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

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Full report, methods are in accordance with test guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Principles of method if other than guideline:

Identification and quantification of metabolites of 14C-MDI in urine, bile and faeces following an inhalation dose. Additionally comparison of metabolic profile of urine before and after hydrolysis and quantification of haemoglobin adducts.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-methylenediphenyl diisocyanate

EC Number:

202-966-0

EC Name:

4,4'-methylenediphenyl diisocyanate

Cas Number:

101-68-8

Molecular formula:

C15H10N2O2

IUPAC Name:

1,1'-methylenebis(4-isocyanatobenzene)

Constituent 2

Reference substance name:

benzene, 1,1'- methylenebis[4-isocyanato-

IUPAC Name:

benzene, 1,1'- methylenebis[4-isocyanato-

Details on test material:

Unlabeled test substance:
- Name of test material (as cited in study report): diphenylmethane 4,4'-diisocyanate, casnr. 101-68-8 , monomeric MDI.
- Physical state: solid
- Analytical purity: 98% (Sigma-Aldrich)

Radiolabelled test substance:
- Name of test material (as cited in study report): 14C-diphenylmethane 4,4'-diisocyanate (Amersham)
- Lot/batch No.: CTL ref no. Y00122/026
- Radiochemical purity (if radiolabelling): >97% following repurification
- Specific activity (if radiolabelling): 4.48GBq/mMole
- Locations of the label (if radiolabelling): ring labelled
- Storage condition of test material: desiccated, at -20°C

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Biological Services Section
- Weight at study initiation: 312-356g
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): yes
- Acclimation period: >4 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.3°C
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

other: Inhalation; condensation aerosol, head-only

Details on exposure:

TYPE OF INHALATION EXPOSURE: head only
The routes and rates of excretion and the tissue distribution of radioactivity in the male rat (6 groups of four Wistar-derived rats) following a 6 hour inhalation exposure (condensation aerosol, head-only) to 14C-MDI at a nominal concentration of 2 mg/m³ was investigated.

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Each test atmosphere was generated using a condensation aerosol. A saturated vapor was generated and delivered into the 46 liter exposure chamber at nominal flow rates of 10 l/min. The test subsance concentration and the minimum of 12 air changes was regulated with diluting air at 2 l/min.
- Method of holding animals in test chamber: poIycarbonate tubes , with latex collar fitted around each animal's neck

TEST ATMOSPHERE
- MDI concentration (Particulate mass concentration was measured gravimetrically): 2.01mg/l +/-0.03
The mean dose received was 5 KBq, which was equivalent to 15.6µg per animal.
- no MDA was detected in the test atmosphere (atmospheric concentrations of MDI were determined by analysis of a filter sample)
- Particle size distribution (aerodynamic particle size distribution was measured using a Marple Cascade Impactor)
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 0.73µm/ 1.76
Analysis of the radioactiv dose preparation by liquid scintillation counting.

Duration and frequency of treatment / exposure:

6 hour(s)

Doses / concentrations

Dose / conc.:

2 mg/m³ air

No. of animals per sex per dose / concentration:

Males: 4 Females: 0

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces, tissues, cage washes, bile, blood, lung lavage
- Time and frequency of sampling: excreta and cage wash was collected in metabolism cages 0-12, 12-24, 24-36, 36-48 and 48-72h after dosing. Animals were terminated 72h after exposure by exsanguation. A terminal blood sample was taken from all animals. Bile, urine and faeces were collected 168h after dosing in the toxicokinetic study. Lung lavage was obtained at the end of the exposure in the metabolism study.
- Method type(s) for identification in non radioactive study: MS, NMR, co-chromatography with reference standards
- Method type(s) for identification in radioactive tissue distribution study: radiochromatography (GC, HPLC, TLC)
- MDA and MDI metabolites present in urine and haemoglobin adducts were measured after hydrolysis with GC-MS

Identification of metabolites: thin layer chromatography (TLC), HPLC, HPLC-mass spectroscopy (LC-MS), tandem mass spectroscopy (MS/MS), proton nuclear magnetic resonance spectroscopy (2H-NMR).
Quantitation of metabolites: HPLC
Measurement of MDA following hydrolysis of urine and heamoglobine:

TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable):
Base hydrolysis (32% NaOH, 95°C, 2h) and acidic hydrolysis (concentrated HCl, 100°C, 1h) followed by GC-MS analysis.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Toxicokinetic parameters

Toxicokinetic parameters 1

Key result

Test no.:

#1

Toxicokinetic parameters:

other: no evidence of free MDA detected

Toxicokinetic parameters 2

Key result

Test no.:

#1

Toxicokinetic parameters:

other: major urinary metabolite was N,N'-diacetyl-4,4'-diaminobenzhydrol

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

There was no MDA detected in any of the samples analyzed for this study. With the exception of 1 minor metabolite, all low molecular weight metabolites present in urine and bile were identified or characterized as follows:

Metabolite I: N,N'-diacetyl-4,4'-diaminobenzhydrol. This was the major urinary metabolite in both intact and bile duct cannulated rats (1% and 6% of the dose respectively). It was also present in bile (1% of the dose).

Metabolite II: N,N'diacetyl-4,4'-diaminophenylmethane This metabolite was present in urine of intact and cannulated rats (0.5% and 4% of the dose respectively) and was present as the major metabolite in bile (4% of the dose).

Metabolite III: N-acetyl-4,4'diaminophenylmethane

Metabolite IV: N,N'-diacetyl-4,4'-diaminobenzophenone Metabolites III and IV were minor urinary metabolites (< 0.5% of the dose) and were not present in bile.
None of these specified low molecular weight metabolites were found in faeces.

The solvent extract of faeces and the major radioactive component in bile (9% of the dose) was thought to consist of mixed molecular weight polyurea oligomers derived from MDI (metabolite VI). The implication of these results, made by the author, is that a proportion of the MDI dose (10%) is converted to these metabolites via intermediary formation of an amine group which is rapidly acetylated. Both formation and acetylation are most likely to occur within specific cells or compartments. It is not possible from the current data to elucidate whether this stage involves:
- MDA, although none was detected
- bound-MDA, i.e. as a bound intermediate on an enzyme involved in the formation of the metabolites,
- an amine group resulting from reversion of the expected MDI-glutathione conjugates as proposed below:

Lung: MDI + 2GSH → GSH-MDI-SHG
Tissues: GSH-MDI-SHG → GSH-MD-NCO → GSH-MD-NH2 → GSH-MD-NHCOCH3

Reversal of the second glutathione link would lead to the formation of Metabolite III, with subsequent metabolism but without free MDA having been formed at any stage.

Any other information on results incl. tables

In order to better understand the toxicokinetic behaviour of MDI after inhalation exposure, biological samples from the Gledhill (2001a) experiments (urine, faeces, bile) were investigated in a separate study on the metabolism of MDI. Urine, faeces and bile were collected for the identification of metabolites at 6 (in urine and bile only), 12, 24, 36 and 48 h (and for intact animals at 24 hourly intervals until 7 days after the end of exposure). Metabolites present in bile and excreta were identified by LC/MS and/or by co-chromatography with reference standards and quantified.

Identification/quantitation of metabolites:

There was no MDA detected in any of the samples analysed for this study. With the exception of 1 minor metabolite, all low molecular weight metabolites present in urine and bile were identified or characterised as follows:

Metabolite I: N,N'-diacetyl-4,4'-diaminobenzhydrol. This was the major urinary metabolite in both intact and bile duct cannulated rats (1% and 6% of the dose respectively). It was also present in bile (1% of the dose).

Metabolite II: N,N'diacetyl-4,4'-diaminophenylmethane This metabolite was present in urine of intact and cannulated rats (0.5% and 4% of the dose respectively) and was present as the major metabolite in bile (4% of the dose).

Metabolite III: N-acetyl-4,4'diaminophenylmethane

Metabolite IV: N,N'-diacetyl-4,4'-diaminobenzophenone Metabolites III and IV were minor urinary metabolites (< 0.5% of the dose) and were not present in bile.

None of these specified low molecular weight metabolites were found in faeces.

The solvent extract of faeces and the major radioactive component in bile (9% of the dose) was thought to consist of mixed molecular weight polyurea oligomers derived from MDI (metabolite VI). The implication of these results, made by the author, is that a proportion of the MDI dose (10%) is converted to these metabolites via intermediary formation of an amine group which is rapidly acetylated. Both formation and acetylation are most likely to occur within specific cells or compartments. It is not possible from the current data to elucidate whether this stage involves:

- MDA, although none was detected

- bound-MDA, i.e. as a bound intermediate on an enzyme involved in the formation of the metabolites,

- an amine group resulting from reversion of the expected MDI-glutathione conjugates as proposed below:

Lung: MDI + 2GSH → GSH-MDI-SHG

Tissues: GSH-MDI-SHG → GSH-MD-NCO → GSH-MD-NH2 → GSH-MD-NHCOCH3

Reversal of the second glutathione link would lead to the formation of Metabolite III, with subsequent metabolism but without free MDA having been formed at any stage.

Table 1: Quantity of each metabolite present in % radioactivity administered.

	^urine	^faeces
^Metabolite I	^1	^0
^Metabolite II	^0.5	^0
^Metabolite III	^0.3	^0
^Metabolite IV	^0.4	^0
^Metabolite V	^0.2	^0
^Metabolite VI	^0	^56
^total	^2.4	^56

Table 2: Quantity of each metabolite present in % radioactivity administered. Samples from Gledhill study 2003.

	urine	bile	faeces
^Metabolite I	6	1	0
^Metabolite II	4	4	0
^Metabolite III	0	0	0
^Metabolite IV	0	0	0
^Metabolite V	1	0	0
^Metabolite VI	0	9	24
^total	10	14	24

10% of the radioactivity was excreted in urine in 0-24h, further 2% in 24-48h.

Bilary elimination accounted for approximately 14% of the dose in 0-48h after exposure and 34% was eliminated in faeces during the same time period.

MDA and MDI metabolites in urine and heamoglobin following hydrolysis:

Table 3: Amount of MDA and concentration of combined deacetylated metabolite I and IV in urine.

	^MDA (ng/ml)		^metabolites I and IV (ng/ml)
	^basic hydrolysis	^acidic hydrolysis	^basic hydrolysis	^acidic hydrolysis
^6h		^482.7 (75%)		^173.6 (25%)
^12h	^96.7 (31%)	^129.5 (41%)	^NQ	^181.0 (64%)
^24h	^64.3 (45%)	^130.5 (91%)	^NQ	^103.1 (81%)
^36h	^66.8	^107.3	^270.1	^103.1
^48h	^96.5		^380.7	^5.5

^{in brackets the values expressed [C14] nmol equiv. MDI}

^{Radioactivity in haemoglobin:}

^{at the end of the exposure haemoglobin contained 25 µg equiv. MDI/g mainly consisting of metabolites I and IV (as shown by GC-MS analysis after acidic hydrolysis).}

Applicant's summary and conclusion

Conclusions:

The major urinary metabolite was N,N'-diacetyl-4,4'-diaminobenzhydrol (50% of urinary radioactivity). All other identified metabolites were intermediates on the same metabolic pathway (N-acetylation and CH2-hydroxylation).
These metabolites were also identified in bile (7 -28% of bilary radioactivity) although the major components were identified as polyureas derived from MDI.
The major radioactive components in faeces were identified as polyureas derived from MDI.
No free MDA was detected in any of the biomatrices investigated.
The concentration of MDA in haemoglobin was below the limit of quantitation, metabolites I and IV were present at 117g/ml following acidic hydrolysis.
The highest concentration of MDA (483ng/ml) and metabolites I and IV (174ng/ml) were detected following hydrolysis for the 6h urine sample using acidic conditions.

Executive summary:

In order to better understand the toxicokinetic behavior of MDI after inhalation exposure, biological samples from the Gledhill (2001a) experiments (urine, feces, bile) were investigated in a separate study on the metabolism of MDI. Urine, feces and bile were collected for the identification of metabolites at 6 (in urine and bile only), 12, 24, 36 and 48 h (and for intact animals at 24 hourly intervals until 7 days after the end of exposure). Metabolites present in bile and excreta were identified by LC/MS and/or by co-chromatography with reference standards and quantified. The major urinary metabolite was N,N'-diacetyl-4,4'-diaminobenzhydrol (50% of urinary radioactivity). All other identified metabolites were intermediates on the same metabolic pathway (N-acetylation and CH2-hydroxylation). These metabolites were also identified in bile (7 -28% of biliary radioactivity) although the major components were identified as polyureas derived from MDI. The major radioactive components in feces were identified as polyureas derived from MDI. No free MDA was detected in any of the biomatrices investigated.