Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1987-1989
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Qualifier:
according to
Guideline:
EPA OTS 798.4700 (Reproduction and Fertility Effects)
Principles of method if other than guideline:
Twenty-eight 42-day-old pups/sex/group (F0) were exposed to toluene diisocyanate vapour (TDI; 80% 2,4-TDI, 20% 2,6-TDI) by inhalation at 0.0, 0.02, 0.08, or 0.3 ppm, 6 h/day, 5 days/week, for 10 weeks, then mated within groups for 3 weeks, with exposure 7 days/week during mating, gestation, and lactation. F0 maternal animals were not exposed from gestational day (gd) 20 through postnatal day (pnd) 4; maternal exposures resumed on pnd 5. Twenty-eight weanlings/sex/group continued exposure for 12 weeks (starting on pnd 28) and were bred as described above. F0 and F1 parents and ten F1 and F2 weanlings/sex/group were necropsied, and adult reproductive organs, pituitary, liver, kidneys, and upper respiratory tract (target organs) were evaluated histologically in ten/sex/group.
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
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS: 145 virgin female and 145 virgin male albino CDt (Sprague-Dawley) rats (Crl:CDt [SD]BR) - 28 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: housed initially two/same sex during quarantine and then singly, except for the cohabitation periods, for the duration of the study. During the quarantine, prebreed periods, mating, and most of gestation, rats were housed in stainless steel, wire mesh cages including during exposure periods. From gd 19 through parturition, and lactation until weaning, female rats were housed in plastic shoebox cages (19.0 x 10.5 x 8 in.) with Alpha-Drit (Shepherd Specialty Papers, Inc., Kalamazoo, MI) bedding during nonexposure periods. Deotized Animal Cage Boardt (Shepherd Specialty Papers, Inc., Kalamazoo, MI) was placed beneath the stainless steel cages and changed regularly.
- Diet (e.g. ad libitum): Certified Ground Rodent Chowt #5002, Ralston-Purina Company, St. Louis, MO ad libitum except during exposures, feed jars were changed weekly;
- Water (e.g. ad libitum): tap water (Municipal Authority of Westmoreland County, Greensburg, PA) ad libitum except during exposures
The water was delivered by an automatic watering system, with demand control valves mounted on each cage rack for rats in stainless steel cages. Female rats housed in shoebox cages received water by water bottles with stainless steel sipper tubes. Analyses of the feed and water for contaminants and of the feed for nutrient levels indicated all contaminant levels were below the maximum certified standards, and all nutrient levels were above the minimum certified standards.
- Acclimation period: quarantined for approximately 2 weeks, during which time they were weighed, examined by a veterinarian, and representative animals were subjected to fecal examination, serum viral antibody analysis, and histologic examination of selected organs. Results of the physical examination, serology, parasitology, and histopathology were negative for signs of infectious disease.

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

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: TDI vapour was generated using a glass evaporator system similar in design to that described by Carpenter et al. (1975) and as described by Tyl et al. (1999). 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 x 1m x 2.1 m (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.
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 vapour within the range of target exposure concentrations employed.
- Method of holding animals in test chamber: Within each chamber, animal cages were rotated weekly to compensate for any possible variation in chamber exposure conditions (i.e., vapour concentration, temperature, or relative humidity).
- Temperature, humidity, pressure in air chamber: Temperature and relative humidity 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. Temperature measurements obtained from the inside surface of each evaporator during the exposure regimen using a Doric Trendicator Model 400A probe (Doric Scientific Division, San Diego, CA) ranged from 42 to 63°C.
- Air flow rate: Airflow in each chamber was approximately 1000–1500 L/min, with a t99 (theoretically derived time required for the chamber to reach 99% of the equilibrium concentration) of approximately 20 min.
- Air change rate: at least 14 air changes per h
- Other: 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 temperature in the evaporator was monitored and maintained at the lowest level sufficient to vaporize the liquid (46–60°C). 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.

TEST ATMOSPHERE
- Brief description of analytical method used: Two Autostep Isocyanate paper tape monitoring devices (GMD Systems, Inc., Hendersonville, PA), one for 0.00, 0.02, 0.08, and one for 0.3 ppm, were used to measure TDI concentrations in the exposure chamber atmospheres. Both instruments, calibrated using a modified Marcali method for detection of TDI in air, were equally sensitive to detection of the 2,4- and 2,6-TDI isomers. Throughout the 235 days of exposure, generated TDI atmospheres were monitored by placing probes in the breathing zone of the animals approximately six times per each 6-h exposure. Control chamber atmospheres were measured six times daily for the first 11 exposure days and once per day thereafter.
The 2,4- and 2,6-TDI isomer concentrations in the exposure chamber atmospheres were measured prior to the onset of the F0 exposure period and on exposure day 143. Reverse phase high pressure liquid chromatography was used to separate and quantify the 2,4- and 2,6-isomers.
- Samples taken from breathing zone: yes (Samples were collected using glass fiber filters coated with 1-(-pyridylpiperazine), as well as impingers containing N-2-nitrobenzyl-n-m-propylamine in toluene. Samples were obtained through a sampling port located approximately 6 feet above the chamber floor and approximately 6 in. from the inside chamber wall. Probes for sampling were located within 2–3 in. of each other.)
- Other: 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.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 3 weeks (with no change of partners)
- Proof of pregnancy: Observations of vaginal sperm and/or dropped or vaginal copulation plug were considered evidence of successful mating (Hafez, 1970); the date of insemination was designated gd 0.
- After successful mating each pregnant female was caged individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The purity and stability of the test chemical were verified by analysis before, during, and after the study. The test material remained over 99% pure throughout the study. However, there were modest differences between the TDI isomer ratio in the test atmospheres as compared to the test material. The periodic analyses of the chamber atmospheres indicated that the mean analytical values of TDI +/- SD for the low (0.02 ppm), middle (0.08 ppm), and high (0.30 ppm) exposure concentrations were 0.020 +/- 0.003, 0.079 +/- 0.009, and 0.29 +/- 0.023 ppm, respectively. No test chemical was detected in the control atmospheres, with a minimum detection limit of 0.002 ppm.
Percentage of the concentrations of the 2,4- and 2,6-isomers in the chamber atmospheres (sampled on exposure day 143) were 63.4% and 36.6% for the low concentration exposure chamber, 69.2% and 30.8% for the mid concentration exposure chamber, and 75.0% and 25.0% for the high concentration exposure chamber. These data indicated that the 2,4-isomer content of 80% decreased approximately 15%, 10%, and 5% in the low, mid, and high concentration exposure chambers, respectively. Although the test material stability was verified before and after the study, the isomer ratio of TDI in the exposure chamber did not precisely match the 80/20 ratio of the test material. This may relate to the imperfect match of analytical to nominal concentrations, suggesting some loss of the test material during th vapor generation process. Given the very low (sub ppm) range of test concentrations of this reactive material, a modest difference in analytical to nominal ratio and the isomer ratio was not surprising. The differences were unlikely to have had a significant impact on the outcome or interpretation of the study.
Duration of treatment / exposure:
F0: 10 weeks (prebreed exposure period), 3 weeks (mating period) , exposure continued throughout gestation and lactation , except for the following interval: after the exposure on gd 19, each female was transferred to a shoebox cage and was not exposed from gd 20 through pnd 4. Exposures to the dam resumed on pnd 5.
F1: selected weanlings were exposed to TDI by inhalation for 12 weeks, then mated as described above, with exposure continuing during mating, gestation, and lactation.
Frequency of treatment:
5 days/week, 6 h/day (F0 premating period, F1 premating period), then 7 days/week, 6 h days for the remaining time (F0 and F1 mating and gestation, except gd19-pnd4).
Details on study schedule:
- F1 parental animals not mated until 1 week after selected from the F1 litters: After weaning on pnd 21, litters (minus the dam) were maintained for one additional week (until pnd 28) before direct exposures began to the selected F1 weanlings.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 20 weeks

At weaning on pnd 21, 28 F1 weanlings/sex/group were randomly selected to be parents of the F2 generation.
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:
0.08 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.3 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
28 (F0), 28 (F1)
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: To determine appropriate target concentrations of TDI, a range-finding study was performed following the same study design as in the definitive developmental 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 3 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 anesthetized control rats in this study was 7.38.) Based on these results, 1.0 ppm was deemed too toxic for use. The target exposure concentrations for the developmental study were, therefore, 0.00, 0.02, 0.10, and 0.50 ppm.
The rationale for the exposure concentrations of the two-generation toxicity study was based on the developmental toxicity study of TDI at 0.02, 0.10, and 0.50 ppm in the same rat strain (mentioned above). In that study, exposure to TDI vapour for six h/day, for gd 6 through 15 at 0.50 ppm, resulted in reductions in maternal body weight, body weight gain, and feed consumption. Because relatively major maternal indications of toxicity were observed at 0.50 ppm after only 10 days of exposure during gestation, there was concern that 0.50 ppm would result in excessive adult toxicity during the 10-week prebreed exposure and during the mating, gestation, and lactation periods (when exposure continued). Therefore, the top and middle target exposure concentrations were slightly lower for the present study.
- Rationale for animal assignment: Two hundred twenty-four animals were placed on study, 28/sex/group, by randomization procedures stratified by body weight, immediately prior to the start of the prebreed exposure period. All animals were assigned a unique number and were toe clipped and ear notched prior to the start of the study.
Positive control:
No details on positive controls available.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: prebreed exposure: clinical observations recorded daily (mortality checks twice daily) and body weights recorded weekly

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, Dams were weighed on gd 0, 7, 14, and 21. Dams with litters were weighed on pnd 1, 4, 7, 14, and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No (feed consumption was not recorded)
Estrous cyclicity (parental animals):
No details on oestrous cyclicity available.
Sperm parameters (parental animals):
No details on sperm parameters available.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes] Litters were randomly culled to a maximum of eight (with as equal sex ratio as possible) on pnd 4 (F1).
- If yes, maximum of 8 pups/litter (sex/litter as nearly as possible); excess pups were killed and discarded.
- F2 weanlings, ten/sex/group, were selected for necropsy; remaining F2 weanlings were examined externally, euthanized, and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
Pups (F1 generation) were individually counted, sexed, and examined grossly on pnd 0, 1, 4, 7, 14, and 21. They were also weighed individually on pnd 1, 4, 7, 14, and 21.
At weaning on pnd 21, ten F1 weanlings/ sex/group were randomly selected for necropsy; remaining F1 weanlings were examined externally, euthanized, and discarded.
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain]

GROSS EXAMINATION OF DEAD PUPS:
[yes, for external and internal abnormalities]
A gross internal examination was also performed on any pup appearing abnormal or dying on test. As the necropsy of the randomly selected animals revealed no gross abnormalities, all remaining pups of all groups not used for the new parental generation were euthanized and discarded.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after the delivery of F1 litters
- Maternal animals: All surviving animals after weaning of the F1 litters.
All F0 and F1 parental animals in all groups (both generations) were euthanized by severing the brachial blood vessels following anaesthesia with methoxyflurane, and subjected to a complete necropsy.

GROSS NECROPSY
- A complete necropsy and histopathologic examination were conducted for any parental animals dying on test.
- Gross necropsy consisted of examination of the external surfaces; all orifices; cranial cavity; carcass; external and cut surfaces of the brain and spinal cord; the thoracic, abdominal, and pelvic cavities and their viscera; and cervical tissues and organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
Of the 28 male and 28 female adults from the control and high exposure groups, ten/sex/group were selected randomly and subjected to a histopathology examination. Histopathologic evaluation was conducted on the tissues specified below after fixation in buffered neutral 10% formalin, paraffin embedment, and sectioning and staining with hematoxylin and eosin: pituitary, liver, kidneys (two), upper and lower respiratory tract (including nasal turbinates), vagina, uterus, ovaries, testes, epididymides, seminal vesicles, prostate, and other tissues with gross lesions identified as being potentially treatment related. Any of the above organs or tissues showing gross or histopathologic alterations, specifically the upper respiratory tract (including the nose, nasal turbinates, larynx, and trachea), were also evaluated microscopically in ten animals/sex from the other treatment groups.
The fixed (buffered neutral 10% formalin) uteri from any parental female of the F0 or F1 generations failing to produce a litter were stained with potassium ferricyanide for confirmation of pregnancy status; implantation sites (if any) were recorded. This procedure did not affect the subsequent histopathologic examination.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- - Gross necropsy consisted of examination of the external surfaces; all orifices; cranial cavity; carcass; external and cut surfaces of the brain and spinal cord; the thoracic, abdominal, and pelvic cavities and their viscera; and cervical tissues and organs.
A gross internal examination was performed on ten pups randomly selected for each sex from each test group of the F1 and F2 generations. As the necropsy of the randomly selected animals revealed no gross abnormalities, all remaining pups of all groups not used for the new parental generation were euthanized and discarded.

HISTOPATHOLOGY / ORGAN WEIGTHS
Histopathologic evaluation was conducted on the tissues specified below after fixation in buffered neutral 10% formalin, paraffin embedment, and sectioning and staining with hematoxylin and eosin: pituitary, liver, kidneys (two), upper and lower respiratory tract (including nasal turbinates), vagina, uterus, ovaries, testes, epididymides, seminal vesicles, prostate, and other tissues with gross lesions identified as being potentially treatment related. Any of the above organs or tissues showing gross or histopathologic alterations, specifically the upper respiratory tract (including the nose, nasal turbinates, larynx, and trachea), were also evaluated microscopically in ten animals/sex from the other treatment groups.
The fixed (buffered neutral 10% formalin) uteri from any parental female of the F0 or F1 generations failing to produce a litter were stained with potassium ferricyanide for confirmation of pregnancy status; implantation sites (if any) were recorded. This procedure did not affect the subsequent histopathologic examination.
Statistics:
The unit of comparison was the male, the female, or the litter (Weil, 1970). Results of the quantitative continuous variables (e.g., body weights, organ weights, etc.) were intercompared for the three treatment groups and one control group by use of Levene’s test for equal variances (Levene, 1960), analysis of variance (ANOVA), and t-tests. 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. The significance levels for the t-test comparisons were corrected by the Bonferroni method for all reproductive data. Nonparametric data were statistically evaluated using the Kruskal-Wallis test (Sokal and Rohlf, 1969), followed by the Mann-Whitney U test for pairwise comparisons (Sokal and Rohlf, 1969), when appropriate. Frequency data (such as the various indices) were compared using the Fischer’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 statistical significance.
Reproductive indices:
No. mating pairs, Mating index, Fecundity index, Fertility index, Gestational index, Gestational length, days, No. live litters, pnd 0, No. live litters, pnd 21, Live birth index, Survival indices, 4-day (pnd 0—4 precull), 7-day (pnd 4 postcull-7), 14-day (pnd 7-14), 21-day (pnd 14-21), Lactation index (pnd 4-21 postcull)


Offspring viability indices:
pnd 0: No. live litters, No. total pups/litter, No. live pups/litter, % males/litter
pnd 1:No. live litters, Pup body weight/litterb,
pnd 4 (precull): No. live litters, No. pups/litter, % males/litter, Pup body weight/litter, Pup weight gain/litter, pnd 1-4b
pnd 7: No. live litters, No. pups/litterd, % males/litter, Pup body weight/litter, Pup weight gain/litter, pnd 4-7
pnd 14: No. live litters, No. pups/litter, % males/litter, Pup body weight/litter, Pup weight gain/litter, pnd 7-14
pnd 21: No. live litters, No. pups/litter, % males/litter, Pup body weight/litter, Pup weight gain/litter, pnd 14-21

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
F0 males: increased incidence of nasal discharge at 0.3 ppm. F0 females: increased incidence of red-tinged fur about the head at 0.3 ppm. Periocular encrustation, perinasal encrustation and/or red nasal discharge in all exposure groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Final male bw (0.3 ppm, week 14) stat. increased. Male F0 bw gains (0.3 ppm, 1 week) reduced, (weeks 4-5 and 8-9) sig. increased terminal F0 male bw gains sig. increased at 0.02, 0.08, and 0.3 ppm. Females (0.3 ppm, week 18–19) sig. increased bw gain. f
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Final male bw (0.3 ppm, week 14) stat. increased. Male F0 bw gains (0.3 ppm, 1 week) reduced, (weeks 4-5 and 8-9) sig. increased terminal F0 male bw gains sig. increased at 0.02, 0.08, and 0.3 ppm. Females (0.3 ppm, week 18–19) sig. increased bw gain. f
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related gross lesions observed at necropsy.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related histopathologic lesions were limited to the upper respiratory tract, with tissues located deeper in the respiratory tract being less affected. (rhinitis, alterations like dysplasia & hyperplasia of the nasal epithelium in the turbinates.
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
no effects observed
Description (incidence and severity):
There was no effect of treatment on reproductive organs or functions.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There was no effect of treatment on reproductive organs or functions.
Reproductive performance:
no effects observed
Description (incidence and severity):
Reproductive parameters of F0 parents to produce F1 offspring were unaffected by treatment. Gestational length was unaffected by exposure to the test chemical. F1 live birth and survival indices were unaffected by treatment.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Treatment-related clinical signs of toxicity in F0 males included an increased incidence of nasal discharge at 0.3 ppm. F0 females exhibited an increased incidence of red-tinged fur about the head at 0.3 ppm. Periocular encrustation, perinasal encrustation, and/or red nasal discharge were observed in all exposure groups of F0 males and females, including the control group, and appeared to be associated with the inhalation treatment conditions per se rather than exposure to the test chemical vapour.
Two F0 adult animals died during the conduct of this study: one female and one male. One F0 female at 0.02 ppm died of an abnormal pregnancy with uterine bleeding. The cause of death of the one F0 male at 0.3 ppm, found dead on study day 85, was not determined, although the animal had microscopic lesions of the respiratory tract similar to those of other animals in its exposure group.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
During the 10-week prebreed exposure and 3-week mating periods of the F0 animals, male body weights were equivalent across all treatment groups. Final male body weights (week 14) were statistically increased at 0.3 ppm. Male F0 weight gains at 0.3 ppm were reduced for the first exposure week and were significantly increased for treatment weeks 4–5 and 8–9; terminal F0 male body weight gains were significantly increased at 0.02, 0.08, and 0.3 ppm. Female F0 body weights exhibited no significant differences among groups during the prebreed exposure period or during the final exposure week. F0 female weight gains exhibited a similar equivalence across treatment groups for the prebreed exposure period. During the final week of exposure (week 18–19), females at 0.3 ppm exhibited a significantly increased weight gain.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Maternal F0 gestational and lactational body weights and weight gain were equivalent across all exposure groups. Reproductive parameters of F0 parents to produce F1 offspring were unaffected by treatment. Gestational length was unaffected by exposure to the test chemical. F1 live birth and survival indices were unaffected by treatment. F1 litter sizes, sex ratio (% males), and pup body weights and weight gains (by litter and by sex by litter) were equivalent across all treatment groups from lactational day 1 through 21. No treatment-related lesions were observed in the F1 pups, which died during the lactation period.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related gross lesions observed at necropsy.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Treatment-related histopathologic lesions were limited to the upper respiratory tract, with tissues located deeper in the respiratory tract being less affected. In both F0 males and females at 0.3 ppm, the most frequently observed lesions were rhinitis and alterations (dysplasia and hyperplasia) of the respiratory (nasal) epithelium in the nasal turbinates. Increased incidences of rhinitis were also observed in nasal turbinates of F0 males and females at 0.08 and 0.02 ppm, relative to the F0 control males (one) and females (none). At 0.02 ppm, three F0 males exhibited rhinitis, one minimal multifocal and two mild multifocal; three F0 females at 0.02 ppm also exhibited rhinitis, one minimal multifocal and two mild (one each focal and multifocal).

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
adult toxicity
Sex:
male/female
Basis for effect level:
other: not determined
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
0.3 ppm
Basis for effect level:
other: the NOAEL for reproductive toxicity was at least 0.3 ppm
Remarks on result:
other: Generation not specified (migrated information)
Dose descriptor:
NOAEL
Remarks:
postnatal toxicity
Effect level:
0.02 ppm
Basis for effect level:
other: the NOAEL for postnatal toxicity was 0.02 ppm
Remarks on result:
other: Generation not specified (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
perinasal encrustation across all treatment groups in F1 females, the incidence was sig. increased at 0.3 ppm. Incidence of red-tinged fur in F1 males, was sig. increased in F1 females at 0.08 & 0.3 ppm from 17 to 22 weeks (study days 120-155) of exposure
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
consisting of reduced body weights and body weight gains
Sexual maturation:
no effects observed
Description (incidence and severity):
Maternal F1 gestational & lactational bw /bw changes were unaffected for all time points. Gestational length was unaffected. F2 pup live birth & survival indices were unaffected. F2 litter size and sex ration (%males) were unaffected.

Details on results (F1)

VIABILITY (OFFSPRING)
not affected

CLINICAL SIGNS (OFFSPRING)
In F1 males, there were no significant treatment- or concentration-related changes in incidence of clinical observations. Although perinasal encrustation was observed across all treatment groups in F1 females, the incidence was significantly increased at 0.3 ppm. The incidence of red-tinged fur, although occasional in TDI-exposed F1 males, was significantly increased in F1 females at 0.08 and 0.3 ppm from 17 to 22 weeks (study days 120–155) of exposure.
The red-tinged fur is from chromo- dacryorrhea, red-brown material (porphyrin) secreted from the Harderian gland and distributed about the face and neck by normal grooming activity (Seely, 1987). It is a relatively non-specific indication of stress. It is likely that this stress-related finding is associated with the longer duration of females’ exposure to TDI.

BODY WEIGHT (OFFSPRING)
During the 12-week prebreed exposure of the F1 animals randomly selected to be parents of the F2 generation, the males at 0.3 ppm exhibited reduced body weights relative to the controls for the first 4 weeks of exposure. For weekly intervals 0–1 and 1–2, as well as the final week of exposure (week 15–16), body weight gains were significantly reduced at 0.3 ppm. F1 males at 0.02 ppm exhibited significantly increased body weight gain relative to controls for weekly intervals 12–13 and 13–14 of the mating period. The F1 females exhibited reduced body weights at 0.3 ppm for the first 2 weeks of exposure, as well as week 6 of exposure. There were no significant differences among groups for F1 female weight gain.

SEXUAL MATURATION (OFFSPRING)
Maternal F1 gestational and lactational body weight and body weight changes were unaffected for all time points measured. Gestational length was unaffected by exposure. F2 pup live birth and survival indices were unaffected by treatment. F2 litter size and sex ratio (% males) were unaffected by treatment. At 0.3 ppm, F2 pup body weight per litter exhibited reductions (males, females, and all pups) beginning on pnd 14 and persisting through day 21 for male pups. Body weights of female pups and all pups/litter were also reduced on lactation day 14 at 0.08 ppm. Pup body weight gains per litter (males, females, and all pups) were reduced at 0.08 and 0.3 ppm for pnd 4 to 7, and at 0.3 ppm pup body weight gain reductions persisted (males, females, and all pups) through pnd 14.

ORGAN WEIGHTS (OFFSPRING)
no details available

GROSS PATHOLOGY (OFFSPRING)
Three F1 females were sacrificed prior to scheduled sacrifice and included one animal at 0.08 ppm on study day 94 due to traumatic injury, and one animal each at 0.3 ppm and 0.08 ppm due to early deliveries during exposure.
After delivery (F1 males) or weaning (F1 females) of the F2 litters, all surviving F1 parental animals were necropsied. No gross treatment-related lesions were observed. Selected tissues were examined histologically on ten animals per sex from high exposure and control animals. The one target tissue, the upper respiratory tract, including nasal turbinates, larynx, and trachea, was also examined microscopically from ten animals/sex from all groups. As with F0 parents, histologic lesions were limited to the upper respiratory tract (nasal cavities, larynx, and trachea). Although dysplasia and/or hyperplasia of the nasal respiratory epithelium were present in F1 parents at 0.08 and 0.3 ppm, the frequency of occurrence was not significantly increased relative to the control frequency. Rhinitis was observed with increased frequencies in all TDI-exposed groups of F1 males and females (with no rhinitis observed in the control animals). Seven of the ten F1 males and four of the ten F1 females examined at 0.02 ppm exhibited rhinitis; in the seven males with rhinitis, six were classified as minimal multifocal and one as moderate multifocal; in the four females with rhinitis, one was minimal focal, one was mild multifocal, and two were moderate multifocal (Table 4). Mononuclear cell infiltration of liver tissue, although present in controls and significantly increased in F1 females at 0.3 ppm, was not deemed treatment related. No treatment-related gross lesions were observed in F2 pups that died during lactation, or in the ten/sex/group subjected to necropsy at weaning.

HISTOPATHOLOGY (OFFSPRING)
Necropsy of ten randomly selected F1 pups/sex/group indicated no treatment-related gross findings.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

    TABLE1        
    Chamber Analyses        
   Target concentrations (ppm)     
  0.00 0.02 0.08 0.3
Analytical concentrations (ppm)a        
Paper tape method <MDLS 0.020±0.0026 0.079 ± 0.0088 0.29 ± 0.023
Corrected by modified Marcali method <MDL 0.018±0.0023 0.070 ± 0.0077 0.23 ± 0.020
A/T ratio'        
Paper tape method 1.00 0.99 0.97
Corrected by modified Marcali method 0.90 0.88 0.77
Nominal Concentrations (ppm)d 0.045 ± 0.0049 0.127 ± 0.017 0.39 ± 0.027
A/N ratioe        
Paper tape method 0.44 0.64 0.74
Corrected by modified Marcali method 0.38 0.56 0.60
2,4-Isomer/2,6-isomer ratio7 1.7:1.0 2.2:1.0 3.0:1.0
Temperature (°C)g 24.1±1.17 24.7 ± 0.99 24.2 ± 0.92 25.0 ± 1.02
Relative humidity (%)g 49.5±4.80 46.0 ± 4.18 47.6 ± 4.49 47.6 ± 5.33
aGrand mean of 230 (0.02 ppm) or 235 (0.08 and 0.3 ppm) daily means ± standard deviation.      
bLess than the minimum detection limit of 0.002 ppm.         
cAnalytical to target concentration ratio.            
dGrand mean of 229 (0.02 ppm), 234 (0.08 ppm), or 235 (0.3 ppm) daily means ± standard deviation.      
eAnalytical to nominal concentration ratio.            
fIsomer ratio of the test chemical is 4.0:1.0 (80%/20%); these data are from "grab" samples taken during the exposures (see text).   
gGrand mean of 233 (0.0 ppm), 230 (0.02 ppm) or 234 (0.08 and 0.3 ppm) daily means for temperature and relative humidity ± standard deviation.
Significant treatment-related histologic findings were limited to changes in the upper respiratory tract, including minimal to moderate rhinitis at all exposure levels in F0 and F1 adult males and females. At 0.02 ppm, there were three (of ten) F0 males, seven F1 males, three F0 females, and four F1 females; at 0.08 ppm, there were eight (of ten) F0 males, five F1 males, six F0 females, and four F1 females; at 0.30 ppm, there were nine (of ten) F0 males, nine F1 males, nine F0 females, and nine F1 females, relative to the control incidence of no F1 males and F0 and F1 females, and one F0 male, out of ten/sex/group examined in each generation.

Rhinitis is a typical response of the rodent to an irritant material, and similar effects have been reported in response to well-known irritants such as chlorine (Barrow et al., 1979), acrolein (Feronet al., 1978), sulfur dioxide (Giddens and Fairchild, 1972), and acetaldehyde (Kruysse et al., 1975). The rat is regarded as the most sensitive of the commonly used laboratory species in its response to these irritants.

Because the rhinitis is localized in the most anterior portion of the upper respiratory tract, the proximate site of contact of TDI, this finding can be viewed as an essentially localized, nonspecific response to exposure to an irritating vapour. Other histologic findings only at 0.3 ppm, specifically dysplasia and/or hyperplasia of the nasal respiratory epithelium, were indicative of more severe irritant effects of TDI.

       TABLE2              
       productive Parameters              
   F0 (TDI, ppm)          F1 (TDI, ppm)     
  0.0 0.02 0.08 0.30 0.0 0.02 0.08 0.30
No. mating pairs 28 28 28 28 28 28 28 28
Mating index" 96.4 100.0 96.4 100.0 89.3 100.0 92.9 100.0
Fecundity index" 88.9 78.6 96.3 89.3 88.0 75.0 80.8 89.3
Fertility index"-' 85.7 78.6 92.9 89.3 78.6 75.0 75.0 89.3
Gestational index" 100.0 95.5 100.0 100.0 100.0 100.0 100.0 100.0
Gestational length, days 21.9 ± 0.3c 21.9 ± 0.4 21.9 ± 0.3 22.0 ± 0.06 21.9 ± 0.3 22.0 ± 0.6 22.0 ± 0.4 22.2 ± 0.5
No. live litters, pnd0 24 21 26 25 22 21 21 25
No. live litters, pnd21 23 20 25 24 21 21 20 25
Live birth index 97.7 ± 6.31c 97.0 ± 5.96 98.2 ± 4.71 98.7 ± 3.41 97.4 ± 4.67 95.2 ± 10.21 96.8 ± 7.76 98.5 ± 4.31
Survival indices"                
4-day (pnd0—4precull) 94.6 ± 20.35c 93.8 ± 21.71 93.9 ± 19.80 94.5 ± 20.12 94.3 ± 21.30 97.3 ± 3.95 93.9 ± 21.70 97.3 ± 6.16
7-day (pnd4postcull-7) 100.0 ± 0.00 100.0 ± 0.00 100.0 ± 0.00 100.0 ± 0.00 100.0 ± 0.00 100.0 ± 0.00 100.0 ± 0.00 100.00 ± 0.00
14-day (pnd7-14) 100.0 ± 0.00 100.0 ± 0.00 99.5 ± 2.50 99.5 ± 2.55 100.0 ± 0.00 100.0 ± 0.00 100.0 ± 0.00 100.0 ± 0.00
21-day (pnd14-21) 100.0 ± 0.00 99.4 ± 2.80 100.0 ± 0.00 99.5 ± 2.55 99.4 ± 2.73 100.0 ± 0.00 100.0 ± 0.00 100.0 ± 0.00
Lactation index (pnd 4-21 postcull) 100.0 ± 0.00 99.4 ± 2.80 99.5 ± 2.50 99.0 ± 3.53 99.4 ± 2.73 100.0 ± 0.00 100.0 ± 0.00 100.0 ± 0.00
Indices                
Mating index(%) = (Number of females with copulation plugs/ Number of females cohabited) *100                 
Fecundity index(%) = (Number of pregnancies / Number of plug-positive females)*100
Fertility index (female)(%) =(Number of females pregnant / Number of females cohabited)*100                 
Fertility index (male)(%) = (Number of males shown to be fertile / Total number of males mated)*100                
Gestational index(%) = (Number of females with live litters / Number of females pregnant)*100             
Live birth index(%) = (Number of live pups at birth / Total number of pups born)*100                
Survival indices(%) = (Number of live pups indicated on postnatal day / Total number of live pups on previous index day)*100 ; from 7-day through lactation index, based on postcull survivors.  
Lactation index(%) = (Number of pups surviving 21 days / Total number of live pups at 4 days(postcull))*100         
Fertility index is the same for both males and females; mating was 1:1 with no change in partners
Data are presented as mean ± SD
No reproductive parameters were affected during either generation (F1 or F2). During the breeding phases for the F0 and F1 females, there were no treatment-related changes in gestational body weights or lactational body weights. F2 (but not F1) litters exhibited reduced body weights at 0.3 ppm for lactational days 14 and 21, and at 0.08 ppm for lactational day 14 only. Lactational body weight gains were reduced in F2 (but not F1) litters at 0.08 ppm for lactational days 4–7 and at 0.3 ppm for lactational days 4–7 and 7–14. Reduced pup body weight gain per litter at 0.08 and 0.3 ppm, observed during the lactation period (only for the F2 generation), initially occurred during the interval lactational days 4–7, when removal of the dams from the nest was reinstated for exposures (beginning on lactational day 5) in all groups. Removal of the dams for more than 6 h/day reduced pups' access to their food supply (the lactating dam) and was most likely compounded by the dams’ discomfort at 0.08 and 0.3 ppm upon return to the nest after the daily exposures. The effects at 0.08 ppm were resolved during the last week of lactation. There were no effects on F1 or F2 offspring body weights or weight gains at 0.02 ppm.
TABLE 3
Offspring Litter Size, Sex Ratio, Body Weights, and Weight Gain during Lactation
   F1 offspring (TDI, ppm)          F2 offspring (TDI, ppm)     
0.0 0.02 0.08 0.30 0.0 0.02 0.08 0.30
pnd 0                
No. live litters 24 21 26 25 22 21 21 25
No. total pups/litter 13.8±2.51° 14.1 ± 2.14 13.1 ± 3.19 14.2±3.53 13.9 ± 3.35 12.8 ± 3.36 13.6±2.91 13.6±2.66
No. live pups/litter 13.5±2.30 13.6±1.94 13.0±3.34 14.0±3.49 13.5 ± 3.38 12.4 ± 3.65 13.1 ± 3.05 13.4±2.74
% males/litter 49.6±12.9 48.0±12.4 45.6 ± 14.5 50.7 ± 15.7 48.6 ± 13.3 50.8 ± 15.3 48.6 ± 20.2 46.5±12.5
pnd 1                
No. live litters 23 21 26 24 22 21 21 25
Pup body weight/litterb 7.20±0.72 7.06 ± 0.78 7.04 ± 0.75 6.73 ± 0.71 7.08 ± 1.06 7.09 ± 1.03 7.04 ± 0.70 7.10±0.75

pnd 4(precull)

               
No. live litters 23 20 25 24 21 21 20 25
No. pups/litter 13.3 ± 2.40 13.5 ± 1.96 13.0±2.94 14.3± 2.18c 13.2±3.35 12.1 ± 3.65 13.2±2.86 13.0±2.74
% males/litter 50.3 ± 13.0 47.8±12.3 45.5 ± 14.6 53.0 ± 11.9 48.8 ± 13.7 51.4±15.8 50.1 ± 20.0 46.1±12.5
Pup body weight/litter 10.19±1.00 9.71 ± 1.12 9.96 ± 1.37 9.46 ± 1.27 10.19±1.82 10.48 ± 1.88 9.93 ± 1.13 10.28 ± 1.28
Pup weight gain/litter, pnd 1-4b 2.99 ± 0.73 2.60 ± 0.80 2.92 ± 0.74 2.72 ± 0.70 3.09 ± 1.02 3.39 ± 1.07 2.89 ± 0.83 3.18 ± 0.74
pnd 7                
No. live litters 23 20 25 24 21 21 20 25
No. pups/litterd 8.0 ± 0.00 8.0 ± 0.00 7.9 ± 0.28 8.0 ± 0.00 7.8 ± 1.09 7.5 ± 1.50 7.8 ± 1.12 8.0 ± 0.20
% males/litter 50.0 ± 6.5 48.8 ± 3.8 46.2 ± 9.5 50.5 ± 4.5 50.8 ± 5.4 50.4 ± 7.7 51.9±14.2 50.3 ± 3.9
Pup body weight/litter 14.89 ± 1.25 14.42±1.56 14.91 ± 1.59 14.02 ± 1.66 15.38 ± 2.41 15.25 ± 2.13 14.23 ± 1.66 14.68 ± 1.46
Pup weight gain/litter, pnd 4-7 4.70 ± 0.60 4.71 ± 0.72 4.95 ± 0.66 4.57 ± 0.78 5.19 ± 0.92 4.77 ± 0.72 4.29±1.08** 4.39 ± 0.93*
pnd 14                
No. live litters 23 20 25 24 21 21 20 25
No. pups/litter 8.0 ± 0.00 8.0 ± 0.00 7.9 ± 0.33 8.0 ± 0.20 7.8 ± 1.09 7.5 ± 1.50 7.8 ± 1.12 8.0 ± 0.20
% males/litter 50.0 ± 0.0 48.8 ± 3.8 45.9 ± 9.5 50.7 ± 3.9 50.8 ± 5.4 50.4 ± 7.7 51.9±14.2 50.3 ± 3.9
Pup body weight/litter 28.14±2.17 27.74 ± 3.21 28.72 ± 2.42 26.71 ± 2.67 29.79 ± 3.56 29.35 ± 3.50 27.51 ± 2.24* 27.33 ± 2.13*
Pup weight gain/litter, pnd 7-14 13.25 ± 1.92 13.32±2.10 13.81 ± 1.53 12.69±1.80 14.41 ± 1.64 14.10±1.94 13.28 ± 1.25 12.66±1.42**
pnd 21                
No. live litters 23 20 25 24 21 21 20 25
No. pups/litter 8.0 ± 0.00 8.0 ± 0.00 7.9 ± 0.33 7.9 ± 0.28 7.7 ± 1.10 7.5 ± 1.50 7.8 ± 1.12 8.0 ± 0.20
% males/litter 50.0 ± 6.5 48.4 ± 4.0 45.9 ± 9.5 51.0±4.0 50.5 ± 5.69 50.4 ± 7.7 51.9±14.2 50.3 ± 3.9
Pup body weight/litter 46.17±3.87 45.74 ± 4.59 46.99 ± 3.86 44.51 ± 4.88 49.03 ± 5.79 48.97 ± 6.20 46.18 ± 3.68 45.47± 3.64e
Pup weight gain/litter, pnd 14-21 18.04 ± 2.19 18.00±1.92 18.27 ± 2.10 17.76±2.66 19.23 ± 2.88 19.62 ± 2.99 18.67 ± 1.83 18.14±1.81
aData are presented as mean ± S.D.
bPup body weight and weight gain in grams, sexes combined.
cThe mean number of pups/litter was higher on pnd 4 than on pnd 0, since one litter with seven live pups, present on pnd 0 (so n= 25) had no live pups on pnd 4 (so n = 24).
dLitters culled to eight pups on pnd 4.
ePup body weight per litter was significantly reduced (p < 0.05) for male pups only, not for female or total pups per litter.
*p< 0.05 versus control group value.
**p< 0.01 versus control group value.

Continued inhalation exposure to TDI vapour for two generations in CDt (Sprague-Dawley) rats resulted in parental toxicity at 0.02, 0.08, and 0.3 ppm, evidenced by occasional body weight and weight gain depression and clinical signs of toxicity at 0.08 and 0.3 ppm, and histologic changes in the nasal cavities in both sexes and both generations at 0.02, 0.08, and 0.3 ppm. Postnatal toxicity, consisting of reduced body weights and body weight gains, occurred only in F2 litters at 0.08 and 0.3 ppm. There was no effect of treatment on reproductive organs or functions. No adult no observable adverse effect level (NOAEL) was identified, although the rhinitis observed at 0.02 ppm was considered a mild, nonspecific response to an irritant. The reproductive NOAEL was at least 0.3 ppm, and the postnatal toxicity NOAEL was 0.02 ppm in rats, under the conditions of this study.

Table 4 F0         
Incidence of Rhinitis in F0 and F1 parental animals                     
   F0(TDI, ppm)          Fl(TDI, ppm)        
  0.0 0.02 0.08 0.30 0.0 0.02 0.08 0.30
Males                        
No. examined 10 10 10 10 10 10 10 10
Rhinitis 1 3 8 9 0 7 5 9
Minimal 0 1 0 0   6 3 2
Focal   0       0 0 1
Multifocal   1       6 3 1
Diffuse   0       0 0 0
Mild 1 2 4 6   0 1 3
Focal 0 0 0 0     0 0
Multifocal 1 2 4 3     1 3
Diffuse 0 0 0 3     0 0
Moderate 0 0 4 3   1 1 4
Focal     0 0   0 0 0
Multifocal     4 2   1 1 4
Diffuse     0 1   0 0 0
Females                        
No. examined 10 10 10 10 10 10 10 10
Rhinitis 0 3 6 9 0 4 5 9
Minimal   1 2 2   1 4 5
Focal   0 0 0   1 0 0
Multifocal   1 2 2   0 4 5
Diffuse   0 0 0   0 0 0
Mild   2 3 4   1 1 3
Focal   1 0 0   0 0 0
Multifocal   1 2 4   1 1 3
Diffuse   0 1 0   0 0 0
Moderate   0 1 3   2 0 1
Focal     0 0   0   0
Multifocal     0 3   2   0
Diffuse     1 0   0   1
Note. The upper respiratory tracts, including the nose, nasal turbinates, larynx, and trachea, from ten parental animals/sex/group/generation, were examined histopathologically. The findings for rhinitis are presented by incidence, degree (minimal, mild, or moderate), and distribution (focal, multifocal, or diffuse).

Applicant's summary and conclusion

Conclusions:
The study on toluene diisocyanate was performed according to the OECD Guideline 416 without deviations and according to the good laboratory practice principles, it is considered to be of high quality (reliability Klimisch 2). The criteria of validity of the test system are fulfilled. There was no reproductive toxicity, reproductive organ pathology, or effect on gestation or lactation at any exposure concentration. Postnatal toxicity and reduced body weights and weight gains during lactation occurred only in F2 litters at 0.08 and 0.3 ppm.
Executive summary:

The toxicity to reproduction of toluene diisocyanate was investigated in CD rats according to OECD TG416 (Märtins, 1989). The test substance was administered to the rats via inhalation (0, 0.02, 0.08 or 0.3 ppm, 6 h/day, 5 day/week) for a total of 10 weeks, then mated within groups for 3 weeks, with exposure 7 days/week during mating, gestation, and lactation. F0 maternal animals were not exposed from gestational day (gd) 20 through postnatal day (pnd) 4; maternal exposures resumed on pnd 5. Twenty-eight weanlings/sex/group continued exposure for 12 weeks (starting on pnd 28) and were bred as described above. F0 and F1 parents and ten F1 and F2 weanlings/sex/group were necropsied, and adult reproductive organs, pituitary, liver, kidneys, and upper respiratory tract (target organs) were evaluated histologically in ten/sex/group. Adult toxicity was observed in both sexes and generations at 0.08 and 0.3 ppm, including occasional reductions in body weights and weight gain, clinical signs of toxicity at 0.08 and 0.3 ppm, and histologic changes in the nasal cavities at 0.02, 0.08, and 0.3 ppm (including rhinitis, a nonspecific response to an irritating vapour, at all concentrations). There was no reproductive toxicity, reproductive organ pathology, or effect on gestation or lactation at any exposure concentration. Postnatal toxicity and reduced body weights and weight gains during lactation occurred only in F2 litters at 0.08 and 0.3 ppm. Therefore, under the conditions of this study, a no observed adverse effect level (NOAEL) was not determined for adult toxicity; the NOAEL for reproductive toxicity was at least 0.3 ppm, and the NOAEL for postnatal toxicity was 0.02 ppm.