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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

MDI MT is not a reproduction toxicant.

Link to relevant study records

Reference

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

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

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

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.

Dose / conc.:

0 ppm (nominal)

Dose / conc.:

0.02 ppm (nominal)

Remarks:

0.15 mg/m3

Dose / conc.:

0.08 ppm (nominal)

Remarks:

0.58 mg/m3

Dose / conc.:

0.3 mg/m³ air (nominal)

Remarks:

2.18 mg/m3

Dose / conc.:

0 ppm (analytical)

Dose / conc.:

0.02 ppm (analytical)

Dose / conc.:

0.079 ppm (analytical)

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.

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)

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: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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.

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

Dose descriptor:

NOAEC

Effect level:

0.3 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no impact on fertility

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

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: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Dose descriptor:

LOAEC

Effect level:

0.3 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

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

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

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

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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.

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

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

0.3 ppm

System:

other: no significant effects compared to control

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

 

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

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. 

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

0.3 ppm

Study duration:

subchronic

Experimental exposure time per week (hours/week):

42

Species:

rat

Quality of whole database:

reliable

Additional information

There are no fertility toxicity studies available for MDI MT. A read across with data from the source substances TDI (mixed isomers) was performed. MDI MT and TDI toxicity are driven by their NCO functionality causing portal of entry effects. As MDI MT and TDI do not contain any other functional groups that effect their toxicological properties and the NCO-functionality is seen as driver of toxicity a read across with TDI data for the endpoint reproductive toxicity  is warranted.

The toxicity on fertility of TDI (mixed isomers) was investigated in a two generation study in rats performed under GLP (Tyl et al. 1989). 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 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 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. The effects seen in this study limited to the respiratory tract (poral of entry), no adverse effects on reproduction were indicated.

The effects observed are in line with the hypothesized MoA for MDI MT predicts local effects on the respiratory tract and no significant systemic exposure to unreacted isocyanate (NCO) and no effects on fertility. Evidence suggests that the isocyanate (NCO) group is scavenged at the portal of entry and is not systemically available in unbound reactive form. Based on structural similarities of MDI MT and TDI, the isocyanate-functionality seen as driver of toxicity, a read across of the TDI data for the endpoint fertility is warranted. Accordingly, the target substance MDI MT like the source substance TDI (mixed isomers) is not classified as a reproduction toxicant by EU GHS 1272/2008 CLP.

 

To increase the MDI data base a series of toxicity to reproduction studies is planned for the source substance 4,4’-MDI. A combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test (OECD 422) is scheduled to start Q1 2023. A test proposal for an Extended One-Generation Reproductive Toxicity Study (OECD 443) was send Q3 2021 to ECHA for approval. To fulfil the data requirements for the MDI MT REACH registration (REACH Annex VIII, 10-100 tonnage).  The new data will be used in a read across approach with MDI MT and MDI MT dossier will be updated when the new MDI data are available.

Effects on developmental toxicity

Description of key information

MDI is not a developmental toxicant.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

1) The concentration of pMDI in the test atmosphere were determined by gravimetry. 2) Lung lavage was not performed in rats exposed to 12 mg pMDI/m3 air. 3) All animals were examined for histopathological changes at 12 mg pMDI. 4) Opthalmology was perform

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

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


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


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

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

whole body

Details on exposure:

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



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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

Details on mating procedure:

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

Duration of treatment / exposure:

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

Frequency of treatment:

once daily; 6h a day

Duration of test:

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

Dose / conc.:

0 mg/m³ air (nominal)

Dose / conc.:

1 mg/m³ air (nominal)

Dose / conc.:

4 mg/m³ air (nominal)

Dose / conc.:

12 mg/m³ air (nominal)

No. of animals per sex per dose:

25 mated females per group.

Control animals:

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

Details on study design:

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

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

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

Maternal examinations:

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


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


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


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


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

Ovaries and uterine content:

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

Fetal examinations:

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

Statistics:

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

Indices:

See "Further details on study design"

Historical control data:

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

Details on maternal toxic effects:

Maternal toxic effects:yes

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

Key result

Dose descriptor:

NOAEC

Remarks:

Maternal toxicity

Effect level:

4 mg/m³ air (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other: effects on respiratory tract indicating respiratory irritation

Key result

Abnormalities:

no effects observed

Localisation:

other:

Description (incidence and severity):

no maternal abnormalities observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

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

Key result

Dose descriptor:

NOAEC

Effect level:

4 mg/m³ air (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

fetal/pup body weight changes

other:

Remarks on result:

other: fetal effects are secondary

Key result

Abnormalities:

no effects observed

Localisation:

other:

Description (incidence and severity):

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

12 mg/m³ air (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

no

Relevant for humans:

no

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

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

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

Conclusions:

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

Executive summary:

Gamer et al. (1994) performed a reliable developmental toxicity study with pMDI (which contains about 50 % mMDI). The study was performed according OECD Guideline 414. Female rats were exposed  to  concentrations of at concentrations of 0, 1, 4 and 12 mg/m³. At concentrations of 1 or 4 mg/m³, no signs of maternal toxicity and no substance-induced adverse effects on the gestational parameters or the fetuses were recorded. Maternal toxicity was substantiated by mortality, damage to the respiratory tract, reduced body weight development and reduced mean gravid uterus weights at 12 mg/m³.  At this concentration clear signs of developmental (embryo-/feto-) toxicity in the form of reduced placental and fetal body weights and an increased occurrence of fetal skeletal (and overall) variations and retardation were recorded. However, no substance-induced teratogenic effects were observed up to and including the highest concentration (12 mg/m³). As the observed fetotoxic effects and adverse effects on the embryonic development are considered as minor signs of developmental toxicity and as these effects occur at the concentration inducing maternal toxicity, they are considered to be secondary to maternal toxicity, the NOAEC for maternal and fetal toxicity is 4 mg/m³. Consequently, pMDI is considered not to be a developmental toxicant.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

4 mg/m³

Study duration:

subacute

Experimental exposure time per week (hours/week):

30

Species:

rat

Quality of whole database:

reliable, guideline studies

Additional information

Developmental toxicity / teratogenicity:

There are no  screening or reproductive/ developmental toxicity studies available for MDI MT. A read across with reliable study data from the source substances 4,4’-MDI and pMDI is performed to fill data gaps for the endpoint developmental toxicity.

A reliable guideline study (OECD Guidance 414) is available for the source substance pMDI. Gamer et al. (1994) performed a reliable developmental toxicity study with pMDI (which contains about 50 % mMDI). The study was performed according to OECD Guideline 414. Female rats were exposed  to  concentrations of 0, 1, 4 and 12 mg/m³. At concentrations of 1 or 4 mg/m³, no signs of maternal toxicity and no substance-induced adverse effects on the gestational parameters or the foetuses were recorded. Maternal toxicity was substantiated by mortality, damage to the respiratory tract, reduced body weight development and reduced mean gravid uterus weights at 12 mg/m³.  At this concentration clear signs of developmental (embryo-/feto-) toxicity in the form of reduced placental and foetal body weights and an increased occurrence of foetal skeletal (and overall) variations and retardation were recorded. However, no substance-induced teratogenic effects were observed up to and including the highest concentration (12 mg/m³). As the observed fetotoxic effects and adverse effects on the embryonic development are considered as minor signs of developmental toxicity and as these effects occur at the concentration inducing maternal toxicity, they are considered to be secondary to maternal toxicity, the NOAEC for maternal and foetal toxicity is 4 mg/m³. Consequently, pMDI is considered not to be a developmental toxicant.

A reliable guideline study (OECD Guidance 414) is available for the source substance 4,4’-MDI (Buschmann et al. 1996). Female rats were exposed to concentrations of 1, 3, and 9 mg/m³. The lung weights in the high-dose group were significantly increased compared to the sham-treated control animals. Treatment did not influence any other maternal and/or foetal parameters investigated (maternal weight gain, number of corpora lutea, implantation sites, pre- and post-implantation loss, foetal and placental weights, gross and visceral anomalies, degree of ossification), although a slight but significant increase in litters with foetuses displaying asymmetric sternebra(e) (within the limits of biological variability) was observed after treatment with the highest dose of 9 mg/m³. Conservatively, a no effect level of 3 mg/m³ for developmental and maternal toxicity was determined.

The findings of the studies were essentially the same with regard to the maternal and foetal NOAECs. Since the highest exposure concentration in the study on pMDI caused greater toxicity than in the study with 4,4’-MDI resulting in foetal effects (albeit secondary to maternal toxicity), this study is regarded as being of higher value and used as the key study for the assessment of developmental toxicity. It is widely recognized that maternal conditions that cause decreased uterine-placental blood flow, decreased nutrient circulation, anaemia, altered acid-base balance, hypoxia, obesity and others have been shown to contribute to foetal developmental toxicity. Some common developmental effects resulting from these maternal conditions include supernumerary ribs, delayed foetal growth, and skeletal variations, such as delayed ossification. For both 4,4’-MDI and pMDI, the only developmental effects noted are minor skeletal variations and slightly inhibited foetal growth and are only observed at concentrations that also induce significant reductions in maternal food consumption and body weight gain, and respiratory toxicity (e.g. laboured breathing, increased lung weights). Reduced foetal weight and delayed ossification are common foetal manifestations induced by maternal toxicity (Banerjee and Durloo, 1973; Woo and Hoar, 1979; Tyl, 2012; Nitzsche, 2017) which suggests that the foetal effects noted in the 4,4’-MDI and pMDI studies are secondary.

While maternal toxicity should not automatically negate a foetal effect, there is also sufficient mechanistic data to support a lack of human relevance for fetotoxicity of MDI substances. OECD “Guidance Document on Inhalation Toxicity Studies” (no. 39) describes the rodent-specific physiological effects of respiratory irritants on inhalation developmental toxicity studies (OECD, 2018). In short, rodents have a respiratory reflex that reduces respiration and body temperature and subsequently results in foetal hypoxia, hypercapnia, hypothermia and malnutrition. As the foetus is more sensitive to hypothermia and hypoxia, developmental defects and delays can develop. Isocyanates are known respiratory irritants and are included as an example of substances that induce the rodent-specific irritation reflex known to cause developmental effects. Thus, effects noted in these studies are not relevant to human exposure and MDI substances should not be classified as developmental toxicants.

In summary, for the developmental toxicity endpoint, the foetal effects observed with 4,4’-MDI and pMDI are consistent and secondary to maternal toxicity as a consequence of respiratory irritation. Respiratory irritation, in turn, is consistent with the hypothesized MoA and direct electrophilic reactions of bioaccessible NCO groups. The hypothesized MoA predicts local effects in the lungs and no significant systemic exposure to unreacted NCO since it reacts with biological nucleophiles before being absorbed as GHS/protein adducts. Minor fetal effects related to growth were considered secondary to the maternal irritation and toxicity.  No direct toxicity was noted in the developmental toxicity study which is consistent with the observed lack of systemic toxicity in combined chronic toxicity and carcinogenicity studies on 4,4’-MDI and pMDI as well as the proposed mechanism for MDI absorption toxicokinetics.

As the source substances 4,4’-MDI, pMDI and the target substance MDI MT contain sufficient monomeric MDI, the driver of toxicity, similarities in reactions with extracellular nucleophilic biomolecules at the site of contact are assumed. As the higher molecular weight non-monomeric content of the UVCB substance MDI MT do not contains reactive centres and is consequently inert and thus do not contribute to the observed toxicity, it is reasonable to assume that using read across to the source substances 4,4’-MDI and pMDI is warranted. Accordingly, the target substance like the source substances 4,4’-MDI and pMDI is not classified as a developmental toxicant by EU GHS 1272/2008 CLP.

Mode of Action Analysis / Human Relevance Framework

Isocyanates (including MDI MT) are highly reactive substances that exhibit strong reactivity with biological nucleophiles. As the MDI substance enters the lung, NCO groups react with biological nucleophiles at the MDI/lung fluid interface which results in pulmonary irritation and inflammation.  The foetal effects observed with the source substances 4,4’-MDI and pMDI are consistent and secondary to maternal toxicity in rats as a consequence of respiratory tract irritation. In OECD Guidance document on inhalation toxicity studies (2018) rodent-specific physiological effects of respiratory irritants on inhalation developmental toxicity studies were addressed. Rodents have a respiratory reflex that reduces respiration and body temperature and subsequently results in foetal hypoxia, hypercapnia, hypothermia and malnutrition. As the foetus is more sensitive to hypothermia and hypoxia, developmental defects and delays can develop. Isocyanates are known respiratory irritants and are included as an example of substances that induce the rodent-specific irritation reflex known to cause developmental effects. Thus, effects noted in these studies are not relevant to human exposure and MDI substances should not be classified as developmental toxicants.

Justification for classification or non-classification

Accordingly, the target substance MDI MT like the source substance TDI (mixed isomers) is not classified as a reproduction toxicant by EU GHS 1272/2008 CLP.

Accordingly, the target substance MDI MT like the source substances 4,4'-MDI and pMDI is not classified as a developmental toxicant by EU GHS 1272/2008 CLP.