4,4'-methylenediphenyl diisocyanate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study falls short of highest standards concerning aspects of protocol or reporting. Acceptable literature publication based on full study report . Also poster presentation with minimal description of method and report

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

1996

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

1994

Reference 3

Reference Type:

publication

Title:

Untersuchungen zur chronischen Toxizität/Kanzerogenität von 4,4'-Methylendiphenyl-Diisocyanat (MDI). Band 1 - 3

Author:

Hoymann HG, Buschmann J, Heinrich U & Bartsch W

Year:

1995

Bibliographic source:

Fraunhofer-Institut für Toxikologie und Aerosolforschung, Forschungsbericht 116 06 084

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

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-isocyanato-4-[(4-isocyanatophenyl)methyl]benzene

Constituent 2

Reference substance name:

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

IUPAC Name:

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

Details on test material:

SOURCE: Bayer A.G. Leverkusen
PURITY: no data ; 4,4'- MDI (Desmodur 44 M Schuppen)
ANY OTHER INFORMATION: no data

Test animals

Species:

rat

Strain:

other: Wistar Crl: (WI)BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga, Germany
- Age at study initiation: No data
- Weight at study initiation: No data
- Fasting period before study: No data
- Housing: The rats were housed prior to conception in groups of two, and thereafter individually in cages with softwood bedding.
- Diet (e.g. ad libitum): Altromin 1314 N
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: No data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 + 2°C
- Humidity (%):40-70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): The light intensity was 150 lux (600 lux with additional light for working procedures).


IN-LIFE DATES: No data

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: yes, exposed in inhalation chambers to fresh air only.

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Exposure chamber
- Method of holding animals in test chamber: Animals were exposed by whole-body inhalation in inhalation chambers.
- System of generating particulates/aerosols: The MDI aerosol was generated using an evaporation condensation technique consisting of the following steps: atomisation of the liquidised MDI, complete evaporation of the droplets, and recondensation of the gaseous MDI by turbulent mixing with dilution air.
- Temperature, humidity, pressure in air chamber: No data
- Method of particle size determination: The aerosol in the exposure chambers was monitored continuously by means of light-scattering photometers, and a "Bemer" low-pressure impactor was used for the determination of the size distribution of the MDI aerosol.

TEST ATMOSPHERE
- Brief description of analytical method used: A technique (no details given) for sample collection and analysis of MDI in both particle and gas forms which guaranteed effective and continuous monitoring of the exposure atmosphere was applied.

- Samples taken from breathing zone: no data

VEHICLE (if applicable)
- Composition of vehicle: Clean air

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The MDI aerosol in the exposure chambers was monitored continuously by means of light-scattering photometers, and a "Bemer" low-pressure impactor was used for the determination of the size distribution of the MDI aerosol. A technique for sample collection and analysis of MDI in both particle and gas forms which guaranteed effective and continuous monitoring of the exposure atmosphere was applied.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: Overnight
- Proof of pregnancy: vaginal smear was considered p.c. 0 (post conception).

Duration of treatment / exposure:

10 days during gestational days 6 to 15

Frequency of treatment:

once daily; 6h a day

Duration of test:

6 to 15 post conception (p.c.); 5 days post-treatment without exposure

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 1, 3 and 9 mg/m3
Basis:
other: actual weekly concs

No. of animals per sex per dose:

26 inseminated females per group

Control animals:

other: yes, exposed in inhalation chambers to fresh air only

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: No data


BODY WEIGHT: Yes
- Time schedule for examinations: pc 1, 6 -15 and 20.


FOOD CONSUMPTION: Yes
- Food consumption: on days 6, 10, 15 and 20 p.c.


WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: on p.c. 20
- Organs examined: Number of corporea lutea, implantation sites, live and dead fetuses, early and late resorptions, pre- and postimplantation loss, and fetal and placental weights.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes

Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

- Other: pre- and postimplantation loss, and fetal and placental weights.

Fetal examinations:

- External examinations: Yes: all foetuses were thoroughly examined for gross anomalies
- Soft tissue examinations: Yes: visceral anomalies
- Skeletal examinations: Yes: half of the foetuses for skeletal anomalies and the degree of ossification after bone staining with alizarin red.

- Head examinations: No data

Statistics:

Parametric data: ANOVA and subsequent two-tailed Dunnett's test with the dam/litter as unit. Incidences: two-tailed Fisher's exact test based on both litters and fetuses. Mean values, standard deviations and sample sizes were calculated and tabled. Incidences were alternatively shown as number of fetuses affected/number of litters with affected fetuses. Differences were considered significant at p<0.05 (*) or 0.01 (**).

Indices:

No data.

Historical control data:

No data.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
MATERNAL TOXICITY EFFECTS BY DOSE LEVEL:
Mortality and day of death: no mortalities, no other clinical symptoms in the dams
Body weights: no effect on body weight gain.
Food consumption: decreased in all treated groups during exposure, with statistically significance from day 6 to 10 p.c. in the high and mid dose group and from day 10 to 15 p.c. in all treated groups. Food consumption returned to normal after exposure ceased.
At necropsy: no treatment-related effects, however
Absolute and relative lung weights: statistically significant increased in the top dose (parameter not investigated in the low- and mid-conc groups).
Reproductive data: 1/26 animal of the control group and 2/25 of the high-conc group were not pregnant. Nr of copora lutea, implantation sites, pre- and post implantation loss, fetal and placental weights, gross and visceral anomalies, degree of ossification and life fetuses per dam: no statistically significant differences between the groups.
Dose level (mg/m3): 0/ 1/ 3/ 9
No of mated dams: 26/ 26/ 25/ 25
No of gravid females: 25/ 26/ 25/ 23
No of corpora lutea: 19.4 (2.6)/ 18.9 (3.6)/ 19.1 (2.3)/ 19.9 (3.2)
No of implantation sites (mean +/- SE): 17.8 (1.5)/ 17.0 (2.1)/ 17.3 (3.6)/ 16.7 (2.3)
No of live foetuses per dam (mean +/- SE): 17.0 (1.6)/ 16.0 (2.1)/ 16.1 (3.6)/ 16.7 (2.3)
No of postimplantation losses (mean +/- SE): 0.9 (0.9)/ 1.0 (1.2)/ 1.2 (1.4)/ 0.7 (0.7)
Placental weights (mean +/- SE): 0.57 (0.05)/ 0.59 (0.05)/ 0.60 (0.08)/ 0.58 (0.06)

Effect levels (maternal animals)

1

Key result

Dose descriptor:

LOAEC

Effect level:

<= 9 mg/m³ air

Basis for effect level:

organ weights and organ / body weight ratios

other: Lung

Remarks on result:

other: 6h/day exposure (days 6-15 p.c.)

Remarks:

Lung weights not recorded in mid and low-dose groups

2

Dose descriptor:

other: LOAEL embryo/fetotoxicity

Effect level:

9 mg/m³ air

Basis for effect level:

other: effect type not specified

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Weight of live fetuses: a) males: comparable in all groups; b) females: comparable in all groups
Sex ratio: no significant difference
Gross anomalies (No of fetuses/ No of litters): no significant difference.
Details:
Total No investigated: 424/25; 415/26; 403/25; 385/23
Subcutaneous hemorrhages: 5/5; 5/5; 9/8; 7/6
Cyanosis: 0; 2/1; 0; 0
Runt: 0; 0; 0; 1/1
Total anomalies: 5/5; 7/6; 9/8; 8/7
Visceral anomalies (investigated: unilateral shortened incisor, asymmetric thymus, dilated renal pelvis, dilated ureter, blood in resp. tract, blood in peritoneum, free blood in brain, stomach, ocular region, renal pelvis, ureter, pericardium, thoracic cavity, subcutaneous hemorrhages on 203; 200; 193 and 186 fetuses): no relevant significant differences.

Slight dilatation of renal pelvis: low dose group: significantly increased number of fetuses with this anomaly (within the limits of biological variability of the strain; no dose-response relationship; not statistically different if comparison on a per litter base)

Degree of ossification: no relevant significant difference (exposure groups display decrease in incomplete or missing ossification).
Incomplete ossification of nasal bones: in the mid conc group: decrease in the number of fetuses (within tan. Variation; no dose-response relation). Incomplete ossification of sacral vertebral centers: in the mid and high dose groups: decrease in the number of fetuses (within tan. Variation; no dose-response relation; not statistically different if comparison on a per litter base).

Skeletal anomalies: (investigated: bipartite hyoid, accessory cervical rib(s), accessory lumbar rib(s), shortened rib(s), wavy rib(s), tandard-shaped vertebral centra(e), bipartite vertebral centra(e), asymmetric sternebra(e), bipartite sternebra(e) on 221; 215; 210 and 199 fetuses). Asymmetric sternebra(e): in the high-conc group: slight and significant increase in litters with fetuses displaying this anomaly. However: the relevance of this is limited by the fact it represents a minor variation (common in the strain used) and the observed incidence is still within the limits of biological variability of the rat strain used (no further data available hereon, at least not in the publication).
Accessory lumbar rib(s): mid dose group: sign.decrease in the number of fetuses with this anomaly (within boil. variation; no dose-response relation; not statistically different if comparison on a per litter base).

Effect levels (fetuses)

Dose descriptor:

NOAEC

Effect level:

3 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

other: minor increase in skeletal variation (asymmetric sternebrae) at the highest dose level.

Fetal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: sternum

Description (incidence and severity):

slight increase in asymmetric sternebra(e) that is within limits of biological variability

Overall developmental toxicity

Developmental effects observed:

yes

Lowest effective dose / conc.:

9 mg/m³ air (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

Table 1 (continued from details on maternal toxic effects)

	Dose level
	0 mg/m^3	1 mg/m^3	3 mg/m^3	9 mg/m^3
No of mated dams	26	26	25	25
No of gravid females	25	26	25	23
No of corpora lutea (mean +/- SE)	19.4 (2.6)	18.9 (3.6)	19.1 (2.3)	19.9 (3.2)
No of implantation sites (mean +/- SE)	17.8 (1.5)	7.0 (2.1)	17.3 (3.6)	16.7 (2.3)
No of live foetuses per dam (mean +/- SE)	17.0 (1.6)	16.0 (2.1)	16.1 (3.6)	16.7 (2.3)
No of postimplantation losses (mean +/- SE)	0.9 (0.9)	1.0 (1.2)	1.2 (1.4)	0.7 (0.7)
Placental weights (mean +/- SE)	0.57(0.05)	0.59(0.05)	0.60(0.08)	0.58(0.06)

Applicant's summary and conclusion

Conclusions:

Gravid rats were exposed by whole-body inhalation to clean air (control) and to 1, 3, and 9 mg/m3 monomeric MDI, respectively, for 6 hr per day from Days 6 to 15 post conception Treatment caused a dose-dependent decrease in food consumption in all substance-treated groups during exposure, returning to normal values after cessation of treatment. 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 fetal 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) was observed after treatment with the highest dose of 9 mg/m3. Although the relevance of an increase of this minor anomaly in doses which cause toxic effects in dams (reduced food consumption, increased lung weights) is limited and the number observed is within the limits of biological variability, a substance-induced effect in the high-dose group cannot be excluded with certainty. A no embryotoxic effect level of 3 mg/m3 was determined.

Executive summary:

The available study data for the endpoint developmental toxicity are consistent with the hypothesized MoA and based on the high reactivity of the NCO group. The hypothesized MoA predicts local effects in the lungs and no significant systemic exposure to unreacted NCO since it reacts with biological nucleophiles before being absorbed as GHS/protein adducts. No systemic toxicity was noted in the developmental toxicity study This is consistent with the observed lack of systemic toxicity in combined chronic toxicity and carcinogenicity studies on 4,4’-MDI and pMDI as well as the proposed mechanism for MDI absorption toxicokinetics.

All substances of the MDI category share similar chemical features namely that they a) all contain a significant amount of mMDI, and b) contain at least two NCO functional groups per molecule which is bound to an aromatic ring and this ring is connected to a second aromatic ring by a methylene group. It is the NCO value (driven by the bioaccessible groups on monomeric MDI and low molecular weight constituents (e.g. three-ring oligomer) which is responsible for chemical and physiological reactivity and subsequent toxicological profile. As reactive NCO groups are a common feature of all substances of the MDI category, it is predicted that these have a similar reactivity profile and a read across within the category is warranted (detailed information on the Mode of Action is available in Category Justification Document).