Isocyanic acid, polymethylenepolyphenylene ester, 2-ethyl-1-hexanol-blocked

Carcinogenicity

Description of key information

Although the endpoint carcinogenicity is not a data requirement under Annex VIII regulation (10 – 100 t/a) this endpoint was assessed for the target substance MDI MT. Since the source substances 4,4’-MDI, pMDI and the target substance MDI MT share structural similarities and contain sufficient monomeric MDI, the driver of toxicity, similarities in reactions leading to inflammation and irritation to the respiratory tract is assumed. Findings from valid carcinogenicity studies in rats revealed formation of pulmonary tumours after chronic inhalation of 4,4’-MDi and pMDI. These study results were interpreted as the result of a non-specific irritating effect of 4,4’-MDI and pMDI with corresponding typical consecutive reactions in the lungs (DFG, 2008). As the target substance MDI MT contains sufficient monomeric MDI, the driver of non-specific irritation of the respiratory tract, a read across and an assessment of the endpoint carcinogenicity is warranted.

There are two reliable chronic and carcinogenicity studies available for the source substances 4,4’-MDI and pMDI.

Reuzel et al. (1994a) evaluated the chronic toxicity and carcinogenicity of pMDI. In the study rats were exposed for 6 hours/day, 5 days/week for 2 years to pMDI aerosol concentrations of 0, 0.2, 1.0 or 6.0 mg/m3 (analytical conc.: 0, 0.19, 0.98, 6.03 mg/m³). This GLP reliability 2 key study was conducted according to OECD Guideline 453 (Combined Chronic Toxicity /Carcinogenicity Studies). Histopathology of the organs/tissues investigated showed that exposure to 6.0 mg/m³ was related to the occurrence of pulmonary tumors in males (6 adenomas and 1 adenocarcinoma) and females (2 adenomas). Therefore, pMDI was carcinogenic in rats after long-term inhalation to aerosol concentrations of 6.0 mg/m³. It was also concluded that exposure to polymeric MDI at concentrations not leading to recurrent lung tissue damage will not produce pulmonary tumors.

Hoymann et al. (1995) investigated the chronic toxicity and carcinogenicity of 4,4’-MDI in a long-term inhalation study over a maximum of 24 months including satellite groups with 3, 12, and 20 months exposure. Female Wistar rats in groups of 80 animals were exposed for 17 hours/day, 5 days/week to 0, 0.23, 0.70, and 2.05 mg/m³ 4,4’-MDI in aerosol form. A dose-dependent impairment of the lung function in the sense of an obstructive-restrictive malfunction with diffusion disorder, increased lung weights, an inflammatory reaction with increased appearance of lymphocytes (but not of granulocytes) in the lung in the high dose group as a sign of specific stimulation of the immune system by 4,4’-MDI, a moderately retarded lung clearance in the high dose group as well as dose-dependent interstitial and peribronchiolar fibrosis, alveolar bronchiolisation and a proliferation of the alveolar epithelium as well as a bronchiolo-alveolar adenoma (at 2.05 mg/m³) were reported.

Based on the available non-human data it can be concluded that 4,4’-MDI and pMDI were carcinogenic in rats after long-term inhalation to aerosol concentrations. Pulmonary tumors (limited incidence of mainly benign adenomas) occurred only at sites of contact (respiratory tract).

Human data in the form of cancer mortality epidemiology studies in cohorts of industrial workers exposed to diisocyanates for up to 40 years did not show an association between occupational exposure to isocyanates and increased risk of lung cancer or other cancers (Sorahan and Nichols, 2002, Mikoczy et al. 2004, Schnorr et al. 1996).

A non-genotoxic mechanism of 4,4’-MDI and pMDI is consistent with the absence of mutagenicity and the hypothesized MoA for the development of pulmonary tumors. This is supported by the tumor spectrum with a limited incidence of mainly benign adenomas, the lack of a local invasive growth, as well as the presence of only microscopic changes (adenomas were only a few millimeters in size) towards the end of the study. The findings of the chronic inhalation studies in rats were interpreted as the result of a non-specific irritating effect of 4,4’-MDI and pMDI with corresponding typical consecutive reactions in the lungs (DFG, 2008). 

As the source substances 4,4’-MDI, pMDI and the target substance MDI MT contain sufficient monomeric MDI, the driver of toxicity, similarities in reactions leading to inflammation and irritation to the respiratory tract is assumed. As the higher molecular weight non-monomeric content of the UVCB substance MDI MT do not contains reactive centers and is consequently inert and thus do not contribute to the observed toxicity, it is reasonable to assume that using read across to the source substances 4,4’-MDI and pMDI is warranted . As the source substances are classified with category Carc Cat.2 (H351) EU GHS 1272/2008 CLP using the read across approach, a similar classification for the target substance is followed and the target substance MDI MT is classified as Carc Cat.2 (H351) EU GHS 1272/2008 CLP.

Key value for chemical safety assessment

Carcinogenicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 1985 - June 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards, well documented and acceptable for assessment.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

- some omitted exposures; clinical chemistry, urinalysis and haematological samples only taken at the end of the study

Principles of method if other than guideline:

Details of deviations:
Omitted exposures due to public holidays or to technical maintenance or repair of inhalation equipment there were no exposures during 21 days. (day 45, 100, 119, 197-198, 204, 290, 293, 323, 326, 331, 342,562-563, 569, 675, 678, 688, 692-693, 716, 727).
Clinical chemistry, urinalysis samples and haematological examination samples only taken at the end of the study (1 yr satellite group, 2 yr main group).

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

- AGE: at the start of the study the animals were approximately 6 weeks old.
- WEIGHT AT STUDY INITIATION: mean weight of male rats was 179 g and of female rats 141 g. The rats were weighted just prior to the start of the study and then weekly during the first 13 weeks and every 4 weeks afterwards. At the end of the exposure time, surviving rats were killed and weighted at the day of scheduled autopsy.
- NUMBER OF ANIMALS: 280 males and 280 females randomly alocated to four groups. Each group, composed of 70 males and 70 females. Each group was subdivided into one satellite group of 10 rats/sex and a main group of 60 rats/sex.
- STRAIN: Cpb:WU Wistar

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

- EXPOSURE CHAMBERS-EXPOSURE CONDITIONS: Animals were exposed to the test atmospheres in H1000 multitiered inhalation chambers (capacity about 2.3 m3) manufactured by Hazleton Systems, Inc.. During exposure the rats were housed individually in wire mesh stainless-steel cages. Total airflow through the chambers was on average between 35 and 45 m3/hr depending on the volume of air needed to reach the required concentration of polymeric MDI in the atmosphere. The temperature and relative humidity in the chambers were generally between 20 and 25°C and between 40 and 70 %, respectively. Except for exposure to polymeric MDI, control rats were treated similarly to test animals including housing in an H1000 multitiered inhalation chamber.
- GENERATION OF TEST ATMOSPHERES: Test atmospheres were generated by atomizing polymeric MDI liquid into droplets by using compressed air in a nebulizer designed by TNO. The nebulizer consisted of an atomizer and a glass jar. The atomizer coded DR 0 11 was purchased from Lechler (Germany). Before use, the nozzle of the atomizer was slightly modified by reducing empirically the internal diameter of the nozzle orifice. The nebulizer was operated at a pressure of approximately 2.5 bar. A baffle was fitted approximately 4 cm below the nozzle orifice to remove the larger droplets from the spray The smaller droplets followed the upward airflow and were emitted through the outlet port and passed through a cyclone with a diameter of 9 cm. The remaining particles larger than 5 µm were impinged onto the wall of the cyclone to obtain an aerosol of which 95% of the particles were smaller than 5 µm. The aerosol was then passed through a manifold pipe system constructed of polyvinyl chloride tubing, having a length of approximately 10 in and an internal diameter of 4.5 cm. Air-operated vacuum pumps (air movers, AIRNAC, Milford, CT; TD series, 110 size) adjacent to the top of the respective inhalation chambers moved polymeric MDI aerosol from the delivery system to the inlet of the inhalation chamber, where it was diluted in air from the main air supply of the inhalation chamber. By varying the operating air pressure to the vacuum pump the amount of withdrawn aerosol could be adjusted to the desired concentration of test material within the chamber.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- ANALYSIS OF POLYMERIC MDI IN THE TEST ATMOSPHERES: Gravimetry was used as primary standard for assessing the polymeric MDI aerosol concentration in the test atmospheres. Test atmosphere samples were collected using closed filter-type collectors provided with glass fiber filters (Sartorius SM 13430; diameter, 44 mm). The filters were weighed before and after sampling. From the increase in weight and the volume of test atmosphere drawn through the filter the time-weighted average concentration of polymeric MDI was calculated. Beta attenuation using attenuators from Verewa (Germany) were used in parallel with gravimetry during the first 3 months of the study, and the results were compared with those obtained from gravimetry. In view of the proven reliability of beta attenuation during this period gravimetric determinations were carried out only once every 2 weeks thereafter. The particle size distribution in each of the test atmospheres was determined at weekly intervals using a 10-stage Berkeley quartz crystal microbalance cascade impactor.

Duration of treatment / exposure:

main groups: 2 years; satellite groups: 1 year

Frequency of treatment:

6 hours/day; 5 days/week

Post exposure period:

none

Dose / conc.:

0 mg/m³ air (nominal)

Dose / conc.:

0.2 mg/m³ air (nominal)

Dose / conc.:

1 mg/L air (nominal)

Dose / conc.:

6 mg/m³ air (nominal)

Dose / conc.:

0 mg/m³ air (analytical)

Dose / conc.:

0.19 mg/m³ air (analytical)

Dose / conc.:

0.98 mg/m³ air (analytical)

Dose / conc.:

6.03 mg/m³ air (analytical)

No. of animals per sex per dose:

60 (main groups); 10 (satellite groups)

Control animals:

yes, sham-exposed

Details on study design:

The mean mass median aerodynamic particle size during the study was 0.68, 0.70 and 0.74 µm with geometric standard deviation of 2.93, 2.46 and 2.31 at the level of 0.2, 1.0 and 6.0 mg/m3, respectively. On average the aerodynamic diameter was for at least 93.5 % of the particles smaller than 4.2 µm.

Observations and examinations performed and frequency:

- HEMATOLOGICAL PARAMETERS (red and white blood cell counts, hemoglobin, packed cell volume, differential white blood cell count, prothrombin time) and urinary parameters (appearance, volume, density, pH, protein, occult blood, glucose, ketones, microscopy of the sediment) were measured in all rats of the satellite groups in week 52.
- BIOCHEMICAL BLOOD PARAMETERS (albumin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, urea nitrogen, total protein, creatinine, total bilirubin, Ca, K, Na, inorganic phosphate, cholesterol, triglycerides, and glucose) were measured in blood samples taken at euthanization from all rats of the satellite groups, except for glucose, which was determined in blood samples after overnight fasting in week 52.

Sacrifice and pathology:

- ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): All rats of the satellite groups were randomly killed on two successive days in week 53, and all survivors of the main groups were randomly euthanized on seven successive working days in weeks 105 and 106 by exsanguination from the abdominal aorta under ether anesthesia, autopsied, and examined for gross pathological changes. Adrenals, brain, heart, kidneys, Iiver, Iungs with mediastinal lymph nodes, trachea and larynx, spleen and testes of all rats of the satellite groups and of all survivors of the main groups were weighed. A wide range of organs or samples of organs or tissues were preserved in anaqueous neutral phosphate-buffered 4% formaldehyde solution. The lungs were fixed by intratracheal infusion with the main groups, nose, lungs, mediastinal lymph nodes, and all gross lesions fixative under 10 cm water pressure. The urinary bladder was fixed by infusion of the fixative through the bladder wall in the neck of the bladder. The nose was fixed by infusion of the fixative through the pharyngeal duct. In the 2-year study (main groups) 43 different organs or tissues and all grossly visible lesions were examined by light microscopy of the control and high-concentration animals and of the low- and midconcentration decedents. Moreover, in the low- and mid-concentration survivors of the main groups, nose, lungs, mediastinal lymph nodes, and all gross lesions were subjected to histopathologicaI examination.

Statistics:

Body weights were analyzed by an analysis of covariance (Cochran, 1957) followed by the Dunnett's multiple comparison test (Dunnett, 1955). Analysis of variance (Steel and Torrie, 1960) followed by the Dunnett's multiple comparison test was applied to hematological, biochemical, and organ weight data. Differential white blood cell count data were analyzed by the Mann-Whitney U test (Siegel, 1956a). Incidences of histopathological changes and numbers of deaths were analyzed by the Fisher exact probability test (Siegel, 1956b).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Relevance of carcinogenic effects / potential:

Although lifetime inhalation of PMDI aerosols by rats resulted in a small number of benign adenomas, they are considered to be of unlikely
relevance to man . Such aerosols are not encountered outside of the experimental laboratory. This is discussed in detail in the overall summaries.

Key result

Dose descriptor:

NOAEC

Effect level:

0.2 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

Remarks on result:

other: Effect type: toxicity

Key result

Dose descriptor:

NOAEC

Effect level:

1 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: Effect type: carcinogenicity

Dose descriptor:

LOAEC

Effect level:

6 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: Effect type: carcinogenicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

6 other: mg/m3 (nominal conc.)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Mortality incidences in males were comparable in all groups. Female rats showed negatively concentration-related mortality incidences. The number of animals with palpable masses did not differ between test and control animals. No treatment-related differences in body weights were observed between control and test groups.

Hematological examination of rats at day 357-358 revealed no exposure-related differences between the groups. Biochemical examination performed on days 360, 366 and/or 367 was essentially negative. No alterations were observed from parameters measured in urine of rats exposed to polymeric MDI aerosol for 360 days.

Lung weights were statistically significantly increased in both males and females exposed to 6.0 mg/m³ for 12 or 24 months. No treatment-related gross changes were found in animals exposed for 12 months. Gross examination of animals exposed for 24 months revealed increased incidence of lungs with spotted surface and/or discoloured appearance in male rats exposed to 6.0 mg/m3.

Histopathology (main groups exposed over 2 years):

Nose: increased incidence of rats with a higher degree of basal cell hyperplasia frequently accompanied by hyperplasia of Bowman's glands in the olfactory epithelium in the nose at levels of 1.0 and 6.0 mg/m³. At 6.0 mg/m³ basal cell hyperplasia occurred in 32/60 males and 49/60 females against 14/60 and 4/60 for the corresponding controls.

Lungs: incidences of non-neoplastic findings and tumors are summarized in the following table 1:

Table 1: Number of rats with non-neoplastic findings and tumors in lungs after 2-year exposure to polymeric MDI (main groups)

pMDI (mg/m3)		0	0.2	1.0	6.0
Animal number	M	60	60	60	60
	F	60	60	60	60
Surviving animals	M	38	38	42	36
	F	41	42	48	50
- Macrophages with yellow pigment	M	0	3	21**	60**
	F	0	1	23**	59**
- Localized fibrosis	M	1	0	9*	44**
	F	0	0	4	48**
- Alveolar duct epithelialization	M	1	0	8*	54**
	F	0	0	8*	57*
- Localized alveolar bronchiolization	M	1	1	2	12**
	F	2	3	3	14**
- Mineralized deposits in the bronchial and alveolar region	M	0	1	1	13**
	F	0	0	0	24**
- Pneumonitis	M	13	13	17	28
	F	3	4	3	2
- Adenoma	M	0	0	0	6*
	F	0	0	0	2
- Adenocarcinoma	M	0	0	0	1
	F	0	0	0	0

* p<0.05; ** p<0.01

Mediastinal lymph nodes: increased incidence of rats with an accumulation of macrophages with yellow pigment at levels of 1.0 and 6.0 mg/m³. At 6.0 mg/m³ this finding occurred in 50/60 males and 43/60 females against 0/60 for the corresponding controls.

Other organs: the incidence and distribution of other tumour types was not affected by treatment.

Histopathology (satellite groups exposed over 1 year):

Rats killed after 1 year of exposure had treatment-related histopathological changes in the nasal cavity, lungs, and mediastinal lymph nodes starting at 1.0 mg/m3, but to a lower degree of severity compared to animals exposed over 2 years. There was no microscopic evidence of lung tumors or any other tumors following exposure to polymeric MDI for 1 year.

Conclusions:

In a combined chronic toxicity and carcinogenicity study rats were exposed for 6 hours/day, 5 days/week for 2 years to polymeric MDI aerosol concentrations of 0, 0.2, 1.0 or 6.0 mg/m3 (analytical conc.: 0, 0.19, 0.98, 6.03 mg/m3). Histopathology of the organs/tissues investigated showed that exposure to 6.0 mg/m³ was related to the occurrence of pulmonary tumors in males (6 adenomas and 1 adenocarcinoma) and females (2 adenomas).
Therefore, polymeric MDI was carcinogenic in rats after long-term inhalation to aerosol concentrations of 6.0 mg/m3. It was also concluded that exposure to polymeric MDI at concentrations not leading to recurrent lung tissue damage will not produce pulmonary tumors.

Executive summary:

Reuzel et al. (1994a) evaluated the chronic toxicity and carcinogenicity of pMDI. In the study rats were exposed for 6 hours/day, 5 days/week for 2 years to pMDI aerosol concentrations of 0, 0.2, 1.0 or 6.0 mg/m³ (analytical conc.: 0, 0.19, 0.98, 6.03 mg/m³). This GLP reliability 2 key study was conducted according to OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies). Histopathology of the organs/tissues investigated showed that exposure to 6.0 mg/m³ was related to the occurrence of pulmonary tumors in males (6 adenomas and 1 adenocarcinoma) and females (2 adenomas). Therefore, pMDI was carcinogenic in rats after long-term inhalation to aerosol concentrations of 6.0 mg/m³. It was also concluded that exposure to polymeric MDI at concentrations not leading to recurrent lung tissue damage will not produce pulmonary tumors.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

1 mg/m³

Study duration:

chronic

Species:

rat

System:

respiratory system: lower respiratory tract

Organ:

lungs

Mode of Action Analysis / Human Relevance Framework

The hypothesized MoA of carcinogenicity of MDI is based on the high reactivity of the NCO group. The initiating event is reaction of bioaccessible NCO groups on MDI substances which react with nucleophilic biomolecules at the MDI/lung fluid interface to form MDI conjugates. This depletion of nucleophilic scavenger molecules (i.e. glutathione) allows reaction with alveolar surfactants and destabilisation of the protective surfactant systems of the lungs. Concomitant to the initiating event is alveolar protein exudation, allowing protein content of alveolar lavage fluids to serve as a measure of acute alveolar irritation (Pauluhn et al., 1999, Pauluhn, 2000, Kilgour et al., 2002). Similar to the local irritation effects following acute exposure, the highly reactive NCO-group rapidly reacts with extracellular biological nucleophiles, which leads to effects on lung surfactants and local irritation leading to chronic regenerative cell proliferation and benign tumors. Chronic toxicity is a function of exposure concentration x exposure period (C x t) and likely to be based on recurrent acute effects (acute on chronic inflammation) leading to hyperplasia, proliferation and eventually adenoma and interstitial fibrosis in the lung. The available study data for the endpoint carcinogenicity are consistent with the hypothesized MoA and based on the high reactivity of the NCO group.

Justification for classification or non-classification

According to CLP Regulation (EC) No.1272/2008 the classification of 4,4'-MDI (CAS No.101-68-8) was considered for the classification of MDI MT (CAS No.147993-65-5):

GHS: Carc.2 (H351: suspected of causing cancer by inhalation).

 

Additional information

Regarding relevance of the findings in animal studies to humans, it should be recognized that the animals in the rodent bioassays were exposed to test material in the form of respirable aerosols generated using sophisticated techniques in the laboratory to optimize hazard identification. Such atmospheres are technically very difficult to generate and maintain in stable form, and in practice are not formed outside of the laboratory. For instance, in inhalation toxicity tests, the test substance often needs to be heated to allow for nebulization so that aerosols are formed. As a general rule, particles are described as inhalable or respirable depending on size i.e. mass median aerodynamic diameter (MMAD). Inhalable particles can enter the nose but because of the relatively large size deposit entirely in this region. Finer, respirable particles, (10 µm MMAD or less), can penetrate to lower regions of the respiratory tract and deposit in terminal alveolar regions. As respirable particles are deposited throughout the lung, but the tumours were only seen in the bronchiol-alveolar region, the regional deposition of respirable particles in the lower regions of the lung is a key event in tumour development. 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)).  Therefore, MDI substances to be present in the workplace atmosphere in respirable form described above for prolonged durations is not anticipated under any conditions of intended or foreseeable use and is the most likely explanation for the apparent disparity between the findings of lung cancer in cancer bioassays in rodents but not workers occupationally exposed to diisocyanates.