4,4'-methylenediphenyldiisocyanate and 2,4'-diisocyanatodiphenylmethane and their oligomerisation reaction products with 2,2'-oxydiethanol, propane-1,2-diol and butane-1,3-diol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Method and description are in accordance with test guidelines. Complete test results are available conducted conform with GLP and OECD guideline.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Reference 2

Reference Type:

publication

Title:

Prenatal toxicity if inhaled polymeric methylenediphenyl diisocyanate (MDI) aerosols in pregnant Wistar rats.

Author:

Gamer A, Hellwig J, Doe J and Tyl R

Year:

2000

Bibliographic source:

Toxicological Sciences, 54, 431-440

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

9016-87-9

Cas Number:

9016-87-9

IUPAC Name:

9016-87-9

Constituent 2

Reference substance name:

Isocyanic acid, polymethylenepolyphenylene ester

IUPAC Name:

Isocyanic acid, polymethylenepolyphenylene ester

Details on test material:

SOURCE: Bayer A.G.
PURITY: technical; polymeric MDI (CAS-nr: 9016-87-9)
ANY OTHER INFORMATION: kept in aluminium bottles blanketed with nitrogen in exhaust hoods at room temperature.
Polymeric MDI contains about 50% monomeric MDI.

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Karl THOMAE, Biberach an der Riss, Germany
- Age at study initiation: Females: approx. 76 - 86 days old , Males age not specified.
- Weight at study initiation: approx. 238.9 g (females)
- Housing: Housed singly in wire cages.
- Diet (e.g. ad libitum): Ground Kliba laboratory diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12h each


IN-LIFE DATES: From: 20 April 1993 To: 11 May 1993

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Glass-steel inhalation chamber, volume V=1.4 m3 (BASF)
- Method of holding animals in test chamber: Kept singly in wired cages that were in a glass-steel inhalation chamber.
- Source and rate of air:
- Method of conditioning air: chamber-positive pressure was used.
- System of generating particulates/aerosols: test substance supplied to a 2-component atomizer with a continuous infusion pump and heated with a circulating thermostat. The aerosol was generated by compressed air into an aerosol mixing stage where the aerosol was mixed with conditioned air and then passed through a cyclone separator into the inhalation chamber.
Duration of test/exposure: 10 days exposure during days 6 to 15 post coitum (p.c.).
Particle size: mass median aerodynamic diameter (geometric standard deviation): 1.6 µm (1.8)/ 1.7 µm (1.8)/ 1.01 µm (0.112) and 1.00 µm (0.054)
Duration of test/exposure: 10 days exposure during days 6 to 15 post coitum (p.c.).



TEST ATMOSPHERE
- Brief description of analytical method used: Concentrations of the inhalation atmospheres of the test groups were analysed by HPLC after pre-column derivatisation with 1-(2-Pyridyl)-piperazin.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The nominal concentration was calculated from the mean of the supplied substance and the amount of the supply air. The concentrations of the inhalation atmospheres of the test groups were analysed by HPLC after pre-column derivatisation with 1-(2-Pyridyl)-piperazin.
Concentrations: mean actual (standard deviation): were 1.03 (0.20)/ 4.03 (0.80)/ 12.0 (1.60).
The concentrations of the test groups were monitored continuously with non-calibrated light scattering photometers.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: From about 4 p.m. to 7.30 a.m. of the following day
- Proof of pregnancy: the day that sperm was detected microscopically in the vaginal smear in the morning was designated gd 0. The next day: day 1 post coitum (p.c.)

Duration of treatment / exposure:

10 days during days 6 to 15 post coitum (p.c.)

Frequency of treatment:

once daily; 6h a day

Duration of test:

20 days: 5 pre-test: exposure to fresh air; 5 post-treatment

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 1.0, 4.0 and 12.0 mg/m³
Basis:

No. of animals per sex per dose:

25 mated females per group.

Control animals:

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

Details on study design:

Clinical observations performed and frequency:
Dams: daily for mortality. At least 3 times per exposure day: inspection for behaviour and state of health and once daily during the pre-test and the post exposure observation period
Maternal weights: were recorded on day 0, 2, 6, 9, 12, 15, 17 and 20 p.c. and body weight gains calculated.
Food and water consumption: at the exception of day 0, this was recorded daily with time frame cfr weights.
Surviving females were killed on day 20 p.c.. Uterus, ovaries were removed and the following data recorded: gravid uterine weight, liver and paired lung weights, number of ovarian corpora lutea, number and distribution of implantation sites classified as early or late resorptions, dead fetuses and life fetuses. Uteri form apparently nongravid rats or single-born pregnancies were stained to detect early resorption sites.
FOETI:
Status at day 20 p.c.
life or dead + viability
weight and sex of life fetus
all foetuses were examined for any external findings.
individual placental weights
half of the fetuses were prepared for examination of visceral anomalies (and discarded afterwards); the other half for skeletal anomalies (and retained afterwards)
Evaluation criteria for assessing the changes in fetuses are defined (to complete).

OTHER:
Conception rate: number of pregnant rats divided by number of fertilized females x 100.
Pre-implantation loss (%) = (a-b)/a x 100
Post-implantation loss (%) = (b-c)/b x 100
With a = number of corpora lutea; b = number of implantation sites; c = number of life fetuses.

- Dose selection rationale: On the basis of DRF study, the exposure concentrations for this study was selected.

Examinations

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily at leasr 3 times (workdays) during exposure days. Once during the preflow period and the post exposure observation period.


BODY WEIGHT: Yes
- Time schedule for examinations: 0, 2, 6, 9, 12, 15, 17 and 20 p.c. days.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption: 2, 6, 9, 12, 15, 17 and 20 p.c. days.


WATER CONSUMPTION: Yes
- Time schedule for examinations: 2, 6, 9, 12, 15, 17 and 20 p.c. days.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 p.c.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: dead fetuses

Fetal examinations:

- External examinations: Yes:
- Soft tissue examinations: Yes:
- Skeletal examinations: Yes:
- Head examinations: Yes:

Statistics:

The statistical data were evaluated using the BASF computer system.
Examination of dams and fetuses done by the Dunnett-test.
Food and water consumption, body weight (mean of mean), body weight change, corrected body weight gain (net maternal body opened, number of corpora lutea, number of implantations, number of resorptions and number of live fetuses, proportion of preimplantation loss, postimplantation loss, resorptions and live fetuses in each litter, litter mean fetal body weight and litter mean placental weight.
Female mortality, female pregnant at terminal sacrifice and the number of litters with fetal findings: Fisher’s Exact test was used for a pairwise comparison of each dose group with the control for the hypothesis of equal proportions.
The Wilcoxon-test for comparison of each dose group with the control for the hypothesis of equal medians. This test was performed one-sided and was used for the proportion of fetuses with malformations, variations, retardationa and/or unclassified observations in each litter.
If the results of these tests were significant, labels (* for p≤0.05, ** for p≤0.01 were used.

Indices:

See "Further details on study design"

Historical control data:

Results are compared with historical control range of the performing laboratory.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
The concentration of 12 mg/m³ during days 6-15 pc for 6 h/day resulted in clear signs of maternal toxicity. Maternal toxicity was substantiated by mortality, damage to the respiratory tract, reduced body weight development and reduced mean gravid uterus weights.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEC

Effect level:

4 mg/m³ air

Basis for effect level:

other: developmental toxicity

2

Dose descriptor:

NOAEC

Effect level:

4 mg/m³ air

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
At concentrations of 12 mg/m³, clear signs of developmental (embryo-/foeto-) toxicity in the form of reduced placental and foetal body weights and an increased occurrence of foetal skeletal (and overall) variations and retardation were recorded; however, no substance-induced teratogenic effects were observed up to and including the highest concentration (12 mg/m³).

Effect levels (fetuses)

Remarks on result:

other: no substance-induced effects were observed

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

MATERNAL TOXICITY EFFECTS BY DOSE LEVEL:
Mortality and day of death: 2 females (gravid) of the top dose died; one on day 15 p.c. and one on 18 p.c. (= post-treatment).<br>Other clinical observations: clinical treatment-related signs: only in the top dose: on days 12, 13, 14 and 15: mainly alterations in respiration (2-6 dams), bloody crust around nose (2-3 dams), piloerection (3-25 dams).<br>Body weights: gd 9, 12, 15, 17 and 20: signif. reduced in the top dose group.<br>Food consumption: from gd 6 to 9, 9-12, 12-15, 15-17 and 17-20: in the high-conc group; for gd 6-9 for the mid conc group.<br>Water consumption: in the top dose: cfr food consumption.<br>Body weight gain: pre treatment: no difference across the groups. For the rest of the time intervals measured (days 6-15, 0-20): significantly reduced in the top dose.<br>At necropsy: terminal body weight, carcass weight (body - gravid uterine weight) and net weight change (carcass gd 20 - body weight at gd 6) significantly reduced in the top dose.<br>Mean gravid weight was reduced at top dose but not at significantly level.<br>Absolute and relative liver weights: statistically significant reduced in the top dose<br>Absolute and relative lung weights: statistically significant increased in the top dose<br>Reproductive data: 2 animals, 1 at the mid and 1 and the top dose were not gravid. Of the gravid females one at the low dose and one at the top dose carried fully resorbed litter at termination. All remaining females had live litters.<br>Dose level (mg/m³): 0/ 1/ 4/ 12<br>Number of pregnant females at termination: 25/ 25/ 24/ 22<br>Number of implantation sites/litter (mean +/- SE): 15.0 (2.0)/ 15.2 (2.4)/ ? (2.6)/ 14.3 (3.8)<br>Number of viable litters: 25/ 24/ 24/ 21<br>Number of live foetuses (mean +/- SE): 13.4 (2.9)/ 14.1 (2.4)/ 14.4 (2.5)/ 13.0 (3.6).

FETAL DATA:

Sex ratio/litter: no sign. difference<br>Fetal body weight per litter (calculated as a mean on the basis of litters with live fetuses):<br>a) total: sig. reduced (p<0.01) in the top dose group b) males: idem ; c) females: idem<br>Placental weight/ litter: a) total: sig. reduced in the top dose; b) females: no diff. And c) males: sig.reduced in top dose (p 0.05)<br>Sex ratio: no sign. difference<br>Gross external changes:<br>Fetal external malformations: no sign. differences<br>Total Number live fetuses investigated/Number of litters evaluated: 336/25; 338/24; 345/24; 274/21<br>External malformations: Number of fetuses: 1/ 2/ 0/ 2<br>External malformations: Number of litters: 1/ 2/ 0/ 2<br>Reported : anophtalmia (control); cleft palate (low dose); anasarca (whole body oedema) and filiformed tail: top dose.<br>- Visceral malformations:<br>Number of fetuses: 1/ 0/ 0/ 5<br>Number of litters: 1/ 0/ 0/ 2<br>Reported were: hydrocephalus (control); globular shaped heart and dilatation of the left ventricle (top dose).<br>- Skeletal malformations:<br>Number of fetuses: 6/ 11/ 9/ 10<br>Number of litters:<br>Reported: asymmetric dumb-bell shaped or bipartite thoracic vertebral body(ies), absent thoracic sacral or caudal vertebra(e), bipartite sterne??, cleft sternum, fused and bifurcated rib(s), absent rib(s) at low and mid dose.<br>Fetal external variations: none<br>Visceral variations: (number of fetuses/litter) significantly increased at low dose not at mid and top dose.<br>Number of fetuses: 15/ 34/ 31/ 17<br>Number of litters: 11/ 15/ 12/ 12<br>Skeletal variations:<br>Skeletal variations and skeletal retardations (number of fet/litter) significantly increased in top dose. Significant changes in incidences: irregular sternebra(e) at top dose (number of litters).<br>Bipartite sternebrae at low dose (number of litters and number of fet/litter) and top dose (number of fet/litter)<br>Incomplete ossified vertebral bodies increased at top dose (number of fet/lit).<br>Total fetal skeletal retardations: sign increased at top dose (number of fet/litter)<br>Total fetal variations (number of fet/litter): significant increased (versus control as well as vs historical control?) in all MDI exposed groups (but without dose-response relationship).<br>Details: number of fetuses: 84/ 118/ 116/ 109<br>Number of litters: 23/ 24/ 23/ 21<br>Remark: this is considered to be due to the unexpected low number of fetal variations in the concurrent control group.<br>Total fetal malformations: no significant difference between the different groups and within the historical control range<br>Number of fetuses: 7/ 12/ 9/ 15<br>Number of litters: 5/ 7/ 8/ 8<br>At the top dose there are clear signs of toxicity (mortality, lung damage accompanied by respiratory symptoms and increased lung weights, decreased feed and water consumption and decreased weight gain. Reduced (not statistically significant) gravid uterus weight and observed developmental toxicity with reduced fetal body weights, reduced placental weight, increased incidence of fet/lit with skeletal variations and skeletal retardation are considered by the authors to be due at least in large part to reduced fetal body weights.

No substance induced teratogenic effects were observed up to and including the highest tested concentration (12 mg/m³).

Applicant's summary and conclusion

Conclusions:

The prenatal toxicity of polymeric MDI in pregnant rats was investigated by aerosol inhalation. The exposure in concentration of 12 mg/m³ during days 6-15 pc for 6 h/day resulted in clear signs of maternal toxicity. Maternal toxicity was substantiated by mortality, damage to the respiratory tract, reduced body weight development and reduced mean gravid uterus weights. At this concentration clear signs of developmental (embryo-/foeto-) toxicity in the form of reduced placental and foetal body weights and an increased occurrence of foetal skeletal (and overall) variations and retardation were recorded; however, no substance-induced teratogenic effects were observed up to and including the highest concentration (12 mg/m³).
At concentrations of 1 or 4 mg/m³, no signs of maternal toxicity and no substance-induced adverse effects on the gestational parameters or the foetuses were recorded.
The NOAEC for maternal and foetal toxicity therefore is 4 mg/m³.
The NOAEC for teratogenic effects is 12 mg/m³.