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

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

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.

Objective of study:

metabolism

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

Radiolabelling:

yes

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

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)

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.

Key result

Test no.:

#1

Toxicokinetic parameters:

other: no evidence of free MDA detected

Key result

Test no.:

#1

Toxicokinetic parameters:

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

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.

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).}

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.

Reference 2

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.

Objective of study:

distribution

excretion

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Principles of method if other than guideline:

Investigation of the routes and rates of excretion and the tissue distribution of radioactivity in the male rat following a 6 hour inhalation exposure to [14C]-MDI/kg.

GLP compliance:

yes

Radiolabelling:

yes

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: 291-348g
- 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

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): 3.67mg/l +/-0.76
The received mean total dose of radioactivety was 33.72 kBq per animal, being equivalent to 0.078 mg MDI equivalents per rat.
- 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.): 1.39µm/ 2.01
Analysis of the radioactiv dose preparation by liquid scintillation counting.

Duration and frequency of treatment / exposure:

6 hour(s)

Dose / conc.:

2 mg/m³ air (nominal)

Dose / conc.:

3.67 mg/m³ air (analytical)

Remarks:

Males: (non toxic dose level)

No. of animals per sex per dose / concentration:

Males: 4 Females: 0

Details on study design:

Immediately after the exposure, one group was killed by a lethal injection to determine the received dose. These rats were decapitated and the skin was separately removed from the head and the body. The head, skin from the head, carcass and pelt were analysed separately. The radioactivity in each of these tissues was summed for each animal to determine the received dose.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma, serum, various tissues, cage washes, bile
- Time and frequency of sampling: Four groups were killed at intervals up to 168 hours after exposure and selected tissues taken for analysis in order to investigate tissue distribution of absorbed material. Urine and faeces were also collected from these rats. Rats in the fifth group were surgically fitted with a bile duct cannula prior to exposure and urine, bile and faeces were collected until 48 hours after dosing, when these rats were terminated.
The radioactivity in excreta, bile and tissues was measured.
In a separate exposure experiment, 4 male rats were exposed to a similar 14C-MDI atmosphere for 6 hours before urine, faeces and exhaled CO2 were collected for up to 36 hours.

ANALYSIS OF RADIOACTIVITY: Liquid scintillation counting (Packard Tricarb instrument)

Type:

absorption

Results:

In conclusion, most of the systemically available dose was excreted via bile with a slightly smaller proportion excreted in urine. There was no radioactivity present in exhaled CO2.

Type:

distribution

Results:

Radioactivity was widely distributed, with the respiratory and excretory organs containing the highest concentrations of radioactivity. These declined to low levels of residues in all tissues analysed after 168 hours.

Details on absorption:

Particle size analyses (MMAD 1.39 µm, GSD 2.01) and the amounts of radioactivity found in the lungs (approximately 13% of the dose) demonstrated that the 14C-MDI atmospheres were highly respirable to the rats. Inhalation exposure of rats to 14C-MDI resulted in the rats receiving a combined inhalation/oral dose as radioactivity deposited on the head and pelt during the exposure was ingested during grooming.

During the 168 hour post-exposure period approximately 5% and 79% of the dose were excreted in urine and faeces, respectively (see table2). Over a 48 hour interval after exposure, bile duct cannulated rats excreted approximately 12% of the dose in urine, 14% in bile and 34% in faeces. In conclusion, most of the systemically available dose was excreted via bile with a slightly smaller proportion excreted in urine. There was no radioactivity present in exhaled CO2.

Details on distribution in tissues:

Radioactivity was widely distributed, with the respiratory and excretory organs containing the highest concentrations of radioactivity. These declined to low levels of residues in all tissues analysed after 168 hours.
More in detail, immediately after the exposure period the highest proportions of radioactivity were found in the residual carcass and gastrointestinal content (accounting for 37% and 32% of the dose respectively). The lungs, gastrointestinal tract, liver and respiratory nasal tissue accounted for 13%, 4%, 3% and 1% of the dose respectively. All other tissues measured contained less than 1% of the received dose. The highest concentration of radioactivity (67 µg equiv./g) was present in the respiratory nasal tissue.
The lungs and trachea contained 7 and 3 µg equiv./g respectively, and the olfactory nasal tissue and thyroid each contained approximately 1.0 µg equiv./g respectively. The concentration of radioactivity in each of the other tissues analysed was 0.5 µg equiv./g or less. At 8 hours after the end of exposure the gastrointestinal contents contained the highest proportion of radioactivity, accounting for 48% of the dose. The residual carcass contained 34% of the dose and the lungs 10%. Respiratory nasal tissue and the liver contained 4% and 3% of the dose respectively, and the gastrointestinal tract and stomach contents each contained 2% of the dose. Again, the respiratory nasal tissue contained the highest concentration of radioactivity, 394 µg equiv./g. The lung, trachea and olfactory nasal tissue contained the next greatest concentrations: 5 µg equiv./g in the lungs and 1 µg equiv./g in each of the trachea and olfactory nasal tissue. Other tissues contained radioactivity at 0.5 µg equiv./g or less.
By 24 hours after the end of exposure the highest proportion of radioactivity was present in the residual carcass and the gastrointestinal contents (19% and 13% of the dose respectively). Lungs and liver were the only other tissues containing appreciable amounts of radioactivity, 6% and 2% of the dose respectively.

All other tissues contained radioactivity at or below 1% of the dose. Respiratory nasal tissue, lungs and trachea contained radioactivity at concentrations of 17.3 and 1 µg equiv./g respectively. The concentration of radioactivity in the olfactory nasal tissue was 0.3 µg equiv./g and in the adrenals 0.2 µg equiv./g. All other tissues contained radioactivity concentrations of 0.2 µg equiv./g or less. At 168 hours after exposure, the lungs contained 4% of the administered dose and the gastrointestinal contents contained 1% of the dose. The residual carcass contained 5% of the dose. All other tissues contained less than 1% of the dose. Respiratory nasal tissue, thyroid and the lungs contained the highest concentrations of radioactivity, 3 µg equiv./g, 3 µg equiv./g, and 2 µg equiv./g respectively. The concentration of radioactivity in the adrenals was 0.3µg equiv./g. All other tissues contained less than 0.2 µg equiv./g.

Key result

Details on excretion:

Total excretion in rats 168h after dosing: 86.4% (total recovery: 99.5%), approximately 79% in faeces, 5% in urine and 3% in terminal cage wash. 1% of the dose was present in the GI-tract content.
Total excretion in bile cannulated rats 48h after dosing: 62.16%, approximately 34% in faeces, 12% in urine, and 14% in the bile. At the termination of this experiment the mean percentage of dose present in the gastrointestinal tract contents was 23%.

Key result

Test no.:

#1

Toxicokinetic parameters:

other: systemic dose 25 % of the received dose (animal terminated immediately after exposure (0 minutes)

Key result

Test no.:

#1

Toxicokinetic parameters:

other: systemic dose 32 % of the received dose (animal terminated 168 h after exposure)

Metabolites identified:

not measured

According to the author, the tissue distribution of radioactivity at different time points after exposure, when considered with the excretion data, imply that the systemic doses were mainly due to absorption of radioactive material present in the gastrointestinal tract after ingestion, with pulmonary absorption of radioactivity deposited in the lungs accounting for only a minor, hardly quantifiable portion of the systemic dose.

Immediately at the end of the exposure period 32% of the received dose was present in the gastrointestinal tract, which could be explained by ingestion of radioactivity deposited on the head and the respiratory tract during exposure. The residual carcass analysed immediately at the end of the exposure period contained 37% of the received dose, with 36% of this present on the skin and 1% present in the true residual carcass (without skin). At 168 hours after dosing, the radioactivity in the residual carcass had decreased to 5% of the received dose. This decrease in radioactivity was accompanied by a concomitant increase in the cumulative excretion of radioactivity in the faeces. It was found reasonable by the author, to conclude therefore, that radioactivity present on the skin was ingested during grooming. This conclusion was supported by the excretion, from the bile duct cannuled rats, of 34% of the received dose in faeces. If the exposure to radioactivity was exclusively via the inhalation route, the radioactivity in faeces from these animals would have been negligible.

Table 1: Distribution of radioactivity (%) in rats terminated immediately after cessation of exposure.

^skin from head	^28
^pelt	^8
^head	^15
^carcass	^49

Table 2: Mean excretion of radioactivity in urine, faeces and bile (expressed as % of received radioactivity +/-SD)

^time after dosing [h]	^urine	^faeces	^urine (bile cannulated rats)	^faeces (bile cannulated rats)	^bile
^0-6	^0.39 +/-0.12	^-	^7.57 +/-0.12		^6.25 +/-1.77
^6-12	^1.35 +/-0.29	^36.82 +/-9.61	^1.41 +/-0.12	^17.08 +/-13.4	^2.3 +/-0.43
^12-24	^0.94 +/-0.24	^6.01 +/-4.03	^1.31 +/-0.12	^4.01 +/-4.01	^2.36 +/-0.73
^24-36	^0.68 +/-0.33	^17.03 +/-4.76	^1.51 +/-0.12	^7.47 +/-4.55	^1.98 +/-0.86
^36-48	^0.26 +/-0.02	^2.84 +/-3.05	^0.73 +/-0.12	^5.87 +/-4.213	^0.93 +/-0.74
^48-72	^0.46 +/-0.17	^6.91 +/-1.91
^72-96	^0.38 +/-0.09	^3.65 +/-0.74
^96-120	^0.3 +/-0.11	^2.55 +/-0.51
^120-144	^0.18 +/-0.03	^1.61 +/-0.29
^144-168	^0.151 +/-0.04	^1.34 +/-0.68
^0-48	^-	^-	^12.36 +/-6.89	^34.44 +/-14.83	^13.83 +/-8.5
^0-168	^5.09 +/-1.09	^78.75 +/-17.49

Table 3: Tissue and carcass residues of radioacticity. Values expressed as % of received radioactivity (+/-SD) of n=4 rats. Only tissues with >1% are listed.

	^0h	^8h	^24h	^168h
^GI-tract	^4.173 +/-0.801	^2.391 +/-0.769	^0.992 +/-0.406	^0.141 +/-0.007
^liver	^3.379 +/-0.756	^2.876 +/-0.42	^2.004 +/-0.408	^0.424 +/-0.058
^lungs	^12.771 +/-2.521	^10.15 +/-1.897	^5.558 +/-0.944	^3.558 +/-0.503
^nasal tissue (respiratory)	^1.44 +/-1.873	^4.069 +/-2.603	^0.182 +/-0.247	^0.058 +/-0.01
^residual carcass	^37.106 +/-9.752	^33.56 +/-8.637	^18.539 +/-4.058	^5.001 +/-1.187
^total	^61.063	^54.901	^28.544	^5.159
^GI-content	^31.787 +/-5.133	^47.543 +/-19.356	^13.177 +/-5.487	^0.617 +/-0.13

Conclusions:

Interpretation of results: low bioaccumulation potential based on study results

Executive summary:

Following a 6 hour inhalation exposure to [14C]-MDI, radioactivity was widely distributed. Initially, tissues associated with the respiratory tract contained the highest concentration of radioactivity, but over the 168 hour time course after the exposure period, these declined markedly to leave low residues in all tissues analysed.

Radioactivity deposited on the head and pelt during the exposure was ingested during grooming resulting in a combined oral/inhaled dose, with the ingestion derived dose predominating. Most of the systemically available dose was excreted via bile (14%) with a slightly smaller (12%) proportion excreted in urine.

Reference 3

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: reliable without restriction

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7600 (Dermal Penetration)

Principles of method if other than guideline:

Absorption, distribution and excretion of radioactivity was studied in male Wistar rats following a single dermal and intradermal administration of [14C]-MDI.

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. Karl Thomae, Biberach (FRG)
- Age at study initiation: 8 weeks
- Weight at study initiation: ca. 250-300 g
- Housing: type III Macrolon cages
- Individual metabolism cages: yes, type Metabowl
- Diet (e.g. ad libitum): Kliba lab diet (Klingentalmühle AG, CH-4303, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): yes


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70% relative

Type of coverage:

semiocclusive

Vehicle:

acetone

Duration of exposure:

8h

Doses:

dermal administration: 4 and 0.4 mg/cm2 corresponding to 40 and 4 mg/animal (approx. 140 and 14 mg/kg bw)
intradermal administration: 0.4 mg/animal (1.4 mg/kg bw)

No. of animals per group:

4 dose and exposure group

Control animals:

no

Details on study design:

Dermal administration:
24 h prior to dosing the back shoulders of the rats were clipped free of hair and the area (about 10 cm2) was washed with acetone.
A silicon ring was glued to the skin, the test substance preparation (about 10 µl/cm2) was administered with a syringe which was weighed before and after application. A nylon mesh gauze was then glued to the surface of the silicone ring and a porous bandage used to encircle the trunk of the animal.
Duration of exposure: 8h

Intradermal administration:
24h prior to dosing the back shoulders of the rats were clipped free of hair and the area (about 10 cm2) was washed with acetone. The test substance preparation (about 100 µl) was administered with a syringe which was weighed before and after application . The injection area was sealed with tissue glue in order to avoid leakage of the test substance preparation.

Pretest for intradermal administration:
Using unlabelled test substance in two male rats with intradermal administration of 0.4 mg MDI/animal.

Collection of excreta: after 8, 24, 48, 72, 96 and 120 h if animals were not sacrificed before.
Sacrifice after 8, 24, 120h
After the respective exposure period the protective cover was removed and the exposed skin was washed with a mild soap solution.
At the end of the various collection periods animals were sacrificed and the following specimens/tissues were analysed for remaining radioactivity:
excreta, bloodcells, plasma, lung, heart, spleen, kidneys, adrenals, gonads, muscle, brain, adipose tissue, bone, thyroid, pancreas, stomach contents, stomach, gut contents, gut, liver, carcass, skin (treated and non-treated areas).
Additionally cage wash, skin wash and the protective cover (including the silicone ring) were also analysed for radioactivity.

For immunological investigations, retroorbital sampling of blood was performed after dermal and intradermal administration of non-radioactive MDI (results reported elsewere).

Radioactivity was measured by liquid scintillation counting.

Signs and symptoms of toxicity:

no effects

Absorption in different matrices:

Very limited absorption after dermal administration (0.9%) but considerable absorption after intradermal administration (26%). Due to the reactive nature of the test substance, considerable amounts of radioactivity could be found at the application site which could not be washed off .

Time point:

8 h

Concentrate / Dilution:

dilution

Dose:

4.0 mg/ cm2 (nominal)

Parameter:

percentage

Absorption:

0.21 %

Remarks on result:

other: dermal application, exposure time: 8 h, sacrifice time: 8 h

Time point:

24 h

Concentrate / Dilution:

dilution

Dose:

4.0 mg/cm2 (nominal concentation)

Parameter:

percentage

Absorption:

0.66 %

Remarks on result:

other: dermal application, exposure time: 8 h, sacrifice time: 24 h

Key result

Time point:

120 h

Concentrate / Dilution:

dilution

Dose:

4.0 (nominal) mg/cm2

Parameter:

percentage

Absorption:

0.88 %

Remarks on result:

other: dermal application, exposure time: 8 h, sacrifice time: 120 h

The test substance was stable in the respective carriers.

Excretion, retention and tissue concentrations after dermal application of 14C-MDI:

Table 1: Mean excretion and retention of radioactivity after a single dermal administration of 14C-MDI (% of the radioactivity administered).

nominal dose [mg/cm2]	4			0.4
sacrifice time [h]	8	24	120	8	24	120
actual dose [mg/cm2]	4.6	4.7	4.8	0.42	0.42	0.42
urine	0.01	0.03	0.05	0.01	0.04	0.09
feces	0	0.02	0.13	0	0.05	0.16
cage wash	0	0	0.03	0	0.01	0.06
stomach content	0	0	0.18	n.d.	n.d.	n.d.
gut content	0.02	0.02	0.29	n.d.	n.d.	n.d.
gut	0	0	0.01	n.d.	n.d.	n.d.
carcass	0.17	0.6	0.21	0.13	0.13	0.38
material absorbed	0.21	0.66	0.88	0.14	0.23	0.69
skin (surrounding)	4.75	7.14	0.55	3.17	1.37	1.47
protective cover	65.9	64.3	69.2	44.49	50.03	42.64
skin (application site)	29.6	25.5	32.2	54.26	55.62	61.14
skin wash	0.5	0.3	0.3	0.81	0.82	0.47
Total recovery	101	97.9	103.1	102.9	108.1	106.4
absorbtion [mg/animal]	0.0973	0.03165	0.4299	0.0058	0.0096	0.029
absorbtion [mg/cm2]	0.0097	0.0316	0.043	0.00058	0.00096	0.0029

In all groups, the largest proportion of radioactivity was recovered from the dressing and the skin of the application site. The amount of radioactivity absorbed (including excreta, cage wash, tissues/organs and carcass) increased in time, but remained below 1% at all dose levels. Excretion occured via urine and feces. In similar amounts after 8 and 24h, but to a higher extend in feces after 120h. The radioactivity absorbed was distributed in all organs and tissues. Due to the limited dermal absorption, concentrations of radioactivity in organs and tissues analyzed were considerably below 1 µg Eq/g (except for carcass). Levels of tissue radioactivity were comparable at 8 and 24 h and declined until 120 h after application with highest levels generally being found in carcass, thyroid, muscle, plasma and liver (not shown in table).

Excretion, retention and tissue concentrations after intradermal application of 14C-MDI:

Table 2: Mean excretion and retention of radioactivity after a single intradermal administration of 14C-MDI (% of the radioactivity administered).

nominal dose [mg/cm2]	0.4
sacrifice time [h]	120
actual dose [mg/cm2]	0.515
urine	4.51
feces	17.1
cage wash	0.75
stomach content	0.05
gut content	0.48
gut	0.09
liver	0.32
carcass	2.18
material absorbed	25.87
skin (surrounding)	8.47
skin (application site)	66.45
skin wash	0.11
Total recovery	100.9
absorbtion [mg/animal]	0.1332

The largest proportion of radioactivity was found at the application site. The amount of radioactivity absorbed (including excreta, cage wash, tissues/organs and carcass) during the 120 h observation period amounted to 25.87 % of the dose applied. Excretion occurred mainly via the feces and concentrations of radioactivity in organs and tissues were rather low being below 1 µg Eq/g.

Conclusions:

Very limited absorption after dermal administration (0.9%) but considerable absorption after intradermal administration (26%). Due to the reactive nature of the test substance, considerable amounts of radioactivity could be found at the application site which could not be washed off.

Executive summary:

The absorption, distribution and excretion  of radioactivity was studied in groups of four male Wistar rats following a  single dermal and intradermal administration of ¹⁴C- MDI at nominal dose levels of 0.4 and 4.0 mg/m² for dermal administration and 0.4 mg/animal for intradermal administration. Considering the animals weights, dose levels corresponded to about 14 mg/kg bw and 140 mg/kg bw (dermal administration) and 1.4 mg/kg bw (intradermal administration). In the experiment with dermal application, animals were exposed for 8 hours and scarified 8, 24, or 120 hours after treatment.

After dermal application of ¹⁴C-MDI, mean recoveries of radioactivity from all dose groups were in the range of 97.86 to 108.07% of the total radioactivity administered. Generally the largest proportion of radioactivity was found at the application site and dressing. The total amount of radioactivity absorbed (including excreta, cage wash, tissues/organs and carcass) increased with increasing sacrifice time. Dermal absorption was very low and quantitatively similar at both dose levels; maximally ca. 0.9% of the applied radioactivity was absorbed.

After intradermal administration of ¹⁴C-MDI, the mean recovery of radioactivity was 100.90% of the radioactivity applied. The largest proportion of radioactivity was found at the application site. The total amount of radioactivity absorbed (including excreta, cage wash, tissues/organs and carcass) amounted 26% of radioactivity applied.

Irrespective of the mode of administration of ¹⁴C-MDI, concentrations of radioactivity in tissues and organs generally were low 1 µEq/g at 120 hours after administration.

In summary, the results of this study comparing systemic availability of radioactivity after single dermal and intradermal administration of 14C-MDI clearly demonstrated very limited absorption after dermal administration but considerable absorption after intradermal administration.

Description of key information

 

Oral exposure: No information is available on the toxicokinetic of MDI following oral exposure in animals.

 

Dermal exposure: There are no study data available for the target substance MDI MT. A read across is performed to study data of the source substance 4,4’-MDI. Two in vivo studies on the dermal absorption and distribution of radioactive labelled MDI in rats are available. Both studies appear to be reliable, although they show contradictory results. From the study of Vock and Lutz (1997) it must be concluded that absorption through the skin is not negligible. Following un-occluded application of 14C-MDI 10% of the administered radioactivity was recovered in the epidermis, 20-30% in faeces and 1% in urine. In the GLP-study of Leibold et al. (1999) the largest proportion of radioactivity was recovered from the application site (32/61% for high dose/low dose) and the dressing (69/42%). Dermal absorption was rather limited following semi-occlusive application. Only 0.9% of the applied radioactivity was absorbed at the most. Absorbed radioactivity was excreted via urine and faeces, with the faecal route of excretion becoming more relevant during prolonged observation time. Although addressed in the study report, contamination from grooming probably explains high levels of absorption found by Vock and Lutz, whereas the high amount of competitive extraction of 14C-MDI to the dressing may contribute to low levels of absorption detected by Leibold et al. (1999).

An in vitro study (Clowes et al., 1999) confirmed the low levels of ¹⁴C-MDI absorption obtained by Leibold et al. (1999). No radioactivity was absorbed through human skin during a 54 hours continuous exposure, and only small amounts (maximally 0.23% of applied dose) were absorbed through rat and guinea pig skin. The majority of applied MDI equivalents were found to be associated with the skin.

As the source substance 4,4’-MDI and the target substance MDI MT contain sufficient monomeric MDI, the driver of toxicity, similarities in reactions with extracellular nucleophilic biomolecules at the site of contact are assumed. As the higher molecular weight non-monomeric content of the UVCB substance MDI MT do not contains reactive centres 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 is warranted.

 

Inhalation exposure: There are no inhalation study data available for the target substance MDI MT. A read across is performed to study data of the source substance 4,4’-MDI. In a two-part inhalation metabolism/toxicokinetics/ distribution study performed by (Gledhill, 2003a; Gledhill, 2003b), tissue distribution of radioactivity after nose-only inhalation exposure to radiolabelled 4,4’-MDI, when considered with excretion data, imply that tissue residues resulted from absorption of radioactive material primarily from the gastrointestinal (GI) tract after ingestion of an inhaled dose.  Further, pulmonary absorption of radioactivity deposited in the lungs accounts for only a minor portion of the administered dose. Similar results were obtained in an older study by Centre d`Etudes (1977) where the faecal elimination of 4,4-MDI was greater than the urinary elimination. In both studies, radioactivity was widely distributed with the respiratory and excretory organs containing the highest concentrations. The highest concentration of radioactivity was present in the respiratory nasal tissue (Gledhill, 2003). Due to grooming and respiratory clearance a combined oral/inhaled exposure existed in the study of Gledhill (2003) with 25 to 32 % of the applied dose entered the systemic circulation. According to Gledhill (2003)  no unreacted monomeric 4,4’-MDI were systemically available.

In order to better understand the toxicokinetic behaviour of MDI after inhalation exposure, biological samples from the Gledhill (2001) 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. 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 in bile and faeces were identified as polyureas derived from MDI. No free MDA was detected in any of the biomatrices investigated.

Taken together, these data support that following exposure, most inhaled test material or reaction products (polymerized insoluble particles and other insoluble adducts) are removed via mucociliary transport and swallowed with a small portion absorbed via solubilized macromolecular adducts via lung into blood stream. Systemic toxicity has not been observed in any in vivo study with either 4,4’-MDI or pMDI which is attributed to the lack of systemic bioavailability of the reactive isocyanate functional group. As described by (Wisnewski et al. 2019a) the fraction of monomeric MDI absorbed by the lung is exclusively via MDI adducts, consisting primarily of soluble low molecular weight MDI-glutathione (GSH) adducts or MDI-protein adducts. These soluble low molecular weight MDI-adducts are enzymatically metabolized as described by Wisnewski et al. (2016, 2019b), Bruggeman et al. (1986), Hinchman et al. (1991) and Bartels et al. (2009), which is in line with the results of Gledhill (2003), with the almost exclusively detection of MDI-metabolites with no evidence of  free MDI or MDA.

As the source substance 4,4’-MDI and the target substance MDI MT contain sufficient monomeric MDI, the driver of toxicity, similarities in reactions with extracellular nucleophilic biomolecules at the site of contact are assumed. As the higher molecular weight non-monomeric content of the UVCB substance MDI MT do not contains reactive centres 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 substance 4,4’-MDI is warranted.

Key value for chemical safety assessment

Absorption rate - dermal (%):

0.9

Absorption rate - inhalation (%):

32

Additional information

Other routes of exposure: After intradermal administration of 14C-MDI to rats about 26% of the radioactivity was absorbed. Excretion was mainly via faeces (Leibold et al., 1999). Following intramuscular injection of 14C-MDI less than 25% of the applied dose was recovered in the excreta 120h after application. The amount of faecal elimination was larger if compared to urinary elimination, indicating that for absorbed MDI biliary transport is a significant route of excretion. The remaining radioactivity was found in the carcass.