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

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Equivalent or similar to OECD Guideline 416, GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Principles of method if other than guideline:

Continued inhalation exposure of rats to TDI vapours for 2 generations.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

m-tolylidene diisocyanate

EC Number:

247-722-4

EC Name:

m-tolylidene diisocyanate

Cas Number:

26471-62-5

IUPAC Name:

2,4-diisocyanato-1-methylbenzene, 2,6-diisocyanato-1-methylbenzene

Details on test material:

80:20 % mixture of the 2,4- and 2,6-isomers of TDI, over 99% pure (Dow Chemical USA).

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Kingston , NY)
- Diet (e.g. ad libitum): certified ground rodent chow #5002, Ralton-Purina
- Water (e.g. ad libitum): tap water
- Acclimation period: 2 weeks
- Weight at study initiation:
(P) Males: 200.2-204.7 g; Females: 141.4-146.6 g;
(F1) Males: 127.5-141.2 g; Females: 111.6-121 g
- Age at study initiation: 6 wks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 62-76°F (RT)
- Humidity (%): 40-70%
- Photoperiod: 12hrs dark / 12hrs light

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: Countercurrent air stream

Details on exposure:

Male and female Sprague-Dawley weanling rats F0 (28 animals/sex/group) were exposed to TDI vapour at different concentrations, 6 hours/day, 5 days/week, for 10 weeks.
Animals were paired randomly within groups for 3 weeks to produce the F1 generation. Exposures of females continued through mating and the first 19 days of gestation and were discontinued from gestation day 20 through the fourth day postpartum. Exposures of females resumed on day 5 postpartum and continued through postnatal day 20. Exposures of F0 males were continuous from the mating period through delivery of the first F1 litters. At weaning, 28 weanlings/sex/group F1 were randomly selected to produce the F2 generation. F1 weanlings were exposed to the same TDI protocol as the F0 generation.
In addition, 10 F1 weanlings/sex/group were necropsied for gross lesions. F0 males were necropsied following delivery of the first F1 litters. F0 females were necropsied after the F1 pups were weaned.
The selected F1 weanings were exposed to the same exposure concentration of TDI as their parents for 12 weeks. After their pre-breed exposure, F1 animals were paired as described above to produce the F2 generation. Mating, gestation, lactation, necropsy of the F1 parents and selected F2 weanlings and historic examination of selected F1 adults tissues were performed as described above except that no F2 animals were selected as parents. Remaining non-selected F1 and F2 pups at weaning were euthanised and discarded after the necropsy of the selected pups. Mating 1 male to 1 female.


GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: TDI vapour was generated using a glass evaporator system. Rats were exposed to TDI vapours in 4320 litre chambers.
- Temperature, humidity, pressure in air chamber: Temperature measurements were obtained from the inside surface of each evaporator during the exposure regimen.
- Air flow rate: 1000 l/min (for the 0.0 and 0.3 ppm chambers), or 1500 l/min (for the 0.02 and 0.08 ppm chambers)
- Air change rate: >=14/h


TEST ATMOSPHERE
- Brief description of analytical method used: Throughout the study, TDI atmosphere were monitored by placing probes in the breathing zone of the animals approximately six times per each 6h exposure. Control chamber atmosphere was measured six times daily for the first 11 exposure days and once per day thereafter. Atmospheres were monitored by paper tape devices based upon modified Marcali method.
Two auto step Isocyanate paper tape monitoring devices (GMD) System, 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.
- Samples taken from breathing zone: yes

Details on mating procedure:

Observations of vaginal sperm and/or dropped or vaginal copulation plug were considered evidence of successful mating. Once the animals mated, they were housed individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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. Samples were obtained and reverse-phase HPLC was used to separate and quantify the 2,4- and 2,6-isomers.

Duration of treatment / exposure:

10 week pre breed exposure F0, 12 week pre breed exposure F1
3 week exposure during mating,
19 day exposure during gestation,
(dams not exposed day 20-24), 16 days during lactation.

Frequency of treatment:

pre breed exposure: 6h/day, 5 days/week
during mating: 6h/day, 7 days/week until day 19 of gestation. ; then exposed again 6h/day, 7 days/week to day 20 postnatal. At day 21 litters weaned. Parents for second generation selected and exposed 6h/day, 5 days/week for 12 weeks prior to mating. Exposure during mating and subsequently, as above.

Details on study schedule:

Exposures of females resumed on day 5 postpartum and continued through postnatal day 20. Exposures of P0 males were continuous from the mating period through delivery of the first F1 litters.

At weaning, 28 weanlings/sex/group were randomly selected to produce the F2 generation. F1 weanlings were exposed to the same TDI protocol as the F0 generation. In addition, 10 F1 weanlings/sex/group were necropsied for gross lesions. F0 males were necropsied following delivery of the first F1 litters. F0 females were necropsied after the F1 pups were weaned. Selected tissues from 10 F0 animals/sex/group in the high exposure and control groups were examined for histopathological lesions. Tissues from the upper respiratory tract from 10 animals/sex from the mid- and low-exposure groups also were examined for histopathological lesions.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

0.02 ppm (nominal)

Remarks:

0.15 mg/m3

Doses / concentrations 3

Dose / conc.:

0.08 ppm (nominal)

Remarks:

0.58 mg/m3

Doses / concentrations 4

Dose / conc.:

0.3 mg/m³ air (nominal)

Remarks:

2.18 mg/m3

Doses / concentrations 5

Dose / conc.:

0 ppm (analytical)

Doses / concentrations 6

Dose / conc.:

0.02 ppm (analytical)

Doses / concentrations 7

Dose / conc.:

0.079 ppm (analytical)

Doses / concentrations 8

Dose / conc.:

0.29 ppm (analytical)

No. of animals per sex per dose:

Twenty-eight pups/sex/group

Control animals:

yes

Details on study design:

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.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: pups pnd 0, 1, 4, 7, 14, 21


BODY WEIGHT: Yes
- Time schedule for examinations:
dams gd 0, 7, 14, 21 and pnd 1, 4, 7, 14;
pups pnd 1, 4, 7, 14, 21, 28

Oestrous cyclicity (parental animals):

not assayed

Sperm parameters (parental animals):

not assayed

Litter observations:

Survival indices were calculated at 0, 4, 7 and 14 days after birth and at weaning.

Postmortem examinations (parental animals):

Necropsy:
All F0 and F1 parental animals in all groups (both generations).
Necropsy included: 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:
Selected tissues from 10 F0 animals/sex/group in the high exposure and control groups were examined for histopathological lesions.
Tissues: pituitary, liver, kidneys (2), 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.
Tissues from the upper respiratory tract from 10 animals/sex from the mid- and low-exposure groups also were examined for histopathological lesions.

Postmortem examinations (offspring):

A gross internal examination was performed on any pup appearing abnormal or dying on test, and ten pups randomly selected for each sex from
each test group of the Fl and F2 generations.

Statistics:

The unit of comparison was the male, the female or the litter. Results of the quantitative continuous variables e.g., body weights, food consumption, organ weights, etc. were intercomparted for the 3 treatment groups and one control group by use of Levene’s test for equal variances, analysis of variance (ANOVA) and t-tests. Nonparametric data were statistically evaluated using the Kruskal-Wallis test followed by the Mann-Whitney U-test for pairwise comparisons when appropriate. Frequency data such as the various indices were compared using the Fisher’s exact-test. For all statistical tests, the fiducial limit of 0.05 (two-tailed) was used as the criterion for statistical significance.

Reproductive indices:

The reproductive indices were calculated for F0 and F1 males and females for each breed (F0 to produced F1 litters and F1 to produce F2 litters).

a. Mating index (%) = Number of females with copulation plugs/Number of females cohabited x 100
b. Fecundity index (%) = Number of pregnancies/Number of plug-positive females x 100
c. Fertility index (female) (%) = Number of females pregnant/ Total number of females cohabitated x 100
d. Fertility index (male) (%) = Number of males shown to be fertile/Total number of males mated x 100
e. Gestational index = Number of females with live litters/Number of females pregnant
f. Live birth index = Number of live pups at birth/Total number of pups born

Offspring viability indices:

The viability indices were calculated for F0 and F1 males and females for each breed (F0 to produced F1 litters and F1 to produce F2 litters).
a. 4-Day survival index = Number of pups surviving 4-day (pre-cull)/Total number of live pups at birth
b. 7-Day survival index = Number of pups surviving 7-days/Total number of live pups at 4-days (post-cull)
c. 14-Day survival index = Number of pups surviving 14-days/Total number of live pups at 7-days (post-cull)
d. 21-Day survival index = Number of pups surviving 21-days/Total number of live pups at 14-days (post-cull)
e. Lactation index = Number of pups surviving 21 days/ Total number of live pups at 4-days (post-cull)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs associated with F0 adult toxicity were observed at 0.3 ppm for both sexes (nasal discharge in males and red-tinged fur in females).
Periocular encrustation, perinasal encrustation and read nasal discharge were observed in all exposure groups of F0 males including controls and appear to be associated with the inhalation treatment conditions rather than the test chemical vapor.

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Female F0 body weights showed no significant differences among groups during the pre-breed exposure periods or during the final exposure week. F0 female weight gains showed a similar equivalence across treatment groups for the pre-breed exposure period. During the final week of exposure (week 18-19) females at 0.3 ppm exhibited a significantly increased weight gain. During the mating period of the F0 generation, maternal gestation body weights and weight gain were equivalent across all exposure groups.

During the pre-breed exposure F0 male body weights were equivalent across all treatment groups. From week 10 through week 13, during the mating period, body weights of exposed males did not differ significantly from their controls. Final body weights (week 14) were significantly increased at 0.3 ppm. Male F0 weight gains were reduced only for the first exposure week at 0.3 ppm. Significantly increased weight gains were observed for F0 males for treatment weeks 4-5 and 8-9 also at 0.3 ppm. Terminal body weight gains were significantly increased at 0.02, 0.08 and 0.3 ppm.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathology was performed on selected organs from 10 parental animals (F0) per sex from the high exposure concentration and control animals. Histological evaluation of nasal turbinates, larynx and trachea from 10 parental animals (F0) was performed for all exposure groups. There were no treatment-related lesions observed in the necropsy. 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, hyperplasia) of the respiratory (nasal) epithelium in the nasal turbinates. Significant incidences of rhinitis were also observed in nasal turbinates of F0 males and females at 0.08 ppm. The incidence of rhinitis was not significantly increased at 0.02 ppm for either males and females.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

Clinical signs of toxicity (nasal discharge in males and red-tinged fur in females) were observed in the high-exposure F0 group. Histopathology revealed a significant increase in the incidence of rhinitis in the nasal turbinates of F0 animals (both sexes) exposed to 0.08 and 0.3 ppm and hyperplasia and dysplasia of the respiratory epithelium of F0 males at 0.3 ppm. The incidence of hyperplasia was significantly increased in F0 females at 0.3 ppm.

Effect levels (P0)

1

Key result

Dose descriptor:

LOAEC

Effect level:

0.08 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

other:

Remarks on result:

other: general toxicity: effects on respiratory tract

2

Dose descriptor:

NOAEC

Effect level:

0.3 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no impact on fertility

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.08 ppm

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In F1 males no clinical signs was observed across all treatment groups related to the test chemical exposure. In F1 females perinasal encrustation was observed across all treatment groups, the incidence was significantly increased at 0.3 ppm. The incidence of red-tinged fur, while occasional in TDI-exposed males, was significantly increased in F1 females at 0.08 and 0.3 ppm from 17 to 22 weeks of exposure.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the twelve-week pre-breed exposure of the F1 animals, males exposed to 0.3 ppm exhibited reduced body weight relative to the controls for the first four weeks of exposure. In males exposed with 0.3 ppm body weight gain was reduced in week 1 to 2 and the final exposure week. F1 males exposed to 0.02 ppm showed significantly increased body weight gain relative to the controls for weekly intervals (12-13, 13-14 week) of the mating period.
F1 females showed reduced body weights at 0.3 ppm for the first two weeks of exposure as well as week 6 of exposure. There were no significant differences among groups for F1 female weight gain.
At the F1 breed to produce F2 litters, maternal gestational body weights and lactational body weights were unaffected by exposure.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related lesions observed in the histopathologic examination of selected organs from F1 adults were limited to rhinitis in F1 males at all exposure levels and in F1 females at 0.08 and 0.3 ppm.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Effect levels (P1)

1

Dose descriptor:

LOAEC

Effect level:

0.3 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

2

Key result

Dose descriptor:

LOAEC

Effect level:

0.08 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: rhinitis

3

Key result

Dose descriptor:

LOAEC

Effect level:

0.02 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: rhinitis

Target system / organ toxicity (P1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.02 ppm

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

other:

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Perinatal deaths/missing pubs seen in F1 litters were within the historical control data range and were considered not treatment-related.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant difference in pub body weights or pub lactation body weights were noted for F1 litter compared to the control group.

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

no effects observed

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

There were no treatment-related gross lesions in F1 (adult) animals that were necropsied. F1 adult males had a significant increase in the incidence of rhinitis at all exposure concentrations; in adult females, this increase was apparent only at the two higher doses. In the high-exposure group (males), there was a significant increase in the incidence of submucosal lymphoid infiltrates in both the larynx and the trachea as well as a significant increase in the incidence of intracellular eosinophilic droplets. There were no treatment-related effects in the trachea or larynx of F1 females (adult).
During the 12-week pre-breed exposure of F1 adult animals, animals from the 0.30 ppm group exhibited reduced body weights (both sexes) and weight gain (males only). The only treatment-related clinical signs were observed in F1 females and included perinasal encrustation and red-tinged fur.
No significant different was seen in F1 total born/litters and alive/litters between the control group and treatment groups. No significant different in F1 litter size and sex ratio were noted between the control group and treatment groups on lactation day 4, 7, 14 and 21.

Effect levels (F1)

Key result

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

0.3 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: no significant different compared to control group

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

0.3 ppm

System:

other: no significant effects compared to control

Results: F2 generation

General toxicity (F2)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 0.3ppm body weight / litter group was reduced beginning on postnatal day 14 persisting until day 21.
At 0.08 ppm: mean female pup body weight were reduced at lactation day 14.
At 0.08 and 0.3 ppm: weight gain per litter were reduced for lactation day 4 to 7 and at 0.3 ppm weight gain reductions persisted trough the lactation day 21.

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Key result

Dose descriptor:

LOAEC

Generation:

F2

Effect level:

0.08 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

 

Table 1: Final body weights (week 14) of F0 adult males

ppm	0	0.02	0.08	0.3
Mean +/-SD	557.7 +/-52.75	584.9 +/-51.58	585 +/-51.26	596.4 +/-53.42

 

Table 2: Litter viability F1-generation on days 0 -4 precull

ppm	0	0.02	0.08	0.3
#dead (d 0-4)	17	16	11	7

Applicant's summary and conclusion

Conclusions:

In this study, there was no effect of exposure on any of the reproduction parameters evaluated, so the reproductive NOAEC is greater than 0.3ppm.

Executive summary:

The toxicity on fertility of TDI was investigated in a two generation study in rats performed under GLP (Tyl et al. 1989). F0 rats were exposed by inhalation (whole body) to 80:20 mixture of 2,4 and 2,6 isomers of TDI at concentrations of 0, 0.02, 0.08, 0.3 ppm, for 6 hrs/day, 5 days/week for 10 weeks prior to mating. All animals were exposed 6 h/d 7d/w during 3 week mating period and subsequent in life period. Females were exposed through mating and for first 19 days of gestation. Exposure resumed 5 days postpartum through day 20 postpartum. Exposure to males was continuous through delivery of F1 litters. F1 weanlings selected for mating were exposed to the same TDI protocol except 12 weeks pre-mating.

Continued inhalation exposure to TDI for two generation in rats  results in parental toxicity indicated by reduced body weight, body weight gains, clinical signs and histopathological changes of the upper respiratory tract. Histological changes were mainly seen  in the  nasal cavities accompanied by  rhinitis. In males F1 males rhinitis was found in all treatment groups whereas for F0 animals and F1 females rhinitis found significant at 0.08 ppm and 0.3 ppm. Postnatal toxicity consists of reduced body weights and body weight gains, occurred only in F2 litters at 0.08 ppm and 0.3 ppm. There was no effect of treatment on reproduction and the NOAEC for reproduction was set greater than 0.3 ppm.