4,4'-methylenedicyclohexyl diisocyanate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan - March 2003

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 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

(1995)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

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)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

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 weeks
- Weight at study initiation: males 365 (344-388) g, females 218 (198-233) g
- Housing: singly in Makrolon Type IIIh cages except during their overnight co-housing during the matings
- Diet and Water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): approx. 50
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

nose only

Mass median aerodynamic diameter (MMAD):

ca. 1 µm

Geometric standard deviation (GSD):

2

Remarks on MMAD:

In all exposure groups, the aerosol was highly respirable to rats, i.e., the average mass median aerodynamic diameter (MMAD) was approx. 1 µm, the geometric standard deviation (GSD) was approx. 2.

Vehicle:

air

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. > 92 % 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 1.1 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).

Details on mating procedure:

MATING PROCEDURES: During the following mating period the first F0 male was cohoused with the first female F0 animal within the group and so on over night at a maximum of 12 times during the two-week mating period. As a rule inseminated females were not further co-housed. Insemination was established by investigating vaginal smears prepared in the morning.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Actual target concentrations were measured in each chamber up to 3 times per day per gravimetric analysis. Chamber samples were taken in the viscinity of the breathing zone.

Duration of treatment / exposure:

The test substance was administered to parental (F0) animals two weeks prior to and during their mating, and for females during the resultant pregnancy up day 19 p.c. Males were dosed 28 days at a minimum. For technical reasons (to avoid withdrawal from their pups) females were not treated during lactation.

Frequency of treatment:

6 hours/day, 7 days/week

Details on study schedule:

After a gestation period of about 22 days litters were born and the dams were allowed to rear them up to day 4-6 p.p. Then dams and their pups were killed.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 mg/m³ air

Remarks:

target conc.; analytical conc. 1.06 mg/m³

Doses / concentrations 2

Dose / conc.:

6 mg/m³ air

Remarks:

target conc.; analytical conc. 5.95 mg/m³

Doses / concentrations 3

Dose / conc.:

36 mg/m³ air

Remarks:

target conc.; analytical conc. 34.0 mg/m³

No. of animals per sex per dose:

12 test animals / 12 controls

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 establishes a NOAEL of 6 mg/m³ and serves for dose selection rationale (see chapter repeated dose toxicity: inhalation)

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes, all F0 parental animals
- Time schedule: A cage inspection concerning mortality and morbidity was performed twice daily (once daily on weekends and public holidays). On all FO parental animals a detailed clinical observation (excluding findings on nose and breathing) was done prior to the study and at least once weekly as routine at their cage change. All clinical symptoms were recorded. Any findings such as course of birth e.g. prolonged parturition, morbidity and mortality as well as lactation behavior noticed during this cage side observation were recorded. Furthermore, all rats were clinically observed before and after inhalation exposure especially for symptoms concerning nose and breathing.

DETAILED CLINICAL OBSERVATIONS: Yes, all F0 parental animals
- Time schedule: Prior to the study and at least once weekly as routine at their cage change. This investigation includes the evaluation of the general state of health, behavior, condition of the fur, and the orifices as well as excretory products. During gestation periods females were clinically examined on day 0, 7, 14,20, and during lactation on day 0 and 4 in the same way.

BODY WEIGHT: Yes, all F0 parental animals
- Time schedule for examinations: at study-start (first day of dosing), then weekly up to necropsy for male animals and weekly up to established insemination for female animals. After that female animals were weighed on postcoital days 0, 7, 14 and 20; and on days 0 and 4 after birth of their pups. F0 animals were weighed at the day of necropsy to permit calculations of the relative organ weights.

FOOD CONSUMPTION: Yes, all F0 parental animals
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

Oestrous cyclicity (parental animals):

At necropsy also the number of corpora lutea in the right and left ovary was determined.

Sperm parameters (parental animals):

- Examination on Sperms and Spermatids: Determination of spermatozoa motility and viability, determination of spermatozoa morphology, determination of spermatozoa in epididymis (spermatozoa density per mg epididymis), determination of homogenization resistant spermatid heads in the testis (number of spermatid heads per mg testis)

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring shortly after birth and on day 4 p.p.:
- number of live and dead pups
- sex of the pups
- body weights
- clinical signs
- apparent malformations

Postmortem examinations (parental animals):

SACRIFICE
- Unscheduled Necropsies: Parental animals that died or were killed in moribund condition (under diethyl ether narcosis) during the study were necropsied and macroscopically examined.
- Scheduled Necropsies: When pups were 4 to 6 days old dams were anesthetized with carbon dioxide and killed by exsanguination (at carotid artery) and examined for gross pathology. F0 males were killed as scheduled under carbon dioxide narcosis when they were administered 28 days to a minimum.

GROSS NECROPSY
In F0 females implantation sites were counted and documented. Implantation sites were stained with 10% ammoniumsulfide. At necropsy also the number of corpora lutea in the right and left ovary was determined.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Organ weight determinations of the lungs (with trachea), left epididymis and testes were done during the scheduled necropsy.
- Fixed organs/organ specimen of the F0 parental animals(in 10 % neutral buffered formalin solution): lungs (instilled) with trachea, head, one testis, ovaries with oviducts, one epididymis, seminal vesicles, coagulation glands, prostate, tattooed ears and all organs/organ specimen exhibiting macroscopic changes.
- Histopathological evaluation of testes, epididymides and ovaries of control and high dose rats.

Postmortem examinations (offspring):

GROSS NECROPSY
- Unscheduled Necropsies: Pups that were found dead at birth, that died during lactation as well as those killed (with carbon dioxide) in moribund condition were macroscopically inspected after opening the body cavities, with particular attention on the organs of reproduction except for cases of autolysis or cannibalism. This includes also visible skeletal abnormalities as far as possible. A lung flotation in water was performed during the necropsy of pups found dead on the day of the first litter inspection to determine whether pups had breathed at birth or not. Macroscopically changed organs were fixed in 10% formalin.
- Scheduled Necropsies
When they were 4 to 6 days old pups were killed under carbon dioxide anesthesia and were examined for macroscopical alterations as described above.

Statistics:

Dunnett-Test with a variance analysis; Fisher’s exact CHI-SQUARE (positive ANOVA probability test with a significance levels of alpha=5%).

Reproductive indices:

- Indices: insemination, fertility, gestation

Offspring viability indices:

- Indices: live birth, viability

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No test substance-related effects on the appearance, health or behavior were observed in male or female F0 parental animals at levels of up to 36 mg/m³ at clinical observations done once weekly, where nose and breathing symptoms were not considered.
Respective respiratory symptoms, at 36 mg/m³ changes in breathing behavior and/or serous nasal discharge (nostrils with red encrustation) were noted in the majority of F0 rats. At 36 mg/m³ in single animals also signs of poor general conditions occurred and slightly increased (2 of 24 rats) mortality was observed. One male of the high dose group was found dead during the premating period and one female of the high dose group has to be killed in moribund condition. At 6 mg/m³ only serous nasal discharge and red encrusted nostrils were noted in F0 rats.

Mortality:

mortality observed, treatment-related

Description (incidence):

At 36 mg/m³ one male of the high dose group was found dead during the premating period (however, without any symptoms) and one female, which was sperm-positive, but not pregnant (no implantation sites detected), was killed in moribund condition.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No toxic effect was seen on body weights of F0 rats up to 6 mg/m³. At 36 mg/m³ reduced body weight gain was noted at some time points in both sexes (males: week 5-6: 2.8 g vs. 9.8 g in controls, week 4-5: 10.2 g vs. -1.2 g in controls; females: week 1-2: -11.2 g vs -4.7 g in controls).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food intake of 36 mg/m³ F0 males was transiently reduced.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the testes of control and high dose rats tubular degeneration was seen in the majority of animals (Doses 0 and 36 mg/m³: Focal tubular degeneration 5/12, 8/12; Dif. tubular degeneration 2/12, 0/12). In the epididymides, spermatic debris and oligospermia occurred in almost all rats (Doses 0 and 36 mg/m³: Spermatic debris 12/12, 11/12; Oligospermia 11/12, 10/12), which corroborate with the changes in sperm morphology findings. Mechanical stress on the epididymides and testes caused by the narrowness of exposure restrainers is considered as the cause of the effects observed. The morphology of ovaries of F0 females was not affected.
In summary there were no indication of test substance-related morphological alterations at 36 mg/m³.

Reproductive function / performance (P0)

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Results of sperm analysis were excluded from the study evaluation, because mechanical stress on the epididymides and testes caused by the narrowness of exposure restrainers had induced untypical and test-compound independent low sperm motility (below 30%) and increase in sperm abnormalities (higher than 50%) in all groups which corroborate with testicular and epididymal changes seen histologically.
(Dose 0, 1, 6, and 36 mg/m³)
Sperm motility (first min) %: 21 / 7 / 26 / 16
Sperm motility (fifth min) %: 18 / 6 / 25 / 15
Abnormal sperms %: 59.4 / 87.4 / 60.9 / 66.4
Mean number of spermatids per mg testis: 52773 / 41767 / 41989 / 49707
Mean number of sperms per mg epididymis: 490037 / 138194 / 347913 / 323042

Description (incidence and severity):

The insemination and gestation indices as well as the mean duration of pregnancy did not differ to a toxicologically relevant extent from the pertinent control data at levels of up to 36 mg/m³. There were some F0 females (1-3-0-0 with ascending dose) which had been found to be sperm-positive after the first day of co-housing, but failed to become pregnant. According to experience this can happen, if an inexperienced male, co-housed with a female for the first time, inseminated a female not in estrus. This assumption is obviously correct for one female (No. 161; 1 mg/m³), because this female delivered pups after it had been remated with the same male for one week following the two week co-housing period.
The fertility indices do not indicate any adverse effect on the fertility up to 6 mg/m³. At 36 mg/m³ a slightly reduced (p > 0.05) fertility index was calculated.
No treatment-related changes in mating performance was evident.
There was no adverse effect on the number of implantation site or prenatal loss.
No toxicologically relevant changes occurred in the total numbers of pups born, stillborn pups, the live birth index, the percentage of male pups born, the litter size at birth and the viability index.

(Dose 0, 1, 6, and 36 mg/m³)
Insemination index %: 100.0 / 91.7 / 100.0 / 100.0
Fertility index %: 91.7 / 81.8 / 83.8 / 66.7 (p > 0.05)
Gestation index %: 90.9 / 100.0 / 100.0 / 100.0
Gestation length Days: 22.11 / 22.25 / 22.22 / 22.00
Co-housed females n: 12 / 12 / 12 / 12
Number of implantation sites (per litter): 10.8 / 10.56 / 11.50 / 9.88
Litters alive n: 10 / 9 / 10 / 8
Live birth index %: 98.57 / 99.07 / 97.22 / 96.36
Viability index %: 99.00 / 100.0 / 92.17 / 98.96
Males %: 54.31 / 50.56 / 49.23 /53.90

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

1 mg/m³ air

Sex:

male/female

Basis for effect level:

other: General toxicity: signs of respiratory irritation (breathing behaviour, nose discharge) at next higher dose group (6 mg/m³)

2

Dose descriptor:

NOAEL

Remarks:

Reproduction Toxicity

Effect level:

6 mg/m³ air

Sex:

male/female

Basis for effect level:

other: Reproduction Toxicity: slightly reduced fertility index at next higher dose group (36 mg/m³), a level with clear toxicological effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No remarkable clinical signs were observed in F1 pups during the four day lactation period at levels of up to 36 mg/m³.

Mortality / viability:

no mortality observed

Description (incidence and severity):

Pub viability was uneffected up to 36 mg/m³.
Viability index %: 99.00 / 100.0 / 92.17 / 98.96

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The weights at birth and on Day 4 p.p. of treated pups were not toxicologically relevantly changed compared to controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related macroscopical alterations were noted at pup necropsies up to and including 36 mg/m³.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Generation:

F1

Effect level:

36 mg/m³ air

Sex:

male/female

Basis for effect level:

other: Developmental toxicity: No effects on developmental parameters such as live birth index and viability index and no apparent malformation were found in pups up to the highest exposure level (36 mg/m³)

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Data from IUCLID4

RS-Freetext:
At 36 mg/m3 changes in breathing behavior and/or serous nasal discharge (nostrils with red encrustation) were noted in the majority of F0 rats. At 36 mg/m3 in single animals also signs of poor general conditions occurred and slightly increased (2 of 24 rats) mortality was observed. One male of the high dose group was found dead during the premating period and one female of the high dose group has to be killed in moribund condition. At 6 mg/m3 only serous nasal discharge and red encrusted nostrils were noted in F0 rats.
No toxic effect was seen on body weights of F0 rats up to 6 mg/m3. At 36 mg/m3 reduced body weight gain was noted at some time points in both sexes (males: week 5-6: 2.8g vs. 9.8g in controls, week 4-5: 10.2g vs. -1.2g in controls; females: week 1-2: -11.2g vs -4.7g in controls).
The food intake of 36 mg/m3 F0 males was transiently reduced. 
The absolute (14%) and relative (18%) weights of the lungs were increased in 36 mg/m3 F0 male rats.

PARAMETERS OF REPRODUCTION

At 36 mg/m3 a slightly reduced fertility index was noted. 
No other reproduction parameter was affected.


Dose mg/m3: 0/ 1/ 6/ 36

Insemination index %: 100.0/ 91.7/ 100.0/ 100.0
Fertility index %: 91.7/ 81.8/ 83.8/ 66.7 (p > 0.05)
Gestation index %: 90.9/ 100.0/ 100.0/ 100.0
Gestation length Days: 22.11/ 22.25/ 22.22/ 22.00
Co-housed females n: 12/ 12/ 12/ 12
Number of implantation sites (per litter): 10.8 / 10.56 / 11.50 / 9.88
Litters alive n: 10/ 9/ 10/ 8
Live birth index %: 98.57 / 99.07 / 97.22 / 96.36
Viability index %: 99.00 / 100.0 / 92.17 / 98.96

SPERM ANALYSIS

Results of sperm analysis were excluded from the study evaluation, because mechanical stress on the epididymides and testes caused by the narrowness of exposure restrainers had induced untypical and test-compound independent low sperm motility ( below 30%) and increase in sperm abnormalities ( higher than 50%) in all groups which corroborate with testicular and epididymal changes seen histologically.

Dose mg/m3: 0 / 1 / 6 / 36

Sperm motility, (first min) %: 21 / 7 / 26 / 16
Sperm motility (fifth min) %: 18 / 6 / 25 / 15
Abnormal sperms %: 59.4 / 87.4 / 60.9 / 66.4
Mean number of spermatids per mg testis:
52773 / 41767 / 41989 / 49707
Mean number of sperms per mg epididymis:
490037 / 138194 / 347913 / 323042

HISTOPATHOLOGY

No test compound-related effects were seen in the testes and epididymides of F0 rats.
In the testes of both groups evaluated (control and high dose) tubular degeneration (mainly multi/focal) was seen in the majority of animals:

dose mg/m3: 0 ; 36 

Focal tubular degeneration: 5/12 ; 8/12
Dif. tubular degeneration: 2/12 ; 0/12

In the epididymides, spermatic debris and oligospermia occurred in almost all rats.

dose mg/m3: 0 ; 36 

Spermatic debris: 12/12 ; 11/12
Oligospermia: 11/12 ; 10/12

The morphology of the ovaries of F0 females was not affected.

Data On PUPS (F1):

dose mg/m3: 0 / 1 / 6 / 36

- Live birth index %: 98.57 / 99.07 / 97.22 / 96.36
- Viability index %: 99.00 / 100.0 / 92.17 / 98.96
- Males %: 54.31 / 50.56 / 49.23 /53.90

No remarkable clinical signs were observed during the four day lactation period up to 36 mg/m3. No effect on body weights and no macroscopical alterations were noted at pup necropsies up to 36 mg/m3.

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): "In a reproduction/developmental toxicity screening test according to OECD TG 421 (...) Wistar rats (12 animals/sex/dose) were exposed to 4,4´-methylenedicyclohexyl diisocyanate aerosol. The rats were exposed nose-only daily for 6 hours/day to concentrations of 0, 1, 6 and 36 mg/m³ (= target concentrations). F₀ male and female rats were exposed for 2 weeks (premating exposure period), which was continued during the approximately 2 week mating period. Males were exposed for at least 28 days (prior to necropsy) whereas the exposure of the F₀ females continued during the pregnancy up to day 19 post coitum (p.c.). Exposure of the F₀ females was suspended up to the day of necropsy on day 4 - 6 p.p. (post partum), i.e., the time points at which F₁ pups were sacrificed. In all exposure groups, the aerosol was highly respirable to rats, i.e., the average mass median aerodynamic diameter (MMAD) was ≈ 1 μm, the geometric standard deviation (GSD) was ≈ 2. Clinical signs as changes in breathing behavior and/or serous nasal discharge were documented for F₀ animals of the 6 and 36 mg/m³ groups. One male of the high dose group was found dead during the premating period and one female of the high dose group had to be killed in moribund condition. No effect on body weights gain, food consumption and necropsy findings, were observed at ≤ 6 mg/m³. Significant increases of absolute and relative weights of the lungs were detected at 36 mg/m³ in male rats. No effects of 4,4´-methylenedicyclohexyl diisocyanate on reproductive parameters such as insemination index, gestation index and length and the number of implantation sites were described. At 36 mg/m³ a slightly reduced fertility index was noted (0, 1, 6, 36 mg/m³: fertility index: 91.7, 81.8, 83.8, 66.7* % (*= p > 0.05)). No remarkable clinical signs were seen in any F₁ pubs during the 4 day lactation period and body weight gain was comparable to the control animals. In the testes of both groups evaluated (control and high concentration F₀ animals) tubular degeneration (mainly multi/focal) was seen in the majority of animals. In the epididymides, spermatic debris and oligospermia occurred in almost all rats. These histopathological findings are concordant with the results of the spermatological evaluation of all animals (no or very low sperm motility and high percentage of abnormal sperms in control rats and in all concentration groups). Nevertheless the findings seen in the testes are not substance-related because they were found also in the control animals and they are dose independent. Mechanical stress on the epididymides and testes caused by the narrowness of exposure restrainers seems to be responsible for the effects seen in all 4,4´-methylenedicyclohexyl diisocyanate and air exposed animals.

Based on these findings, the NOAELs were considered to be 1 mg/m³ in males and females for general toxicity. Due to a slightly reduced fertility index at 36 mg/m³, 6 mg/m³ is the NOAEL for reproductive toxicity in rats."