Registration Dossier

Diss Factsheets

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
Reliability:
1 (reliable without restriction)
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)
Deviations:
no
Principles of method if other than guideline:
Contiued inhalation exposure of rats to TDI vapours for 2 generations.
GLP compliance:
yes

Test material

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
Details on test material:
80:20 % mixture of the 2,4- and 2,6-isomers, 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 euthansised 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 atomosphere 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 Autostep 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,
3 week exposure during mating,
19 day eposure 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 / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 0.02, 0.08, 0.30 ppm (0, 0.15, 0.58, 2.18 mg/m3)
Basis:
other: target concentration by inhalation
Remarks:
Doses / Concentrations:
0, 0.2, 0.79, 0.29 mg/m3
Basis:
other: analytical concentration by inhalation
No. of animals per sex per dose:
Twenty-eight pups/sex/group
Control animals:
yes, concurrent no treatment
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:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
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, but there were no
effects on body weight. 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)

open allclose all
Dose descriptor:
LOEC
Effect level:
0.02 ppm
Sex:
male/female
Basis for effect level:
other: minimal irritation to respiratory tract
Dose descriptor:
NOEC
Effect level:
0.08 ppm
Sex:
male/female
Basis for effect level:
other: slight decrease in fetal bw
Remarks on result:
other: Generation: embryotoxicity (migrated information)
Dose descriptor:
NOAEC
Effect level:
0.3 ppm
Sex:
male/female
Basis for effect level:
other: no impact on fertility
Remarks on result:
other: Generation: fertility (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

There were no treatment-related gross lesions in F1 animals that were necropsied. F1 males had a significant increase in the incidence of rhinitis at
all exposure concentrations; in 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.

During the 12-week prebreed exposure of F1 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.
F2 pup body weights and weight gain per litter were reduced at 0.080 and 0.30 ppm during lactation.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

F0:

F0 females exhibited no differences among groups for body weight gains.

In F0 males, body weight gain was reduced at 0.3 ppm only for the first exposure week, with increased body weight gains for the fifth and ninth treatment weeks also at 0.3 ppm. Terminal F0 male body weight gains were significantly increased at 0.02, 0.08 and 0.3 ppm (data not shown).

Final body weights (week 14) were significantly increased at 0.3 ppm (6.5% Table 1).

Clinical signs of toxicity (nasal discharge in males and red-tinged fur in females) were observed in the high-exposure F0 group.

There were no treatment-related gross lesions observed in necropsy.

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 also significantly increased in F0 females at 0.3 ppm. Treatment-related histopathologic lesions were limited to the upper respiratory tract with tissues located deeper in the respiratory tract being less affected.

F1-pups:

The Fl litters exhibited equivalent litter sizes and body weights per litter throughout lactation (data not shown). Perinatal deaths (postnatal days 0-4) were decreased (survival was increased) at 0.3 ppm (Table 2).

There were no treatment-related gross lesions in F1 animals that were necropsied.

F1 males had a significant increase in the incidence of rhinitis at all exposure concentrations; in 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. During the 12-week prebreed exposure of F1 animals, animals from the 0.3 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. F2 pup body weights and weight gain per litter were reduced at 0.08 and 0.3 ppm during lactation.

F1 -adult:

Males at 0.3 ppm exhibited reduced body weights (Table 3) and weight gain, females at 0.3 ppm also exhibited reduced body weights (Table 3) but not weight gain. Though final body weight were not reduced in both genders.

Treatment-related clinical signs were observed in Fl females (but not in Fl males) at 0.08 and 0.3 ppm (perinasal encrustation and red-tinged fur).

Histopathology revealed lesions limited to rhinitis in Fl males at all exposure levels and in Fl females at 0.08 and 0.3 ppm.

F2-pups:

F2 pup body weights and weight gain per litter were slightly reduced (<8%) at 0.08 and 0.3 ppm during lactation. Only in the high dose group reduced bw was persisting through day 21 (7%)

Perinatal deaths were reduced (survival was increased) at 0.02 and 0.3 ppm, but lactational survival indices were unaffected by treatment.

There were no treatment-related gross lesions in F2 animals that were necropsied.

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.
For other observations such as rhinitis and body weight changes the LOEC was 0.02 ppm