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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan 2003 - March 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(2001)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-methylenedicyclohexyl diisocyanate
EC Number:
225-863-2
EC Name:
4,4'-methylenedicyclohexyl diisocyanate
Cas Number:
5124-30-1
Molecular formula:
C15H22N2O2
IUPAC Name:
1,1'-methylenebis(4-isocyanatocyclohexane)
Details on test material:
- Active ingredient: >/= 99.5%

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Wistar Hsd Cpb:WU (SPF)
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 13-15 weeks
- Weight at study initiation: females 201-245 g
- Housing: Starting from gestation day 0 females were singly accommodated in in Makrolon Type III cages.
- Diet and Water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): approx. 55 */- 20
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
ca. 1 µm
Geometric standard deviation (GSD):
2
Remarks on MMAD:
The aerosol was highly respirable to rats, i.e., the average mass median aerodynamic diameter (MMAD) was ca. 1 µm, the geometric standard deviation (GSD) was ca. 2.
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Mode of exposure: Animals were nose-only exposed to the aerosolized test article in restrainers made of Plexiglas. The type of exposure is comparable with a directed-flow exposure design (Moss and Asgharian, Respiratory Drug Delivery IV, 1994, 197-201).
- Exposure apparatus: Chambers used are commercially available (TSE, Bad Homburg, Germany) and the performance as well as their validation has been published (e.g. Pauluhn, Journal of Applied Toxicology, 14, 55-62, 1994). Each segment of the aluminum inhalation chamber has the following dimensions: inner diameter = 14 cm, outer diameter = 35 cm (two-chamber system), height = 25 cm (internal volume = about 3.8 I).
- Generation of aerosol: In order to increase the efficiency of the generation of respirable particles and to prevent larger particles from entering the chamber a preseparator/baffle system was used. Under dynamic conditions the various concentrations of the test substance were nebulized into the baffle (pre-separator) which entrained the substance into the intake of the cylindrical inhalation chamber. For nebulization a binary nozzle (maintained at a temperature of 30°C) and conditioned compressed air (15 L/min and 10 µL/min test substance; dispersion pressure approximately 600 kPa). The test substance was fed into the nozzle by a digital pump (Hamilton Microlab M). This atmosphere was diluted further with 45 L/min prior to entering the inhalation chamber. The targeted concentrations were achieved by using air extraction/substitution dilution cascades.
- Inhalation chamber equilibrium concentration: The test atmosphere generation conditions provide an adequate number of air exchanges per hour (> 200 x, continuos generation of test atmosphere). Under such test conditions used chamber equilibrium is attained in less than one minute of exposure (t99% = 4.6 x chamber volume/chamber airflow). The ratio between the air supplied and exhausted was chosen so that approximately 90% of the supplied air is removed via the exhaust system.
- Conditioning the compressed air: Compressed air was supplied by Boge compressors and was conditioned (i.e. freed from water, dust, and oil) automatically by a VIA compressed air dryer. Adequate control devices were employed to control supply pressure.
- Exhaust air treatment: The exhaust air was purified via cotton woll and HEPA filters.
- Temperature and humidity measurements were performed by the computerized Data Acquisition and Control System using FTF11-Sensoren (Fa. Elka Electronic, Lüdenscheid, Germany).

TEST ATMOSPHERE
- The integrity end stability of the aerosol generation and exposure system was monitored using a RAM-1 (MIE, Bedford, MA, USA) and FhG (Fraunhofer Institute, Hannover, Germany) real-time aerosol photometer.
- Samples taken from breathing zone: yes
- Brief description of analytical method used: gravimetric analysis of filter samples (filter: Glass-Fibre-Filter, Sartorius, Göttingen, Germany; digital balance).
- Particle size distribution: The particle-size distribution was analyzed using a BERNER-Type Aeras low-pressure critical orifice cascade impactor. > 85.8 % of the particle mass had an aerodynamic diameter - MMAD (Mass median aerodynamic diameter): The respirability of the aerosol was adequate, i.e. the mass median aerodynamic diameter (MMAD) was 1.3 µm at 1 mg/m³, 1.1 µm at 6 mg/m³ and 1.0 µm at 36 mg/m³ (geometric standard deviation (GSD) approx. 2).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test substance concentration was determined by gravimetric analysis. Chamber samples were taken in the viscinity of the breathing zone.
Details on mating procedure:
The animals were mated by placing two females overnight into a type III cage together with one male rat. If sperm was detected in the vaginal smear taken on the morning following mating, this day was regarded as day 0 of gestation.
Duration of treatment / exposure:
6 hours/day from days 6 to 19 p.c.
Frequency of treatment:
once daily
Duration of test:
Day 0 until cesarian section and sacrifice at Day 20 of gestation
Doses / concentrationsopen allclose all
Dose / conc.:
1 mg/m³ air
Remarks:
target conc.; analytical conc. 1.06 mg/m³
Dose / conc.:
6 mg/m³ air
Remarks:
target conc.; analytical conc. 5.99 mg/m³
Dose / conc.:
36 mg/m³ air
Remarks:
target conc.; analytical conc. 33.2 mg/m³
No. of animals per sex per dose:
27 inseminated females at 0, 1, and 6 mg/m³; 32 inseminated females at 36 mg/m³
Control animals:
other: conditioned air
Details on study design:
- Dose selection rationale: An orientating aerosol inhalation pilot study with 5 male and 5 female rats exposed nose-only for 1 week to target concentrations of 1, 6 and 36 mg/m³ established a NOAEL of 6 mg/m³ and served for dose selection rationale (cp chapter repeated dose toxicity: inhalation)

Cesarean sections were performed on gestation day 20 without knowledge of treatment groups.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes, all females
- Time schedule for examinations: From days 0 to 20 p.c. all animals were inspected twice daily (before and after inhalation exposure, on days without inhalation exposure once daily, only), and all findings were recorded.

BODY WEIGHT: Yes, all females
- Time schedule for examinations: The body weights of the animals were determined on day 0 p.c. and daily from day 6 to day 20 p.c. Corrected body weight gain was calculated by subtracting the weight of the uterus on day 20 p.c. from the body weight gain over the period from day 0 to day 20 p.c.

FOOD CONSUMPTION: Yes, all females
- The feed consumption of the animals on gestation days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, and 18-20 was determined based on the differences in weight of feed provided and feed, which remained unconsumed.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes, all females
The females were subjected to gross pathological examination at the time of cesarean section on day 20 p.c. without knowledge of treatment groups.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes, for all females
Examinations included:
- Number of corpora lutea
- Number of implantations (in females without visible implantation sites after staining of the uterus with a solution of 10 % ammoniumsulfide)
- Uterine weights
- Individual weight and appearance of the placentas
- Number of early resorptions (only implantation site visible), late resorptions (fetal or placental remnant visible), and dead fetuses (fetuses without signs of life, but without maceration)
- Number of live fetuses
Fetal examinations:
- Sex of live fetuses
- Individual weights of live fetuses
- External malformations: Yes, all per litter
- Visceral malformations: Yes, about half per litter
- Findings in abdominal, pelvic, and thoracic organs as well as skeletal and cartilage findings with the addition of cartilage staining: Yes, about half per litter
Statistics:
Dunnett-Test with a variance analysis; Fisher’s exact CHI-SQUARE (positive ANOVA probability test with a significance levels of alpha=5%)
Indices:
- Indices: Fertility and Gestation Index

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Appearance, behaviour, and mortality of the females were unaffected at the 1 mg/m³ level, while increased or decreased respiratory rates, laboured breathing, irregular respiration, and sounds consistent with rhinitis occurred each before and after inhalation at the 36 mg/m³ level. Furthermore, rough fur, piloerection (one female, one day only), serous nasal discharge, and reddish encrusted nostrils each before and after inhalation, and reddish encrusted nose (one female, one day only) occurred in the 36 mg/m³ group. Laboured breathing after inhalation, piloerection before and after inhalation, paleness before and after inhalation and and reddish encrusted nostrils after inhalation occurred in single females at the 6 mg/m³ level; several females had serous nasal discharge before (9 females) and after inhalation (17 females) at this dose level.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant transient body weight loss (approx. 5%) after start of treatment (days 6-7 p.c.), resulting in a decreased body weight gain during the treatment period (days 6-19 p.c., approx. 24%) and during gestation (days 0-20 p.c., approx. 15%) occurred at the 36 mg/m³ level. Mean corrected body weight gain was also marginally decreased (approx. 14%) at this dose level.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Decreased feed intake (days 6-9 p.c.: approx. 22%; days 9-12 p.c.: approx. 8%) was observed at the 36 mg/m³ level.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related gross pathological findings occurred at levels up to and including 36 mg/m³. No gross pathological fmdings were evident in the females at the dose level of 1 mg/m³ and 36 mg/m³.
Histopathological findings: non-neoplastic:
not examined

Maternal developmental toxicity

Details on maternal toxic effects:
GENERAL REPRODUCTION DATA
The gestation rate, appearance of placentas, placental weights, postimplantation loss, number of live fetuses, fetal sex distribution, and fetal weights were unaffected by treatment with the test material up to and including the dose level of 36 mg/m³.

The number of inseminated females with implantation sites in % of those inseminated, and the mean number of implantation sites were lower at 36 mg/m³, and a slight increased preimplantation loss occurred in this group, whereas the mean number of corporalutea did not differ to meaningful extent, indicating a homogeneous distribution regarding this parameter. The values in the 36 mg/m³ dose group were comparable to the normal range of scattering for the rat strain used, and statistical significance was not evident for these findings, so that treatment related unobserved early postimplantation loss (which would not be detectable by staining the uterus, and would appear as preimplantation loss) is unlikely.

Females with implantations in % of those inseminated (doses 0, 1, 6, and 36 mg/m³): 77.8 / 96.3 / 74.1 / 71.9
Mean values per female with implantation sites (doses 0, 1, 6, and 36 mg/m³):
- corpora lutea: 15.0 / 14.4 / 14.1 / 14.7
- preimplantation loss: 1.8 / 1.4 / 1.7 / 2.8
- implantations: 13.2 / 13.0 / 12.4 / 11.8

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 mg/m³ air
Basis for effect level:
clinical signs

Results (fetuses)

Details on embryotoxic / teratogenic effects:
FETAL DATA
The postimplantation loss, number of live fetuses, fetal sex distribution, and fetal weights were unaffected by treatment with the test material up to and including the dose level of 36 mg/m³.

Means per female (doses 0, 1, 6, and 36 mg/m³)
- post implantation loss: 0.7 / 0.8 / 1.3 / 1.0
- number of live fetuses: 12.6 / 12.2 / 11.2 / 10.8
- fetal weights g: 3.51 / 3.53 / 3.40 / 3.55
- sex of live fetuses (% males): 55.3 / 51.8 / 50.5 / 51.8

FETAL MALFORMATIONS
A treatment related effect cannot completely be excluded for the marginally increased incidence (1.2 % versus 0.9 % in historical controls) of ventricular septal defects of the heart (common finding) at the level of 36 mg/m³, despite of a lower overall number of fetuses with malformations at this dose level, as this dose showed clear maternal toxicity.

(doses 0, 1, 6, and 36 mg/m³)
- Number of fetuses per group: 264 / 317 / 224 / 248
- Number of fetuses with malformations: 9 / 5 / 5 / 4
- malformed fetuses per group (%): 3.4 / 1.6 / 2.2 / 1.6
- Number of litters per group: 21 / 26 / 20 / 23
- Number of litters with malformations: 6 / 4 / 5 / 4
- malformed litters per group (%): 28.6 / 15.4 / 25.0 / 17.4
- ventricular septal defect of the heart: 1 / 1 / 1 / 3
- number of litters affected by this malformation: 1 / 1 / 1 / 3
The highest values of total numbers of fetuses or litters with malformations were found in the control group (3.4 % of affected fetuses, 28.6 % affected litters).

FETAL EXTERNAL AND VISCERAL DEVIATIONS
Meaningful external or visceral deviations of fetuses were not assumed at dose levels up to and including 6 mg/m³. A treatment related effect for the significantly increased incidence of slight dilation of lateral brain ventricle(s) (11.3% affected fetuses, 56.5% affected litters) at the 36 mg/m³ level is unlikely, as the value lay within the range of historical control data and data of different unaffected study groups used (up to 12.7% affected fetuses, up to 65.5% affected litters).

(doses 0, 1, 6, and 36 mg/m³)
- Number of fetuses per group: 264 / 317 / 224 / 248
- Number of fetuses with deviations: 59 / 60 / 48 / 53
- fetuses with deviations per group (%): 22.3 / 18.9 / 21.4 / 21.4
- Number of litters per group: 21 / 26 / 20 / 23
- Number of litters with deviations: 18 / 20 / 18 / 20
- litters with deviations per group (%): 85.7 / 76.9 / 90.0 / 87.0
- slight dilation of lateral brain ventricle (s): 11 / 14 / 17 / 28
- number of litters affected by this deviation: 5 / 7 / 8 / 13

FETAL SKELETAL DEVIATIONS INCLUDING CARTILAGINOUS DEVIATIONS
Summarizing all skeletal including cartilaginous tissue findings, no treatment related fmdings occurred at levels up to and including 36 mg/m³.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
6 mg/m³ air
Sex:
male/female
Basis for effect level:
other: See Remarks

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
36 mg/m³ air
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects

Any other information on results incl. tables

Data from IUCLID4

RS-Freetext:
MATERNAL DATA:

Appearance, behavior, and mortality of the females were unaffected at the 1 mg/m3 level, while increased or decreased respiratory rates, labored breathing, irregular
respiration, and sounds consistent with rhinitis occurred each before and after inhalation at the 36 mg/m3 level. Furthermore, rough fur, piloerection (one female,
one day only), serous nasal discharge, and reddish encrusted nostrils each before and after inhalation, and reddish encrusted nose (one female, one day only) occurred in the 36 mg/m3 group. 
Labored breathing after inhalation, piloerection before and after inhalation, paleness before and after inhalation and and reddish encrusted nostrils after inhalation occurred in single females at the 6 mg/m3 level; several females had serous nasal discharge before (9 females) and after inhalation (17 females) at this dose level.
 
Decreased feed intake (days 6-9 p.c.: approx. 22%; days 9-12 p.c.: approx. 8%), statistically significant transient body weight loss (approx. 5%) after start of treatment (days 6-7 p.c.), resulting in a decreased body weight gain  during the treatment period (days 6-19 p.c., approx. 24%) and during gestation (days 0-20 p.c., approx. 15%) occurred at the 36 mg/m3 level. Mean corrected body weight gain was also marginally decreased (approx. 14%) at this dose level.

No treatment related gross pathological findings occurred at levels up to and including 36 mg/m3.

The gestation rate, appearance of placentas, placental weights, postimplantation loss, number of live fetuses, fetal sex distribution, and fetal weights were unaffected by treatment with the test material up to and including the dose level of 36 mg/m3.

GENERAL REPRODUCTION DATA

The mean number of implantation sites were lower at 36 mg/m3, and a slight increased preimplantation loss occured in this group, whereas the mean number of corporalutea did not differ to meaningful extent, indicating a homogeneous distribution regarding this parameter. The values in the 36 mg/m3 dose group were comparable to the normal range of scattering for the rat strain used, and statistical significance was not evident for these findings, so that treatment related unobserved early postimplantation loss (which would not be detectable by staining the uterus, and would appear as preimplantation loss) is unlikely.

Dose mg/m3:          0/ 1/ 6/ 36
Females with implantations in % of those inseminated:  - 77.8 / 96.3/  74.1 / 71.9
mean values per female with implantation sites
- corpora lutea: 15.0 / 14.4 / 14.1 / 14.7
- preimplantation loss: 1.8 / 1.4 / 1.7 / 2.8
- implantations: 13.2 / 13.0 / 12.4 / 11.8

FETAL DATA 

Dose mg/m3:          0/ 1/ 6/ 36
Means per female
- post implantation loss: 0.7 / 0.8 / 1.3 / 1.0
- Number of live fetuses: 12.6 / 12.2 / 11.2 / 10.8 
- fetal weights g: 3.51 / 3.53 / 3.40 / 3.55
- sex of live fetuses (% males): 55.3 / 51.8 / 50.5 / 51.8

FETAL MALFORMATIONS

A treatment related effect cannot completely be excluded for the marginally increased incidence (1.2% versus 0.9% in historical controls) in of ventricular septal defects of the heart (common finding) at the
level of 36 mg/m3, despite of a lower overall number of fetuses with malformations at this dose level, as this dose showed clear maternal toxicity.

Dose mg/m3:          0/ 1/ 6/ 36

- Number of fetuses per group: 264 / 317 / 224 / 248
- Number of fetuses with malformations: 9 / 5 / 5 / 4
- malformed fetuses per group (%): 3.4 / 1.6 / 2.2 / 1.6
- Number of litters per group: 21 / 26 / 20 / 23
- Number of litters with malformations: 6 / 4 / 5 / 4
- malformed litters per group (%): 28.6 / 15.4 / 25.0 / 17.4
- ventricular septal defect of the heart: 1 / 1 / 1 / 3
- number of litters affected by this malformation:
1 / 1 / 1 / 3

FETAL EXTERNAL AND VISCERAL DEVIATIONS

Meaningful external or visceral deviations of fetuses were not assumed at dose levels up to and including 6 mg/m3. A treatment related effect for the significantly increased incidence of slight dilation of lateral brain ventricle(s) (11.3% affected fetuses, 56.5% affected litters) at the 36 mg/m3 level is unlikely, as the value lay within the range of historical control data and data of different unaffected study groups used (up to 12.7% affected fetuses, up to 65.5% affected litters).

Dose mg/m3:          0/ 1/ 6/ 36

- Number of fetuses per group: 264 / 317 / 224 / 248
- Number of fetuses with deviations: 59 / 60 / 48 / 53
- fetuses with deviations per group (%): 22.3 / 18.9 / 21.4 / 21.4
- Number of litters per group: 21 / 26 / 20 / 23
- Number of litters with deviations: 18 / 20 / 18 / 20
- litters with deviations per group (%): 85.7 / 76.9 / 90.0 / 87.0  
- slight dilation of lateral brain ventricle (s):
11 / 14 / 17 / 28
- number of litters affected by this deviation:
5 / 7 / 8 / 13

Applicant's summary and conclusion

Executive summary:

4,4´-Methylenedicyclohexyl diisocyanate has been assessed in the OECD HPV programme, 2005.

Cited from SIAR of SIAM 20 (Paris, April 19 -22, 2005): "The prenatal toxicity of 4,4´-methylenedicyclohexyl diisocyanate in pregnant Wistar rats was investigated by aerosol inhalation according to OECD TG 414 (...). Mated female rats (at least 27/dose) were exposed nose-only to target concentrations of 0, 1, 6 and 36 mg/m³ for 6 h/day from day 6 to day 19 p.c. Particle size determination of the aerosol yielded MMADs within the respirable range (≈ 1 μm, the geometric standard deviation (GSD) ≈ 2). Exposure to 4,4´-methylenedicyclohexyl diisocyanate aerosols caused no premature death. Clinical signs of respiratory tract irritation (i.e. decreased respiratory rates, labored breathing, irregular respiration and sounds consistent with rhinitis) became apparent at 36 mg/m³. Furthermore, rough fur, serous nasal discharge, reddish encrusted nostrils, occurred in the 36 mg/m³ group. (...) At 6 mg/m³ only single females evidenced clinical signs of beginning respiratory irritation. Reduction in food consumption occurred at 36 mg/m³, which was accompanied by a delay of body weight development during the treatment period (approximately 24 % below controls) and during gestation (approximately 15 % below controls). Gross-pathologically no treatment-related findings in the dams occurred at any concentration. The gestation rate, appearance of placentas, placental weights, postimplantation loss, number of live fetuses, fetal sex distribution, and fetal weights were unaffected by treatment up to and including the concentration of 36 mg/m³. With regard to the results on malformation a marginally increased incidence of ventricular septal defects of the heart (three fetuses out of three litters, 1.2 % of total fetuses) occurred at the 36 mg/m³ level, which lay only marginally above the upper normal range (0.9 % affected fetuses) of scattering of the rat strain used. The incidence and type of external or visceral deviations were unaffected by treatment at levels up to and including 6 mg/m³. A statistically significantly increased incidence of slight dilation of lateral brain ventricles occurred at 36 mg/m³. A treatment related effect for this anomaly is unlikely, as the incidence is normally broadly scattering between unaffected study groups, and because the value (11.3 % affected fetuses, 56.5 % affected litters) lay within the range of historical control data and data of different unaffected study groups of the rat strain used (up to 12.7 % affected fetuses, up to 65.0 % affected litters).

Nevertheless, a NOAEL of 6 mg/m³ was determined in this study with regard to developmental toxicity."