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Key value for chemical safety assessment

Effects on fertility

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:
Studies on "Read Across" chemicals were performed using Guideline protocols under GLP.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

General considerations

In accordance to ECHA Chapter R.7a (Version 4.0, July 2015, page 24 “Selection of the appropriate route of exposure for toxicity testing“) “…toxicity data obtained using the appropriate route of exposure…” should be preferred for the assessment. This is particularly relevant for reactive substances like MDI, for which significant differences in metabolism can be demonstrated dependent on the route of exposure (see toxicokinetics). Route-to-route extrapolation therefore is not applicable for hazard and risk assessment purposes. Since inhalation of respirable aerosols is the most relevant exposure for workers (and consumers) this hazard assessment for toxicity to fertility is focusing on the inhalation route of exposure.

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 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 assumption/conclusion that there is a low level of uncertainty with respect to 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.   a read-across to fertility studies of other aromatic and aliphatic diisocyanates, and

3.   an evaluation of quantity and form of systemic availability

 

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 weeks 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 observedat 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.

 

Read across to fertility studies of other aromatic (and aliphatic) diisocyanates

In the absence of 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, these testing requirements for toxicity to fertility can be covered by an end-point specific read-across to data of other aromatic (TDI; toluene diisocyanate) and aliphatic (HDI; hexamethylene diisocyanate) diisocyanates.

This read-across in accordance to Annex XI for general rules for adaptation of the standard testing regime 1.5 is justified by the similar toxicological properties of all diisocyanates based on the two common functional isocyanate (NCO) groups. 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. No systemic effects were noted in acute, subacute and chronic bioassays of all three substances.

 

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 of mg/m3. (NOEC fertility 2.03 mg/m3, NOEClocal 0.034 mg/m3; Astroff 2000).

 

When tested in inhalation toxicity assays, MDI is predominantly an aerosol and TDI and HDI are predominately vapours. Nevertheless, similar effect levels for respiratory irritation were identified in chronic rat studies, taking into consideration the various experimental spacing’s 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.

mg/m3/ ppm

NOEAC

LOAEC

Literature

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

 

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).

Taken together read-across to fertility data of other aromatic (TDI) and aliphatic (HDI) diisocyanates is substantiating a common toxicological mode of action of local irritation at the respiratory tract in absence of systemic toxicity and toxicity to fertility. Since similar experimental test designs will apply for inhalation fertility studies with MDI, additional information on toxicity to fertility is unlikely to be expected from such a study.

 

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), can be expected to be considerably low.

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 humans excretion of conjugated MDI in urine begins 30min after onset of exposure with a half life of maximally 4h (Lewalter 1994). Due to this 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. Systemic toxicity including toxicity to fertility at or below irritating doses is therefore unlikely to occur.

 

Conclusion

The existing DNEL for MDI is based on strong irritation of the respiratory tract. This low DNEL (0.05 mg/m3) ensures that there will be no exposure at levels at which, based on the evidence available, there is a realistic possibility of reproductive toxicity.

The available evidence indicates that there is a low level of uncertainty and includes

(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) the absence of any concern for toxicity to fertility based on an end-point specific read-across to fertility studies of other aromatic and aliphatic diisocyanates, and

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

In accordance with section 3 of Annex XI in some cases, it is not be 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 can not 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.

 

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.

Short description of key information:

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. For MDI there is sufficient weight of evidence from several independent sources of information leading to the assumption/conclusion that there is a low level of uncertainty with respect to 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. a read-across to fertility studies of other aromatic and aliphatic diisocyanates, and

3. an evaluation of quantity and form of systemic availability

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).