reproductive toxicant according to Regulation (EC) No 1272/2008 (CLP). For MDI, there is evidence that the strong respiratory irritation restricts exposure and ensures that there will be no exposure at levels at which there is a realistic possibility of reproductive toxicity.

The available evidence indicates

(1)   the absence of any type of systemic toxicity including toxicity to reproductive organs in subchronic and chronic inhalation studies in rats at concentrations resulting in irritation to the respiratory tract,

(2)    a low systemic dose of MDI which is exclusively present in form of macromolecular adducts and

(3)   the absence of evidence for toxicity to fertility based on an end-point specific weight-of-evidence approach using fertility studies of other aromatic and aliphatic diisocyanates.

In accordance with section 3 of Annex XI in some cases, it is not necessary to generate missing information, because risk management measures and operational conditions which are necessary to control a well-characterized risk will also be sufficient to control other potential risks, which will not therefore need to be characterized precisely. In essence, and based on the weight of evidence presented, there is no evidence which would support the conclusion that the current risk management measures which are in place to protect from respiratory tract irritation are inadequate to address toxicity to fertility. Potential risk due to toxicity to fertility that needs to be clarified and a realistic possibility that the requested information may lead to improved risk management measures cannot be anticipated. Scientifically and taking into consideration the already implemented protection measurements, there is no indication for an information need for the protection of human health pursuant to substance evaluation. Based on these considerations the performance of an additional animal study would not be in agreement with animal welfare principles.

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
0.2 mg/m³
Study duration:
chronic
Species:
rat
Quality of whole database:
Supporting studies on chemicals with a comparable mode of action were performed using Guideline protocols under GLP.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

General Considerations:

Data availability

Regarding fertility, while there are no guideline fertility studies available on any category members, numerous toxicokinetic (Section X.X) and repeated dose studies (X.X) indicate that toxic effects are limited to site of contact. Additional fertility studies from other diisocyanates that are not included in the current category but have a comparable toxicological mode of action (direct nucleophilic reactions with biological nucleophiles) have also demonstrated a lack of systemic toxicity and effects on fertility in guideline 2-generation reproduction studies. 

 

Mode of action of diisocyanates

The most relevant route of exposure for MDI (and other diisocyanates) is via inhalation. Due to the chemical reactivity of the isocyanate functional group diisocyanates will seek nucleophiles at the point of deposition, and e.g. the lung contains a relatively high level of non-protein sulfhydryl groups such as glutathione as well as proteins with accessible -SH, -OH, -NH and -COOH groups for interaction with the isocyanate functional group (Wisnewski et al. 2005, Kelly 1999). The primary toxic effects in response to both acute and repeated inhalation exposures (as vapour or aerosol) for diisocyanates are therefore focused on the portal of entry, the respiratory tract. Other systemic organ systems or tissues would be affected only at exposure concentrations that produce sufficient lung toxicity. The propensity of diisocyanates to cause lung effects consequently would led to secondary effects such as hypoxia that could influence development endpoints. This major mode of action of diisocyanates is also confirmed for MDI (see below). Due to the common primary toxic effect on the port of entry read across for systemic toxicity (toxicity that is related to systemic availability of the substance), is a reasonable approach for diisocyanates. This also includes the endpoint reproductive toxicity. Even if secondary toxicity at doses/concentrations with major port of entry toxicity cannot be excluded based on all available data it seems to be very unlikely to observe effects on reproductive/ developmental toxicity at an MDI exposure concentration that does not lead to port of entry toxicity. This is confirmed by the available data on MDI in rats.

Most appropriate route of administration

In accordance to ECHA Guidance Chapter R.7a (Version 6.0, July 2017) toxicity data obtained using the appropriate route of exposure should be preferred for the assessment the potential hazard of a substance to human beings. Due to the high reactivity of the functional NCO-group, isocyanates readily react with biomolecules at the point of first contact. Due to the varying composition of biological surfaces in the lung, the gastro-intestinal tract and on the skin, different primary reaction products are formed. Based on such significant differences in toxicokinetics route-to-route extrapolation therefore is not a reliable approach for hazard and risk assessment purposes of isocyanates. Since inhalation of respirable aerosols/vapour is the most relevant exposure for workers (and consumers), the hazard assessment in general and for reproductive/developmental toxicity in particular shall focus on the inhalation route of exposure.

Effects on fertility

According to Annex X (Section 8.7.3.) an extended One- (or a Two Generation) Reproductive Toxicity Study in one species on the most appropriate route of administration, having regard to the likely route of human exposure, is a standard information requirement. Standard information requirements shall be considered in conjunction with Annex XI, which allows variation from the standard approach, where it can be justified. Therefore, a registrant may adapt the required standard information set out in column 1 of Annex X according to the general rules contained in (column 2 and/or) Annex XI.

In absence of an extended One- (or a Two Generation) Reproductive Toxicity Study for MDI substances, a weight of evidence is presented in accordance to Annex XI for general rules for adaptation of the standard testing regime 1.2. For MDI there is sufficient weight of evidence from several independent sources of information leading to the conclusion that there is no evidence of a potential toxicity to fertility.

The justification for this weight of evidence for MDI is based on three independent sources of information:

1.   data of repeated dose toxicity studies including detailed assessment of reproductive organs

2.   an evaluation of quantity and form of systemic availability and,

3.    fertility studies of other aromatic and aliphatic diisocyanates

 

1. Data of repeated dose toxicity studies including detailed assessment of reproductive organs

In a subchronic inhalation toxicity study with pMDI 30 males and 30 females each, were exposed to 0, 4.1, 8.4 and 12.3 mg respirable pMDI/m³ for 6h/day, 5 days/week for 13 weeks (followed by a 4-week post-treatment). Reproductive organs and tissues were macroscopically and gross pathologically assessed at the autopsy. A detailed histopathological assessment was performed of 10 rats/sex of the control group and 20 rats/sex of the high-concentration group at the end of the exposure (week 14) and of 10 rats/sex of the control and high-concentration group at the end of the posttreatment period (in week 18). Histopathological assessment included e.g. adrenals, epididymides, mammary glands, seminal vesicles, testes and uterus. Neither gross examination at autopsy nor histopathological assessment revealed any treatment related systemic effects. The only treatment related effects were confined to the respiratory tract (increased relative lung weight ratios in the mid- and high concentration groups, histopathological and microscopic changes in all pMDI exposed groups) (Reuzel et al. 1994, see repeated dose toxicity).

Data on macroscopy, gross pathology and histopathology in the reproductive organs of both sexes can furthermore be derived from a 24 months chronic inhalation toxicity and carcinogenicity study of respirable pMDI aerosol in rats. In this study 70 rats/sex/group (each group subdivided into one satellite group of 10 rats/sex and one main group of 60 rats/sex; exposure of the satellite group was limited to 1 year) had been exposed at concentrations of 0, 0.2, 1.0 and 6 mg/m³ for 6h/day, 5 days/week. In the satellite groups histopathological examinations was carried out of a number of organs and of all gross lesions of all rats of the 2-year study (e.g. including epididymides, mammary glands, ovaries, prostate, seminal vesicles, testes and uterus). Compound-related changes were exclusively found in the respiratory tract (NOAEC 0.2 mg/m³) but no treatment related findings on reproductive or any other systemic organ effects were reported (Reuzel et al. 1994b; see repeated dose toxicity).

In a further chronic inhalation study with respirable aerosols of 4,4’-MDI, 80 female rats per dose group were whole-body exposed to atmospheres of 0.23, 0.70 or 2.05 mg/m³ for 17 hours/day, 5 days /week for up to 24 months. Full pathological examination was done on 20 rats/dose of a 12 months exposure group and 20 rats/dose of a 24 months exposure group. Reproductive organs assessed included e.g. adrenals, ovaries, uterus, vagina and mammary gland. Again compound-related changes were found in the respiratory tract (LOAEC 0.23 mg/m³), but no treatment related findings on reproductive or any other systemic organ effects were reported (Hoymann et al. 1995; see repeated dose toxicity).

These studies demonstrate that even with a chronic/lifetime exposure duration, effects from pMDI/4,4´-MDI aerosol are confined to the lungs. Effects on systemic organs including reproductive organs were not observed at exposure concentrations revealing respiratory tract toxicity.

As confirmed by recent literature (Mangelsdorf et al. 2003, Ulbrich & Palmer 1995, Janer et al. 2007, Dent 2007, Sanbuissho et al. 2009) in rodents histopathological examinations in repeated dose toxicity studies of reproductive tissues are of high value and high sensitivity for evaluation of reproductive toxicity in males and females. For example, the review of Mangelsdorf et al (2003) outlines that of the endpoints investigated for detecting adverse effects of chemicals on male reproduction in animal species, the most sensitive proved to be histopathology of the testes. Only in some cases sperm motility was found to be more sensitive than histopathology. The above parameters showed a higher sensitivity than fertility parameters. Summarizing these reviews, histopathological changes in the reproductive organs in repeated dose toxicity studies are highly indicative of effects on fertility. In this respect repeated dose toxicity studies for MDI are considered in this weight of evidence, since they provide sensitive and sufficient information to evaluate toxicity on fertility if histological examination of the reproductive organs is available. 

2. Evaluation of quantity and form of systemic availability

As described above the high reactivity of the functional NCO groups of MDI results in irritation of the respiratory tract occurring at low effect levels (LOAEC chronic 1 mg/m³, Reuzel et al. 1990), in absence of systemic toxicity. These low effect levels in humans and experimental animals translate in a low OEL (e.g. MAK 0.05 mg/m3).

By means of plausibility the quantities of systemically available MDI can be estimated by default values for the specific conditions in an animal experiment or at the workplaces (ECHA guidance Chapter R.8):

based on a default respiratory volume of 0.29 m3/kg bw for a 6h exposure for rats and the LOAEC for respiratory tract irritation of 1 mg/m3, a maximal bioavailable dose of only 0.29 mg/kg bw/day can be extrapolated for rats under the worst-case assumption of 100% bioavailability via the lungs. This worst-case assumption is put into perspective by an experimentally identified systemic bioavailability in rats of only 25-32 % of the applied dose (Gledhill 2003, see toxicokinetics). Therefore, systemic doses in a potential fertility assay, at concentrations resulting in respiratory tract irritation (MTD), are too low to expect systemic toxicity.

As a worst case for workplaces complying with the OEL of 0.05 mg/m3an internal dose can be calculated as 0.0048 mg/kg bw/day for workers, based on a respiratory volume of 6.7 m3/person for an 8h shift and a body weight of 70 kg.

Data on the metabolic fate of inhaled MDI provide convincing evidence that after inhalation, systemic circulation of absorbed MDI is exclusively in the form of macromolecular adducts. MDI is absorbed predominantly as a glutathione adduct (Gledhill et al. 2005, see toxicokinetics) which is rapidly excreted via the kidneys. In human’s excretion of conjugated MDI in urine begins 30min after onset of exposure with a half life of maximally 4h (Lewalter 1994). Due to this limited absorption and fast excretion high concentrations in the systemic circulation are not seen. Some of the systemically circulating and labile MDI-gluthathione-adducts undergoes a transcarbamoylation reaction with other nucleophiles, typicall with amino acids of proteins, to form more stable conjugates. This transcarbamoylation reaction proceeds without formation of free MDA (methylene dianiline) and is e.g. resulting in adducts such as MDI-haemoglobin (Gledhill et al. 2005, Bartels et al. 2009, Pauluhn et al. 2006, see toxicokinetics). For example, in workers (n = 25) exposed to MDI the specific MDI marker ABP-Val-Hyd was detected in 22 of these samples at low concentrations of 0.15-16.2 pmol/g (Gries & Leng 2013).

In conclusion it is evident that a potential systemic bioavailability in animal assays and at workplaces is significantly limited by the low irritation thresholds for respirable MDI aerosols. In addition, systemic unchanged MDI is unlikely to occur since MDI is exclusively detected as macromolecular adducts. Therefore, there is no evidence of systemic toxicity including toxicity to fertility at or below irritating doses.

3. Evaluation of fertility studies of other aromatic (and aliphatic) diisocyanates

While no studies including functional fertility parameters (e.g. hosting a mating protocol including a 10-week pre-mating period or a post-natal evaluation of the F1 generation) are available, guideline reproduction study from other aromatic (TDI; toluene diisocyanate) and aliphatic (HDI; hexamethylene diisocyanate) diisocyanates provide supporting evidence to evaluate this endpoint for the MDI category.

As described above, all diisocyanates share a property based on the common functional isocyanate (NCO) group. Due to the high reactivity of the functional NCO groups towards nucleophilic biomolecules the primary health effect of diisocyanates is irritation at the point of contact, and sensitization. This is reflected in the fact that similar effect levels for respiratory irritation were identified in chronic rat studies, taking into consideration the various experimental spacings of exposure concentrations. Similar effect levels in humans and experimental animals were the basis for identical OELs, e.g. MAK values, on a molar basis (see table 1)

Table 1: Comparison of effect levels for respiratory irritation from chronic 6h/day inhalation studies and MAK values of diisocyanates.

Test Substance

NOAEC

(mg/m3; ppm)

LOAEC

(mg/m3; ppm)

Reference

MAK

MDI

0.2; 0.02

1; 0.096

Reuzel, 1990

0.05/0.005

TDI

-

0.362; 0.05

Owen, 1984

0.035/0.005

HDI

0.035; 0.005

0.175; 0.025

Shiotsuka, 1989

0.035/0.005

 

No systemic effects were noted in acute, subacute and chronic bioassays of MDI, TDI or HDI. Therefore, the maximally tolerated concentration (MTD) on a molar basis which can be applied in an inhalation fertility study with MDI is not expected to significantly vary to the MTD applied in the available TDI and HDI fertility studies (2.9 mg/m3and 0.308 mg/m3, respectively). Given this consistency over all three substances, it is considered valid to use fertility data for TDI and HDI in a weight-of-evidence approach to evaluate this endpoint for MDI and its category members.It should be noted, however, that HDI and TDI provide supporting evidence for functional fertility parameter due to the comparable mode of action and toxicokinetic behaviour, they are not part of the MDI category because of their differing physico-chemical properties.

The toxicity of TDI on fertility was investigated in a two-generation inhalation study in rats (OECD 416). Rats were whole body exposed to vapours of a 80:20 mixture of 2,4´- and 2,6´-isomers of TDI at concentrations of 0, 0.2, 0.8, 2.9 mg/m3for 6 hrs/day for 5 days/week during the pre-mating period and 7 days/week during mating period and subsequent in life period. No effect of exposure on any of the reproductive parameters (e.g. reproductive performance) or organ weights, sexual maturation, viability, or gross- or histopathology were observed in the F0 and F1 generation. The only signs of toxicity were transient irritations of the upper respiratory tract. The NOAEC for fertility was the highest tested concentration of 2.9 mg/m3 (Tyl et al. 1989; see reproductive toxicity).

Local toxicity at the respiratory tract in absence of systemic effects or effects on fertility was confirmed for HDI in a combined reproductive/developmental/neurotoxicity study (OECD TG 422). Rats were whole body exposed to vapour atmospheres of0, 0.034, 0.34, 2.03mg/m3. (NOEC fertility 2.03 mg/m3, NOEC local 0.034 mg/m3; Astroff 2000).

 

Literature:

  • Dent M.P. (2007). Strength and limitations of using repeated-dose toxicity studies to predict effects on fertility. Regulatory Toxicology and Pharmacology 48, 241-258.
  • ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint Specific Guidance (Version 3.0, August 2014)
  • ECHA guidance on information requirements and chemical safety assessment Chapter R.8: Characterization of dose [concentration]-reponse for human health
  • Janer G. et al. (2007). A retrospective analysis of the added value of the rat two-generation reproductive toxicity study versus the rat subchronic toxicity study.Reproductive Toxicology 24, 103-113.
  • Lewalter J., Steinmann-Steiner-Haldenstätt W. (1994). Untersuchung der molekularbiologischen Konsequenzen des 4.4'-Methylenbiphenyldiisocyanat (MDI)-Umgangs. In: R. Kessel (Hrsg.), Verhandlungen der Deutschen Gesellschaft für Arbeitsmedizin und Umweltmedizin e. V., 34 Jahrestagung, Wiesbaden, Gentner Verlag, Stuttgart, 329-334.
  • Mangelsdorf I., Buschmann J., Orthen B. (2003).Some aspects relating to the evaluation of the effects of chemicals on male fertility, Regulatory Toxicology and Pharmacology 37: 356–369.
  • Owen P.E. (1984). The toxicity and carcinogenicity to rats of toluene diisocyanate vapour administered by inhalation for a period of 113 weeks. Addendum Report Volume 1 III Report 10233.
  • Sanbuissho A. et al. (2009). Collaborative work on evaluation of ovarian toxicity by repeated-dose and fertility studies in female rats, The Journal of Toxicological Sciences 34, Special Issue, SP1-SP22
  • Shiotsuka R.N. (1989), Chronic inhalation toxicity and oncogenicity study with 1,6 hexamethylene diisocyanate (HDI) in rats, Bayer Report No. 1157.
  • Ulbrich B. & Palmer A.K. (1995): Detection of effects on male reproduction – a literature survey. J am. College of Toxicology 14, 293-327.
  • Slatter, J.G. et al. (1991), Biotransformation of methyl isocyanate in the rat. Evidence for glutathione conjugation as a major pathway of metabolism and implications for isocyanate-mediated toxicity, Chem. Res. Toxicol. 4:157-161.
  • Day B.W. et al. (1997), Formation, solvolysis, and transcarbamoylation reactions of bis(S-glutathionyl) adducts of 2,4- and 2,6-diisocyanatototoluene. Chem. Res. Tox. 10: 424-431.

Justification for selection of Effect on fertility via inhalation route:

In absence of an extended One- (or a Two Generation) Reproductive Toxicity Study for MDI a weight of evidence is presented in accordance to Annex XI for general rules for adaptation of the standard testing regime 1.2 (see below). Selection of an effect level is based on respiratory irritation as the most sensitive endpoint. It is demonstrated that at or below concentrations resulting in significant irritation to the respiratory tract no effects on fertility can be expected.

Effects on developmental toxicity

Description of key information

The key study performed on polymeric MDI was conducted according to OECD Guideline 414 (Prenatal Developmental Toxicity Study) and under GLP with reliability 1. At concentrations of 1 or 4 mg/m³, no signs of maternal toxicity and no substance-induced adverse effects on the gestational parameters or the foetuses were recorded. The NOAEC for development/teratogenicity is observed at 12 mg/m3 (Gamer et al., 2000). As the observed foetotoxic effects and adverse effects on the embryonic development are considered as minor signs of developmental toxicity and as these effects occur at the concentration inducing maternal toxicity, they are considered to be secondary to maternal toxicity. As a consequence, polymeric MDI is considered not to be a developmental toxicant.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
4 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
There are 3 studies available, one on monomeric MDI and two using polymeric MDI which contains about 50% monomeric MDI. All studies are of reliable quality.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The key study performed on polymeric MDI (which contains about 50% monomeric MDI) was conducted according to OECD Guideline 414 (Prenatal Developmental Toxicity Study) and under GLP with reliability 1 at concentrations of 0, 1, 4 and 12 mg/m3). At concentrations of 1 or 4 mg/m³, no signs of maternal toxicity and no substance-induced adverse effects on the gestational parameters or the foetuses were recorded. Maternal toxicity was substantiated by mortality, damage to the respiratory tract, reduced body weight development and reduced mean gravid uterus weights at 12 mg/m3. At this concentration clear signs of developmental (embryo-/foeto-) toxicity in the form of reduced placental and foetal body weights and an increased occurrence of foetal skeletal (and overall) variations and retardation were recorded; however, no substance-induced teratogenic effects were observed up to and including the highest concentration (12 mg/m³). As the observed foetotoxic effects and adverse effects on the embryonic development are considered as minor signs of developmental toxicity and as these effects occur at the concentration inducing maternal toxicity, they are considered to be secondary to maternal toxicity, the NOAEC for maternal and foetal toxicity is 4 mg/m3. As a consequence, polymeric MDI (pMDI) is considered not to be a developmental toxicant.

In support of this conclusion, a study by Buschmann et al.,1996 (no info on GLP) performed according to OECD 414 using monomeric MDI at concentrations of 1, 3, and 9 mg/m3. The lung weights in the high-dose group were significantly increased compared to the sham-treated control animals. Treatment did not influence any other maternal and/or foetal parameters investigated (maternal weight gain, number of corpora lutea, implantation sites, pre- and post implantation loss, fetal and placental weights, gross and visceral anomalies, degree of ossification), although a slight but significant increase in litters with fetuses displaying asymmetric sternebra(e) (within the limits of biological variability) was observed after treatment with the highest dose of 9 mg/m3. Conservatively, a no embryotoxic effect level of 3 mg/m3 was determined.

Another range-finding study performed on polymeric MDI (which contains about 50% monomeric MDI) at doses of 0, 2, 8 and 12 mg/m³ was conducted according to OECD Guideline 414 (Prenatal Developmental Toxicity Study) and under GLP with reliability 1 (Waalkens-Berendsen et al, 1992). No observed external abnormalities in the foetuses were considered to be treatment-related, leading to a NOAEC (fetotoxicity) of greater than 12 mg/m3, and a maternal toxicity NOAEC of 8 mg/m3 based increased lung weights and decreased food intake.

Therefore the key and supporting studies indicate that MDI and pMDI are not developmental toxicants.

Justification for classification or non-classification

Not classified as a reproductive toxicant according to Regulation (EC) No 1272/2008 (CLP).