Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1986-1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well documented publication, which meets basic scientific principles

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1999
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OTS 798.4350 (Inhalation Developmental Toxicity Screen)
Principles of method if other than guideline:
Mated female CD (Sprague-Dawley) rats, 25/group, were exposed to toluene diisocyanate (TDI) vapour, for six h/day on gestational days (gd) 6 through 15, at 0.00, 0.02, 0.10, or 0.50 p.p.m. Maternal clinical signs, body weights, and feed and water consumption were recorded throughout gestation. At termination (gd 21), maternal body, gravid uterine, and liver weights were recorded. Corpora lutea were counted, and implantation sites were identified: resorptions and dead and live fetuses. All live fetuses were examined for external alterations. One-half of the live fetuses/litter were examined for visceral (including craniofacial) alterations. The remaining intact fetuses/litter were stained with alizarin red S and examined for ossified skeletal alterations.
GLP compliance:
yes
Remarks:
This study was performed in compliance with U.S. Environmental Protection Agency (EPA) TSCA (Toxic Substances Control Act) good laboratory practice regulations (U.S. EPA, 1983).
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Toluene diisocyanate - mixture of 2,4- and 2,6-TDI)], a commercial grade of an 80:20 mixture of the 2,4- and 2,6-isomers (CAS Nos. 584–84–9 and 91–08–7, respectively),
- Chemical name: Benzene, 1,3-diisocyanatomethyl
- Molecular formula (if other than submission substance): C9H6N2O2
- Molecular weight (if other than submission substance): 174.15 g/mol
- Smiles notation (if other than submission substance): CC(C)c1c(N=C(=O))c(C(C)C)c(N=C(=O))c(C(C)C)c1
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type: aromatic diisocyanate
- Physical state: liquid
- Composition of test material, percentage of components: 80:20 mixture of the 2,4- and 2,6-isomers (CAS Nos. 584–84–9 and 91–08–7),
- Isomers composition: 80:20 mixture of the 2,4- and 2,6-isomers (CAS Nos. 584–84–9 and 91–08–7, respectively),

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS: 188 virgin female and 187 virgin male (COBS) CDt (SD)BR outbred albino rats, referred to as the CD (Sprague-Dawley) rat - 63 (males) and 56 (females) days old upon arrival
- Source: Charles River Breeding Laboratories (Kingston, NY)
- Age at study initiation: (P) 42 days; (F1) 28 days
- Weight at study initiation: (P) Males: ca. 200 g; Females: ca. 150 g; (F1) Males: 125 -150 g; Females: 100-125 g
- Housing: individually housed (cage dimensions, 22.5 * 15.5 * 18 cm high) for the duration of the study (except during exposures). For exposures, plug-positive females were transferred to stainless steel wire mesh exposure cages (35 cm * 17 cm * 18 cm high) in cage carriers that were moved onto the chambers. After each exposure, animals were returned to their original cages and “home” room. Paperboard (Deotized Animal Cage Boardt, Shepherd Specialty Papers, Inc., Kalamazoo, MI) was placed beneath the cages.
control data indicated that these animals were in good health and suitable for use.
- Diet (e.g. ad libitum):Certified feed (Purina Certified Ground Rodent Chowt, No. 5002, Ralston Purina Co., St. Louis, MO) ad libitum except during exposures.
- Fasting period: during exposures: feed and water were withheld during exposures.
- Water (e.g. ad libitum): water (Municipal Authority of Westmoreland County, Greensburg, PA) were available ad libitum except during exposures.
- Acclimation period: quarantined for approximately 2 weeks, during which time representative animals were subjected to fecal sampling, aerobic bacteriologic cultures of lung tissue, histologic examination of selected organs, and to serum viral antibody evaluation. Quality control data indicated that these animals were in good health and suitable for use. During acclimation, the rats were housed separately by sex, 2–3 per cage, in stainless steel wire mesh cages (6 * 9 * 7 in. high).

ENVIRONMENTAL CONDITIONS
- Temperature: 62–76°F; room temperature was recorded continuously (Cole-Parmer Hygrothermograph Seven-Day Continuous Recorder, Model No. 8368–00, Cole-Parmer Instrument Company, Chicago, IL).
- Humidity (%): 40–70% , relative humidity was recorded continuously (Cole-Parmer Hygrothermograph Seven-Day Continuous Recorder, Model No. 8368–00, Cole-Parmer Instrument Company, Chicago, IL).
- Photoperiod (hrs dark / hrs light): 12-h, 0530–1750 h for light phase.

All animals were handled and treated at all times in conformance with the National Institutes of Health Guide (NIH, 1985).

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Liquid toluene diisocyanate was metered from a syringe pump (Sage Instruments, Cambridge, MA) into a heated glass evaporator, similar in design to that described by Carpenter et al. (1975).
The resultant vapour was carried into the chamber by a counter-current air stream that entered the bottom of the evaporator. Chamber atmospheres containing toluene diisocyanate were filtered before leaving an exhaust stack.
The four chambers employed in this study were rectangular in shape, constructed of glass and stainless steel (Wahmann Manufacturing Company, Timonium, MD), and measured approximately 2.1m * 1m * 2.1m (height). Total volume in each chamber was approximately 4320 L. An orifice plate was positioned in the exhaust duct of the chamber and was connected to a Dwyer Magnehelic Pressure Gauge. Airflow in each chamber was approximately 1000–1500 L/min (at least 14 air changes per h), with a t99 (theoretically derived time required for the chamber to reach 99% of the equilibrium concentration) of approximately 20 min. The chambers were illuminated with artificial room light. Within each chamber, the animal cages were rotated daily to compensate for any possible but undetected variation in chamber exposure conditions (i.e., concentration, temperature, relative humidity). A vapour distribution study involving six sampling positions each at 0.02 and 1.0 ppm indicated low coefficients of variation (3.0–7.4%) and, therefore, uniform distribution of toluene diisocyanate vapor within the range of target exposure concentrations employed.
- Method of holding animals in test chamber:
- Source and rate of air:
- Method of conditioning air:
- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber: The temperature in the evaporator was monitored and maintained at the lowest level sufficient to vaporize the liquid (46–60°C). Temperature (Minimum-Maximum Thermometer, Abbeon Cal, Inc., Santa Barbara, CA, or Brooklyn Thermometer Co., Inc., Farmingdale, NY) and relative humidity (Airguide Instrument Company, Chicago, IL) gauges were placed inside each chamber during exposures. Chamber temperature, relative humidity, and air- flow rate were recorded every 30 min during each 6-h exposure.
- Air flow rate:
- Air change rate:
- Method of particle size determination:
- Treatment of exhaust air:

TEST ATMOSPHERE
- Brief description of analytical method used: Two Autostep Portable Monitors (GMD Systems, Inc., Hendersonville, PA) were used to monitor the TDI concentration in the exposure chamber atmospheres. The Model 920 isocyanates instrument (0–0.2 ppm range) was used to measure the concentrations in the atmospheres of the control, the 0.02, and 0.1 ppm exposure chambers. A second instrument was specially constructed by the manufacturer to monitor the higher TDI concentrations of the 0.5 ppm target exposure chamber atmosphere.
Both instruments operated using the same basic principles. A volume of air was drawn through a chemically impregnated paper tape. A stain developed on the tape with an intensity proportional to the TDI concentration. The sensitivity to the 2,4- and 2,6-isomers was the same.
The GMD instruments were calibrated prior to exposure using the Marcali method, as modified by Rando and Hammad (1985).
The impinger samples were then analysed by the modified Marcali method, and the results were compared. Correction factors were calculated for the instruments, so that the corrected instrument results agreed with the modified Marcali results.
For analysis of TDI by the modified Marcali method, chamber atmosphere samples were drawn through two midget impingers (in series) using Reciprotor (Waltham Chemical Pump Corporation) pumps equipped with critical orifices.
Impinger efficiencies were 95%, and results were corrected accordingly. The impinger solution was 15 ml of 2.2% acetic acid, 3.5% hydrochloric acid in water. This solution was used to hydrolyze the TDI to its corresponding diamine. The samples were then cooled to 0°C in an icewater bath. A 0.5-ml aliquot (cooled) of 3% sodium nitrite, 5% sodium bromide in water was then added to the sample. These solutions were mixed rapidly using a vortex mixer and then allowed to react for 0.5 min. This reaction diotized the toluene diamine to a diazonium compound. One mL of 10% sulfamic acid was then added to destroy excess nitrous acid. One milliliter of 0.1% naphthylethylenediamine was added. A coloured complex formed. The sample was diluted to 25.0 ml with distilled water and the absorbance measured at 548 nm with a Bausch and Lomb Spectronic 710 UV-Visible Optical Spectrometer.
- Samples taken from breathing zone: yes - Instrument measurements and impinger samples were taken from each of the chamber atmospheres.
A soap film buret (24°C, 735 mm Hg) was used to determine the 1 l/min flowrates of the sampling systems. Sampling times were typically 20, 10, and 6 min for the low, mid, and high concentration atmospheres, respectively.
Each chamber atmosphere was analysed for toluene diisocyanate approximately six times during each 6-h exposure. Daily nominal concentrations (an estimated concentration calculated from the amount of test material delivered and the chamber airflow during the exposure period) were also calculated for each chamber.

For exposures, plug-positive females were transferred to stainless steel wire mesh exposure cages (35 cm x 17 cm x 18 cm high) in cage carriers that were moved onto the chambers. Feed and water were withheld during exposures. Exposures were for 6 h/day, gd 6–15. After each exposure, animals were returned to their original cages and “home” room. The animals were observed daily for clinical signs throughout the study (gd 0–21). Maternal body weights were taken on gd 0, 6, 9, 12, 16, and 21. Maternal feed and water consumption was measured at 3-day intervals, gd 0–3, 3–6, 6–9, 9–12, 12–15, 15–18, and 18–21.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber Analyses
The mean analytical concentrations for the 0.02, 0.10, and 0.50 ppm (0.14, 0.72, and 3.6 mg/m³) chambers were 0.021 +/- 0.0018 ppm (105.0% of target), 0.12 +/- 0.017 ppm (120.0% of target), and 0.48 +/- 0.038 ppm (96.0% of target), respectively. No test chemical was detected in the control chamber (0 ppm) with a minimum detection limit of 1 ppb. The analytical to nominal concentration ratios were 0.44–0.72. Mean temperature was maintained at 22.6 –23.9°C and mean relative humidity at 50.1–54.4% for all four chambers.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: housed in stainless steel wire mesh cages and the paperboard beneath the cages
- M/F ratio per cage: 1:1 (one male:one female)
- Length of cohabitation:
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- Verification of same strain and source of both sexes: [yes / no (explain)]
- Proof of pregnancy: checked twice daily for dropped copulation plugs. The date a copulation plug was found was designated gd 0 (Hafez, 1970).
- Any other deviations from standard protocol:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: one male:one female in stainless steel wire mesh cages and the paperboard beneath the cages checked twice daily for dropped copulation plugs
- Length of cohabitation:
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- Verification of same strain and source of both sexes: [yes / no (explain)]
- Proof of pregnancy: copulation plug referred to as day 0 of pregnancy
- Any other deviations from standard protocol:
Twenty-five mated females were assigned to each experimental group on gd 0 by stratified randomization procedures based on body weight.
Duration of treatment / exposure:
6 h/day
Frequency of treatment:
once daily
Duration of test:
gd 6–15, observation period gd 0 - 21
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.0 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.02 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.1 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.5 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
25 mated females
Control animals:
not specified
Details on study design:
- Dose selection rationale: To determine appropriate target concentrations of TDI for the definitive study, a range-finding study was performed following the same study design as in the definitive study, except that eight inseminated females per group were employed, the exposure concentrations were 0.0, 0.1, 0.5, and 1.0 ppm (analytical concentrations 0.0 [minimum detection limit of 1 ppb], 0.12 +/- 0.013, 0.48 +/- 0.018, and 0.95 +/- 0.093 ppm), and the dams were terminated after the last exposure on gd 15. At termination, immediately after the last exposure, the dams were anesthetized with methoxyflurane (Metofanet, Pittman-Moore) and arterial blood was removed from the abdominal aorta into arterial blood samplers (Carney Medical, Medfield, MA) containing heparin as an anticoagulant. Blood parameters directly measured were pH, pCO2 and pO2 (Corning Model 170 Blood Gas Analyzer, Corning Medical, Medfield, MA); HCO3- (bicarbonate) was calculated by the following formula: 0.031 pCO2 X 10E(pH–6.1). Commercially available quality control samples (Certain, Level I, II, III, Corning Medical, Medfield, MA) were analysed prior to and following every 10 samples, with appropriate calibration checks immediately prior to sample analysis. The dams were then examined for body weight, gravid uterine weight, liver weight, number of ovarian corpora lutea, number of uterine implantation sites (total, early, and late absorptions, and dead implants [there were none]), and live implants. At 1.0 ppm, maternal body weights, weight changes, and liver weights (absolute but not relative) were profoundly and significantly reduced, audible respiration and nasal red discharge were observed; stomach and intestines were gas filled (due to gasping), and adrenal glands were enlarged (most likely due to stress). There were no effects of treatment on any gestational parameters, including pre- or postimplantation loss and percent live implants per litter at any exposure concentration tested. The maternal blood gas results include the following statistically significant changes at 1.0 ppm: reduced pH (1%), markedly increased pCO2 (31%), markedly decreased pO2 (33%), and slightly increased HCO3- (13%). These alterations are characteristic of respiratory acidosis resulting from compromised pulmonary function. Typically, as a result of impaired alveolar ventilation, arterial O2 levels are reduced, CO2 is inefficiently eliminated, and the bicarbonate levels rise to compensate for the increased CO2 levels. (Based on the slight increase in bicarbonate and the effects on pH, pCO2 and pO2, compensation was apparently not achieved.) Since this compensating change takes place over 2–3 days, the alterations are considered to be of a chronic nature. (The use of methoxyflurane as an anaesthetic may have confounded the blood gas results, as the pH of conscious control rats is usually 7.5 and the mean pH of the anaesthetized control rats in this study was 7.38.) Based on these results, 1.0 ppm was deemed too toxic for use in the definitive study. The target exposure concentrations for definitive study were, therefore, 0.00, 0.02, 0.10, and 0.50 ppm.
- Rationale for animal assignment (if not random): random, each animal was assigned a unique number and received a stainless steel Monelt ear tag (Gey Band and Tag Co., Morristown, PA).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed daily for clinical signs throughout the study (gd 0–21).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed daily for clinical signs throughout the study (gd 0–21).

BODY WEIGHT: Yes
- Time schedule for examinations: Maternal body weights were taken on gd 0, 6, 9, 12, 16, and 21.

FOOD CONSUMPTION: Yes
- Maternal feed and water consumption was measured at 3-day intervals, gd 0–3, 3–6, 6–9, 9–12, 12–15, 15–18, and 18–21.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Maternal feed and water consumption was measured at 3-day intervals, gd 0–3, 3–6, 6–9, 9–12, 12–15, 15–18, and 18–21.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21 by carbon dioxide asphyxiation
- Organs examined: The gravid uterus, ovaries (including corpora lutea), cervix, vagina, and abdominal and thoracic cavities were examined grossly. Ovarian corpora lutea of pregnancy were counted, and maternal liver and uterine weights were determined. Sections of maternal liver, kidney(s), and upper and lower respiratory tract (including nasal turbinates) were retained in fixative for possible subsequent histologic examination. The uteri were immediately ligated at their cervical end to prevent the expulsion of conceptuses by myo- metrial peristalsis, externally examined for signs of haemorrhage, removed from the peritoneal cavity, and dissected longitudinally to expose their contents. All live and dead fetuses and resorption sites (early and late) were noted and recorded. Uteri from females that appeared nongravid were placed in a 10% ammonium sulfide solution for detection of early resorptions (Salewski, 1964).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
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
Fetal examinations:
A total of 21–23 litters were examined in each experimental group.

- External examinations: Yes: all live fetuses
- Soft tissue examinations: Yes: 1/2 in each litter was examined for thoracic and abdominal visceral abnormalities
- Skeletal examinations: Yes: 1/2 per litter (the other half)
- Head examinations: Yes: 1/2 per litter (those examined for thoracic and abdominal visceral abnormalities)

All live fetuses were anesthetized by hypothermia and were weighed, sexed, and examined for external malformations, including cleft palate and variations. Approximately one-half of the live fetuses in each litter (even numbered fetuses from litters with an even number of live fetuses; odd numbered fetuses from litters with an odd number of live fetuses) were examined for thoracic and abdominal visceral abnormalities by modification of methods described by Staples (1974). The fetuses examined viscerally were decapitated and their heads were fixed in Bouin’s solution for examination of craniofacial structures by serial freehand sectioning methods modified from Wilson (1965; 1973) and van Julsingha and Bennett (1977). Intact fetuses (not decapitated) in each litter were eviscerated, fixed in ethanol, then processed for skeletal staining with alizarin red S (Crary, 1962; Peltzer and Schardein, 1966) and examined for ossified skeletal malformations and variations. Decapitated fetuses were also processed for skeletal staining but were not examined.
Statistics:
The unit of comparison was the pregnant dam or the litter (Weil, 1970). Results of the quantitative continuous variables (e.g., maternal body weights, liver weights, etc.) were intercompared for the three exposure groups and control group by use of Levene’s test for equal variances (Levene, 1960), analysis of variance (ANOVA), and t-tests with Bonferroni probabilities. The t-tests were used when the F value from the ANOVA was significant. When Levene’s test indicated homogeneous variances, and the ANOVA was significant, the pooled t-test was used for pairwise comparisons. When Levene’s test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances (Brown and Forsythe, 1974), followed when necessary by the separate variance t-test for pairwise comparisons. Nonparametric data obtained following necropsy were statistically treated using the Kruskal-Wallis test (Sokal and Rohlf, 1969), followed by the Mann- Whitney U test (Sokal and Rohlf, 1969) when appropriate. Incidence data were compared using Fisher’s Exact Test (Sokal and Rohlf, 1969). For all statistical tests, the fiducial limit of 0.05 (two-tailed) was used as the criterion for significance.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: No females died, delivered early, aborted or were removed from study. Reduced Body weight gain, Clinical symptoms: audible respiration and red nasal discharge, reduced feed consumption.

Details on maternal toxic effects:
No females died, delivered early, aborted or were removed from study. Pregnancy rate was equivalent across all groups (84–92%), and all pregnant animals bore at least one live fetus at scheduled sacrifice, except for one dam with a fully resorbed litter at 0.02 ppm.
Body weights and weight gain were equivalent across all groups prior to the start of exposures, gd 0–6. Maternal body weight was significantly reduced at 0.50 ppm, only on gd 16, and at 0.02 ppm only on gd 21 (due at least partially to one fully resorbed litter at 0.02 ppm). Maternal weight gain was statistically reduced early in the exposure period, gd 6–9, at 0.02, 0.10, and 0.50 ppm. At 0.50 ppm, it was also reduced for gd 9–12, 12–16, and 6–16 (exposure period). In addition, weight gain was significantly reduced at 0.02 ppm for gd 16–21 (postexposure period) and for gd 0–21 (gestation period), again due, at least partially, to the one fully resorbed litter at this exposure concentration. Treatment-related clinical signs of toxicity were limited to audible respiration in 15 dams of 23 at 0.50 ppm, and red nasal discharge at 0.02 ppm (five dams), 0.10 ppm (three dams), and 0.50 ppm (seven dams during the exposure period). The latter sign did not exhibit a concentration-related incidence, but it was observed only in toluene diisocyanate-exposed dams during the exposure period; in the postexposure period (gd 16–21), the incidence was 1, 1, 2, and 6 dams at 0.0, 0.02, 0.10, and 0.50 ppm, respectively. Maternal feed consumption was significantly reduced at 0.50 ppm for gd 9–12 and 12–15, and for gd 6–15 (exposure period). In addition, feed consumption was reduced at 0.02 ppm for gd 18–21 (in the postexposure period), possibly due to the dam with one fully resorbed litter (eating at a nonpregnant rate) and smaller litter sizes in this group. Maternal water consumption exhibited no statistically significant differences among groups for any interval measured, gd 0–21. Maternal gross observations at necropsy indicated no treatment-related lesions. Maternal body weight at sacrifice was statistically reduced at 0.02 ppm, again most likely related to the one dam in this group with a fully resorbed litter and smaller litter sizes. Gravid uterine weights, body weights at sacrifice corrected for gravid uterine weight, and body weight change (gestational weight gain corrected for the weight of the gravid uterus) were unaffected by treatment, as was absolute or relative liver weight.

The maternal toxicity at 0.50 ppm included reduced maternal weight gain and feed consumption, and clinical signs of toxicity, all observed during the exposure period. Clinical signs were limited to audible respiration exclusively at 0.50 ppm during exposures, and red nasal discharge observed predominantly at 0.50 ppm during and after the exposure period (with no dose–response pattern at 0.02 and 0.10 ppm during exposure and at low incidence; one to two dams at these exposure concentrations in the postexposure period). Concentration-related reduced body weight gains were also observed for all TDI-exposed groups for the first exposure interval, gd 6–9. However, at 0.02 and 0.10 ppm, these effects were clearly transient (and less extreme, p , 0.05, versus the effect at 0.50 ppm, p , 0.001), with no significant effects occurring at 0.02 or 0.10 ppm for subsequent treatment intervals, gd 9–12 or gd 12–16, or for the entire exposure period, gd 6–16.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
0.1 ppm
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
0.1 ppm
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: reduced number of total implants per litter and of viable implants per litter at 0.02 ppm; one poorly ossified cervical centrum 5, exhibited an increased incidence at 0.50 ppm relative to that in controls, indicative of possible minimal fetotoxicity.

Details on embryotoxic / teratogenic effects:
The number of corpora lutea per dam was unaffected by treatment. The number of total implants per litter was reduced at 0.02 ppm, and the number of viable implants per litter was also reduced at 0.02 ppm, probably because of the reduced implant count (as preimplantation loss was not significantly affected by treatment), and because of one fully resorbed litter at 0.02 ppm. The number of nonviable implants per litter (total, early or late resorptions, or dead fetuses) and the percent live fetuses per litter were not significantly changed by treatment. Sex ratio (% males) per litter was unchanged by exposures. Fetal body weight per litter (males, females, or all fetuses) was unaffected by exposure.
There were no significant differences in the incidence of any individual malformation, of malformations by category (external, visceral, or skeletal), or of total malformations. There were no significant differences in the incidence of individual external or visceral variations, of variations by category, or of total variations. Out of 111 skeletal variations observed in the present study, six exhibited statistically significant differences in incidence in one or more toluene diisocyanate-exposed groups relative to the incidence in the control group. Only one of these, poorly ossified cervical centrum 5, exhibited an increased incidence at 0.50 ppm relative to that in controls, indicative of possible minimal fetotoxicity. This finding was observed in the absence of any other indication of developmental toxicity. The five remaining significant findings did not indicate fetotoxicity and did not appear treatment related. They included reductions in the incidence of bilobed thoracic centra 11 and 13 and wavy rib at 0.50 ppm, and reductions in the incidence of some (1– 4) proximal phalanges of the forelimb unossified and of all proximal phalanges of the hindlimb unossified at 0.02 ppm.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 2: Chamber analysis
Exposure Parameters        
Target Concentration, ppm 0.00 0.02 0.10 0.50
Analytical Concentration, ppm a  < MDL b 0.021 ±0.0018 0.12 ± 0.017 0.48 ± 0.038
A/T Ratio c  -   1.05  1.20  0.96
 Nominal Concentration, ppm a  -  0.049 ± 0.0038  0.18 ± 0.017   0.67 ± 0.025
 A/N Ratio d  -   0.44  0.67  0.72 
 Temperature, °C a 22.6 ± 0.43 23.3 ± 0.44  23.5 ± 0.33 23.9 ± 0.19
Relative Humidity, %a  54.4 ± 0.86  50.9 ± 1.65  52.1 ± 0.98  50.1 ± 1.41
aGrand mean of daily means ± standard deviation.
bLess than the minimum detection limit of 1 ppb.
cAnalytical to target concentration ratio.
dAnalytical to nominal concentration ratio.

Table 3:  Maternal Toxicity Parameters       
   TDI vapour, ppm
0.00 0.02 0.10 0.50
No. females on study 25 25 25 25
No. females at scheduled sacrificea 25 (100.0) 25 (100.0) 25 (100.0) 25 (100.0)
No. (%) gravid 22 (88.0) 21 (84.0) 23 (92.0) 23 (92.0)
No. (%) fully resorbed litters 0 (0.0) 1 (4.8) 0 (0.0) 0 (0.0)
No. (%) dams with viable fetuses 22 (100.0) 20 (95.2) 23 (100.0) 23 (100.0)
Maternal Body Weight Changes        
Day 0 to 6 (pre-exposure period) 36.1±8.05b 33.3 ± 7.98 33.7 ± 8.43 40.1 ± 8.29
Day 6 to 9 12.3 ± 3.90 9.2±3.76* 9.4 ± 2.89* 1.8 ± 5.31***
Day 9 to 12 16.7 ± 4.35 16.9 ± 4.74 17.7 ± 4.95 11.3 ± 8.23*
Day 12 to 16 24.1 ± 4.88 20.8 ± 7.10 22.5 ± 6.03 15.8 ± 8.23*
Day 6 to 16 (exposure period) 53.1 ± 7.37 46.4 ± 11.66 49.5 ± 9.96 28.9 ± 17.59***
Day 16 to 21 (post exposure period) 89.1 ± 11.63 76.3 ± 18.67* 88.4 ± 13.23 95.3 ± 18.07
Day 0 to 21 (gestation) 178.3 ± 20.25 156.0 ± 32.02* 171.5 ± 18.42 164.4 ± 28.79
Maternal Feed Consumption (g/animal/day)        
Day 0 to 6 (pre-exposure period) 22.91± 3.434b 21.44 ± 2.909 22.37 ± 4.045 22.28 ± 2.129
Day 6 to 15 (exposure period) 23.10±2.524 22.69 ± 2.123 23.17 ± 2.481 19.62 ± 2.006***
Day 15 to 21 (post exposure period) 26.64 ± 2.537 25.22 ± 2.009 26.60 ± 2.801 25.48 ± 3.267
Maternal Water Consumption (g/animal/day)        
Day 0 to 6 (pre-exposure period) 36.45±7.331b 34.88 ± 8.247 38.71 ± 12.623 41.67 ± 11.249
Day 6 to 15 (exposure period) 43.71 ± 9.743 42.03 ± 9.802 42.77 ± 11.509 39.14±10.374
Day 15 to 21 (post exposure period) 51.71 ± 8.897 54.91 ± 15.653 51.80±11.350 52.39 ± 11.772
aNo females died, aborted, delivered early, or were removed from study.         
bData are presented as mean ± SD.            
*p < 0.05 compared to control.            
**p < 0.01 compared to control.            
***p < 0.001 compared to control.            

Table 4: Maternal Organ Weights
   TDI Vapour, ppm        
  0.00 0.02 0.10 0.50
No. Dams 22 217 23 23
 Initial body weight (g)  223.08 ± 10.89a 220.65 ± 10.58  222.30 ± 9.05  224.16 ± 7.97
Body weight at sacrifice (g)  401.43 ± 24.34  376.63 ± 34.45*   393.80 ± 20.77   388.53 ± 31.40
Gravid uterine weight (g) 106.68 ± 12.22  92.50 ± 28.19  101.31 ± 18.21   101.25 ± 22.40 
 Corrected body weight (g)b  294.74 ± 19.25  284.14 ± 16.41 292.49 ± 15.81 287.28 ± 21.82 
Corrected body weight change (g)c 71.66 ± 14.11   63.49 ± 14.55   70.20 ± 12.43 63.12 ± 17.90 
 Liver weight (g) 14.35 ± 1.58  13.34 ± 2.80  14.33 ± 1.37  13.98 ± 1.53 
 Relative liver weight (%)d 4.86 ± 0.39  4.70 ± 0.5 4.90 ± 0.39 4.87 ± 0.48
aData are presented as mean ± SD.
bCorrected body weight = body weight at sacrifice minus gravid uterine weight.
cCorrected body weight change = corrected body weight minus initial body weight.
dCalculated as a percentage of the corrected body weight.
*p < 0.05 compared to control.

Table 5: Gestational Parameters
   TDI Vapour, ppm     
  0.00 0.02 0.10 0.50
No. dams 22 21 23 23
Corpora lutea/dam 17.4±3.8a 17.6±5.6 17.1±3.6 16.3±2.8
Total implants/litter 15.3±1.8 13.0±3.8* 14.3±3.0 14.3±3.4
% preimplantation loss/litterb 10.2±14.0 18.6±19.0 14.6±18.8 12.1±19.01
Viable implants/litter 14.5±1.7 12.3±4.0* 13.5±2.8 14.0±3.5
Non-viable implants/litter 0.7±0.9 0.7±1.1 0.8±1.1 0.3±0.5
Early resorptions/litter 0.6±0.7 0.6±0.9 0.7±1.0 0.3±0.5
Late resorptions/litter 0.1±0.3 0.1±0.3 0.1±0.4 0.0±0.0
Dead fetuses/litter 0.0±0.0 0.0±0.0 0.0±0.2 0.0±0.0
% live fetuses/litter 95.4±5.4 89.1±23.6 94.6±6.7 97.1±5.7
No. litters 22 20c 23 23
Sex ratio (% male fetuses)/litter 49.8±12.6 44.4±15.6 54.6±16.1 46.2±14.1
Fetal body weights per litter (g)        
All fetuses 5.21±0.21 5.37±0.25 5.37±0.30 5.22±0.37
Male fetuses 5.32±0.23 5.51±0.37 5.51±0.36 5.37±0.39
Female fetuses 5.10±0.21 5.27±0.38 5.20±0.28 5.09±0.36
aData are presented as mean±SD.
bPercent preimplantation loss=[(corpora lutea - total implants)/corpora lutea]*100.
cThe N is reduced because one dam was found to be pregnant by ammonium sulfide staining of the uterus (i.e., a fully resorbed litter).
*p<0.05compared to control.

Table 6: Summarized Incidence and Frequency of Fetal Malformations and Variations
   TDI vapour, ppm     
0.00 0.02 0.10 0.50
Number examined externallya        
Fetuses 320 258 311 322
Litters 22 20 23 23
No significant malformation findings        
Number examined viscerally'b        
Fetuses 164 134 162 168
Litters 22 20 23 23
No significant malformation findings        
Number examined skeletallyc        
Fetuses 156 124 149 154
Litters 22 20 23 23
No significant malformation findings        
Significant variations        
Cervical centrum no. 5 poorly ossified        
No. (%) fetuses 13 (8.3) 18 (14.5) 29 (19.5) 36 (23.4)
No. (%) litters 8 (36.4) 12 (60.0) 14 (60.9) 18 (78.3)*
Wavy rib        
No. (%) fetuses 45 (28.8) 27 (21.8) 16 (10.7) 11 (7.1)
No. (%) litters 11 (50.0) 9 (45.0) 8 (34.8) 4 (17.4)*
All proximal phalanges (hindlimb) unossified        
No. (%) fetuses 61 (39.1) 23 (18.5) 57 (38.3) 58 (37.7)
No. (%) litters 19 (86.4) 9 (45.0)* 18 (78.3) 20 (87.0)
Summarized malformations        
External        
No. (%) fetuses 0 (0.0) 1 (0.4)d 0 (0.0) 0 (0.0)
No. (%) litters 0 (0.0) 1 (5.0) 0 (0.0) 0 (0.0)
Visceral        
No. (%) fetuses 7 (4.3) 7 (5.2) 9 (5.6) 8 (4.8)
No. (%) litters 5 (22.7) 5 (25.0) 6(26.1) 7 (30.4)
Skeletal        
No. (%) fetuses 1 (0.7) 3 (2.4) 2(1.3) 1 (0.6)
No. (%) litters 1 (4.5) 3 (15.0) 2 (8.7) 1 (4.3)
Total malformations        
No. (%) fetuses 8 (2.5) 10 (3.9) 11 (3.5) 9 (2.8)
No. (%) litters 6 (27.3) 7 (35.0) 8 (34.8) 8 (34.8)
Summarized variations        
External        
No. (%) fetuses 16 (5.0) 10 (3.9) 9 (2.9) 12 (3.7)
No. (%) litters 13 (59.1) 7 (35.0) 7 (30.4) 10 (43.5)
Visceral        
No. (%) fetuses 106 (64.6) 93 (69.4) 99 (61.1) 86 (51.2)
No. (%) litters 20 (90.9) 20 (100.0) 22 (95.7) 22 (95.7)
Skeletal        
No. (%) fetuses 156 (100.0) 124 (100.0) 149 (100.0) 154 (100.0)
No. (%) litters 22 (100.0) 20 (100.0) 23 (100.0) 23 (100.0)
Total variations        
No. (%) fetuses 265 (82.8) 220 (85.3) 249 (80.1) 243 (75.5)
No. (%) litters 22 (100.0) 20 (100.0) 23 (100.0) 23 (100.0)
Note: A single fetus may be represented more than once in listing individual defects. Only live fetuses were examined.
aAll fetuses were examined externally.
bApproximately 50% of each litter were examined viscerally (Staples, 1974) and for soft tissue craniofacial defects (Wilson, 1965; van Julsingha and Bennett, 1977).
cApproximately 50% of each litter were examined for skeletal defects after staining with alizarin red S.
dOne fetus (in one litter) at 0.02 ppm exhibited multiple malformations and variations; all four of the external malformations observed (unilateral anophthalmia, cleft palate, unilateral microtia, parasitic twin on ventrum), and 11 of the 24 visceral malformations observed (it was not randomly selected for skeletal examination).
*p< 0.05 compared to control.

Applicant's summary and conclusion

Conclusions:
The study on toluene diisocyanate was performed according to EPA OTS 789.4350 (Inhalation Developmental Toxicity Screen), it is considered to be of high quality (reliability Klimisch 2). The criteria of validity of the test system are fulfilled. There was only one possible indication of minor fetotoxicity at 0.50 ppm with no other indications of developmental toxicity.The NOAEL for maternal and developmental toxicity was 0.1 ppm in this study. No embrytoxicity or teratogenicity was observed at any exposure concentrations employed.
Executive summary:

The developmental toxicity of toluene diisocyanate was investigated in Sprague Dawley rats according to EPA OTS 798.4350 (Inhalation Developmental Toxicity Screen) (Tyl, 1999). The test substance was administered to mated female rats via inhalation (0, 0.02, 0.1 or 0.5 ppm, 6 h/day, from gestation day 6 to 15). Maternal clinical signs, body weights, and feed and water consumption were recorded throughout gestation. At termination (gd 21), maternal body, gravid uterine, and liver weights were recorded. Corpora lutea were counted, and implantation sites were identified. In addition resorptions and dead and live fetuses were recorded. All live fetuses were examined for external alterations. One-half of the live fetuses/litter were examined for visceral (including craniofacial) alterations. The remaining intact fetuses/litter were stained with alizarin red S and examined for ossified skeletal alterations. The most plausible and appropriate interpretation of the initial, brief, minor, and transient decrements in weight gain at 0.02 and 0.10 ppm is that they were due to initial responses of the dams as they acclimated to exposure of an irritating vapour rather than to toxicity per se. There were also significant reductions at 0.02 ppm in maternal weight gain for gd 16–21 (p < 0.05; postexposure period), therefore, for gd 0–21 (p < 0.05, gestational period) and for body weight at sacrifice (p < 0.05; not observed at 0.10 or 0.50 ppm), accompanied by significant reductions in total implants per litter and in viable implants per litter (both p < 0.05) at 0.02 ppm. These effects at 0.02 ppm were due, at least partially, to the presence of a fully resorbed litter with one implant only in this group (the dam and her data were included in the summary tables because she was pregnant). When her data are deleted from the individual data and the revised data statistically compared to the control group values for gd 21 terminal body weight and viable implants per litter, the differences were still statistically significant but less so. It is unlikely that this cluster of interrelated effects is due to exposure to TDI for the following reasons: (1) none of the effects were observed at higher concentrations so it is not dose related; (2) the major effect, reduced body weight gain for gd 16–21, occurred after exposures were over (they ended on gd 15); (3) this last trimester is during the time of rapid fetal growth and weight gain, and so the reduced maternal weight gain was most likely due to the reduced number of fetuses per litter in this group (i.e., the fetuses grew normally but were fewer in number); and (4) the reduction in maternal weight gain for gd 16–21 for 0.02 ppm was enough to affect the gestational weight gain (gd 0–21) for maternal body weight on gd 21 but did not affect maternal weight gain during the exposure period (gd 6–16). The cause of the smaller litters at 0.02 ppm is most likely due to biologic variation, with the viable litter size (12.9) slightly below the current historical control values of 13.30 –16.24 live fetuses per litter. The maternal toxicity at 0.50 ppm consisted of reduced body weights, body weight gains, feed consumption, and clinical signs of toxicity. Water consumption was unaffected. Gestational parameters exhibited no significant treatment-related changes, including pre- and postimplantation loss, sex ratio/litter, or fetal body weights/litter. Incidences of individual malformations, malformations by category (external, visceral, and skeletal), total malformations, individual external and visceral variations, variations by category, and total variations were unaffected. Of 111 skeletal variants observed, only 1, incidence of poorly ossified cervical centrum 5, was increased at 0.50 ppm, indicating possible minimal fetotoxicity, although it occurred in the absence of any other indications of developmental toxicity. Therefore, exposure to TDI vapor by inhalation, during major organogenesis in CD rats, resulted in maternal toxicity and minimal fetotoxicity at 0.50 ppm. No treatment-related embryotoxicity or teratogenicity was observed. The A/D ratio (Fabro et al., 1982), the ratio of the lowest doses resulting in adult and developmental toxicity, is at most 1.0, indicating no preferential susceptibility to the conceptus.

This study confirmed effects of TDI on the maternal respiratory tract, with concomitant reductions in maternal body weight, weight gain, and feed consumption at 0.50 ppm. There was only one indication of possible minor fetotoxicity at 0.50 ppm (increased incidence of reduced ossification in cervical centrum no. 5), with no other indications of developmental toxicity. There was no evidence of maternal or developmental toxicity at 0.02 or 0.10 ppm, and no evidence of embryotoxicity or teratogenicity at any exposure concentration evaluated. The present study has shown that exposure to toluene diisocyanate vapour by inhalation during major organogenesis in CD (Sprague-Dawley) rats results in maternal toxicity and minimal indication of fetotoxicity at 0.50 ppm. The no observable adverse effect level (NOAEL) for maternal and developmental toxicity was 0.10 ppm in this study. No embryotoxicity or teratogenicity was observed at any exposure concentrations employed.