Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-11-14 to 2006-02-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Isophorone diisocyanate of Bayer MaterialScience AG, batch no.LL48/3-55, purity: 99.8%

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Sex: male
- Strain: Hsd/win: NMRI
- Source: Harlan Winkelmann GmbH, Borchen
- Age: 6-12 weeks
- Weight at study initiation: 34 - 40 g
- Number of animals: 6/group/sacrifice timepoint (5 for the 24h positive control group)
- Housing: single in conventional Makrolon Typ II cages
- Diet: standard fixed-furmula diet 3883 (PROVIMI Kliba; Kaiseraugst.; CH) ad libitum
- Water: tab water ad libitum
- Acclimation period: at least 5 days, prior to exposure mice were adapted to exposure tubes on 3 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 2 °C
- Humidity (%): approximately 40 - 60 %
- Air changes (per hr): approximately 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h/ 12h

Administration / exposure

Route of administration:
other: nose-only inhalation (vapor/aerosol)
Vehicle:
air
Details on exposure:
ISOPHORONE DIISOCYANATE EXPOSURE:
- Mice were assigned to four exposure groups and were exposed to the  aerosolized test substance to target concentrations of 
0 air (--), 5(4.3), 15 (16.1) and  40 (39.6) mg/m³ (1 x 6 hr); target concentrations (analytical concentrations)
- number of animals per concentration of test substance and air control: 18 (main study) and additionally 5  (satellite mice for respiratory function measurements)
PARAMETERS ASSESSED 
- Clinical observations: several times on the day of exposure and at  least once daily thereafter
- Body weights: daily
-  Body temperature: rectal body temperature directly after cessation of  exposure; subcutaneous body temperature (transponders) during exposure  (at intervals of 30 min.) up to 3 hours after ceasing exposure
- Respiratory function measurements (restricted to the satellite mice)
CONDUCT OF THE MICRONUCLEUS TEST
- Experimental Group Target Concentration Sacrifice Time
                          (mg/m³)             (hours)
---------------------------------------------------------  
Negative Control           0                 24  
Isophorone Diisocyanate    5                 24   
Isophorone Diisocyanate    15                 24   
Isophorone Diisocyanate    40                 24   
Positive Control   
Cyclophosphamide           20 mg/kg
  
Negative Control           0                    48   
Isophorone Diisocyanate    5                  48   
Isophorone Diisocyanate    15                  48   
Isophorone Diisocyanate    40                   48
  
Negative Control           0                   72   
Isophorone Diisocyanate    5                   72   
Isophorone Diisocyanate    15                   72   
Isophorone Diisocyanate    40                   72     

number of animals in the positive control group: 5   
2000 polychromatic erythrocytes were counted per animal
Duration of treatment / exposure:
1 x 6 hours
Frequency of treatment:
1 time
Post exposure period:
24, 48, 72 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 5, 15, 40 mg/m³ (target concentration)
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
--, 4.3, 16.1, 39.6 mg/m3
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
--, 33, 94.2, 212 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
--, 2.1, 15.9, 44.0 mg/m3
Basis:
other: gravimetric concentration
No. of animals per sex per dose:
test substance: 6
negativ control: 6
positive control: 5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamid (Endoxan) 20 mg/kg single intraperitoneal treatmaent

Examinations

Tissues and cell types examined:
polychromatic erythrocytes of the bone marrow from femur
Details of tissue and slide preparation:
A method described by Schmid (1975) was used to produce the smears (Schmid, W.; The Micronucleus Test; Mutation Res. 31, 9-15, 1975 ; and: Schmid, W. Der Mikrokerntest; Deutsche Forschungsgemeinschaft. Kommission für Mutagenitätsfragen. Mitteilung III, 53-61, 1975). The smears were stained automatically with an Ames Hema-Tek slide Stainer (Mile Company), destrained with methanol, rinsed with deionized water, dried and covered.
Evaluation criteria:
ASSESSMENT CRITERIA:
- A test was considered positive if there was at any time point a  biologically relevant and statistically  significant increase in the  number of 
polychromatic erythrocytes showing micronuclei in comparison to  the negative control.
- A test was considered negative if there was no relevant or significant  increase in the rate of micronucleated polychromatic erythrocytes. 
A test  was also considered negative if there was a significant increase in that  rate which, according to the laboratory's experience was within
the range  of historical negative controls.
- In addition, a test was considered equivocal if there was an increase  of micronucleated polychromatic erythrocytes above the range of attached  
historical negative controls, provided the increase was not significant  and the result of the negative control was not closely related to the  data 
of the respective treatment group. A test was also considered  equivocal, if its result was implausible. In both case, normally a second  test will be
performed.
Statistics:
BIOMETRY:
- Normally 2000 polychromatic erythrocytes were counted per animal.
- Per sacrifice time the Isophorone diisocyanate group with the highest  mean (provided this superceded the negative control mean) and the  
positive control were checked by Wilcoxon's non-parametric rank sum test  with respect to the number of polychromatic erythrocytes having  
micronuclei and the number of normochromatic erythrocytes. A variation  was considered statistically significant if its error probability was  
below 5% and the treatment group figure was higher than that of the  negative control.
- The rate of normochromatic erythrocytes containing micronuclei was  examined if the micronuclear rate for polychromatic erythrocytes was  
already relevantly increased. In this case, the group with the highest  mean was compared with the negative control using the one-sided  
chi2-test. A variation was considered statistically significant if the  error probability was below 5% and the treatment group figure was higher  
than that of the negative control.
- In addition, standard deviations (1s ranges) were calculated for all  the means.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Remarks:
no relevant indications of a clastogenic effect of the test substance under conditions of this study in male mice
Toxicity:
yes
Remarks:
all concentrations: evidence of respiratory tract irritation and clinical symptoms caused by exposure to the test substance observed (see "Add. information on results")
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
ISOPHORONE DIISOCYANATE EXPOSURE
- Target Concentration (mg/m³): 0, 5, 15, 40
- Nominal Concentration (mg/m³): --, 33, 94.2, 212
- Gravimetric Concentration (mg/m³):--,  2.1, 15.9, 44.0
- Analytical Concentration (mg/m³): --, 4.3, 16.1, 39.6 
- The Mass Median Aerodynamic Diameters (MMAD) was <4 µm (MMAD 1.3 µm,  Geometric Standard Deviation 2). At the lowest concentration IPDI  at mospheres consisted of vapor rather than aerosol to an appreciable  extent.
PARAMETERS ASSESSED:
- Mortality: did not occur at any exposure level:
- Clinical Observations:   
Target      Deaths/signs/total  Onset and Duration    
Concentration                       of Signs    
(mg/m³)                   
0                 0 /  0 / 18              --        --   
5                 0 / 18 / 18           0d - 3d           
15                  0 / 18 / 18           0d - 3d           
40                 0 / 18 / 18           0d - 3d          
0d = day of exposure.   
3d: terminal sacrifice   
Values given in the 'Deaths/signs/total' column are as follows:   
1st number        = number of dead animals   
2nd number        = number of animals with signs after exposure cessation   
3rd number        = number of animals exposed   
0 mg/m³: All mice tolerated the exposure/dosing without signs.   
5 mg/m³: Bradypnea, labored breathing patterns, stridor, motility  reduced, high-legged gait, piloerection, eyelids closed.    
15 mg/m³: Bradypnea, labored breathing patterns, breathing sounds,  stridor, motility reduced, high-legged gait, piloerection, hair-coat  
ungroomed, eyelids closed, blepharospasm.   
40 mg/m³: Bradypnea, labored breathing patterns, breathing sounds,  stridor, motility reduced, high-legged gait, piloerection, hair-coat  
ungroomed, eyelids closed, blepharospasm, tremor, prostration,  salivation, cyanosis.
- Body weights: Marked, concentration-dependent decrease in body weights  which was most pronounced at 15 mg/m³ and 40 mg/m³.
- Body temperature: The mean body temperatures were significantly  decreased at the end of the 6-h exposure period in all test  material-exposure 
groups.    
0 mg/m³: 38.1 °C   
5 mg/m³: 33.3* °C  
15 mg/m³: 27.9** °C  
40 mg/m³: 25.6** °C *  = p < 0.05, ** = p < 0.01   
Subcutaneously measured body temperatures were consistent with rectal  temperatures. The subcutenously measured values showed that the 
duration  of hypothermia was more pronounced in 40 mg/m³-group as compared to 15  mg/m³-group.
- Respiratory function measurements (satellite groups):   Moderate effects on respiration were observed in 5 mg/m³, whilst in 15  mg/m³-group and  40 mg/m³-group a maximal depression of respiration was  observed. This was caused specifically by increases in the bradypnoic  period (pause 
between end inspiration and start of expiration).
CONDUCT OF THE MICRONUCLEUS TEST
- As may be seen from the tables below, the ratio of polychromatic to  normochromatic erythrocytes in males was altered by the treatment with  
Isophorone diisocyanate for all sacrifice times at all concentration  groups.
- As may be further deduced from the tables below, no biologically  important or statistically significant variations existed for males  between the 
negative control and the groups treated by inhalation with  isophorone diisocyanate, with respect to the incidence of micronucleated  
polychromatic erythrocytes. 
- Similarly, there could be no biologically significant variation between  the negative control and Isophorone diisocyanate groups in the number of  
micronucleated normochromatic erythrocytes, since normochromatic  erythrocytes originated from polychromatic ones. As expected, relevant  
variations were not observed.
- The positive control, cyclophosphamide, caused a clear increase in the  number of polychromatic erythrocytes with micronuclei. The incidence of  
micronucleated cells represents biologically relevant increases in  comparison to the negative control.

Any other information on results incl. tables

RS-Freetext:
SUMMARY OF RESULTS OF 24 HOURS INTERVAL

Experimental  number of   number of   MNNCE per  MNPCE per
groups        evaluated   of NCE per  2000 NCE   2000 PCE
              PCE         2000 PCE    
                          (1s range)  (1s range) (1s range)
-----------------------------------------------------------
negative
control       12000        1988           2.3       2.0
                           (412)         (2.3)     (0.6)

5 mg/m³       12000        3117           2.1       1.7
                           (997)         (1.2)     (0.8)

15 mg/m³      12000        4598*          3.0       3.0
                          (1617)         (1.0)     (0.6)

40 mg/m³      12000        3679           2.8       4.5
                          (1959)         (3.3)     (2.2)

positive      10000        2515           1.9      26.2**
control                    (768)         (0.8)    (12.4)        

SUMMARY OF RESULTS OF 48 HOURS INTERVAL

Experimental  number of   number of   MNNCE per  MNPCE per
groups        evaluated   of NCE per  2000 NCE   2000 PCE
              PCE         2000 PCE    
                          (1s range)  (1s range) (1s range)
-----------------------------------------------------------
negative
control       12000        1682           2.4       4.3
                           (154)         (1.6)     (2.3)

5 mg/m³       12000        2039           2.8       2.2
                           (635)         (2.5)     (1.5)

15 mg/m³      12000        3874           2.1       3.3
                          (2810)         (0.9)     (2.3)

40 mg/m³      12000        4236*          2.5       3.5
                          (1568)         (0.6)     (1.4)


SUMMARY OF RESULTS OF 72 HOURS INTERVAL

Experimental  number of   number of   MNNCE per  MNPCE per
groups        evaluated   of NCE per  2000 NCE   2000 PCE
              PCE         2000 PCE    
                          (1s range)  (1s range) (1s range)
-----------------------------------------------------------
negative
control       12000        1353           3.2       2.3
                           (209)         (2.3)     (1.8)

5 mg/m³       12000        1800           1.7       1.8
                           (541)         (2.5)     (1.0)

15 mg/m³      12000        3387           2.4       3.7
                          (2810)         (0.9)     (2.3)

40 mg/m³      12000        7168**         2.6       4.5
                          (2577)         (0.7)     (1.8)
-----------------------------------------------------------
*P        < 0.05 in non-parametric Wilcoxon ranking test
**P        < 0.01 in non-parametric Wilcoxon ranking test

Applicant's summary and conclusion

Conclusions:
There were no biologically relevant indications of a clastogenic effect after inhalative exposure of Isophorone diisocyanate in the micronucleus test on the male mouse under the experimental conditions of this study.
Analytical monitoring data of the exposure conditions, clinical signs of respiratory tract irritation, hypothermia and respiratory depression
appear to suggest adequate bioavailability by this route.
Executive summary:

The micronucleus test was conducted to investigate the test substance Isophorone Diisocyanate in male NMRI mice for a possible clastogenic effect on the chromosomes of bone-marrow erythroblasts. Male mice treated with Isophorone Diisocyanate received for 6 hours inhalative target concentrations of 0 (air control), 5, 15 and 40 mg/m3 respectively. The animals were sacrificed 24, 48 and 72 hours after exposure. After inhalative treatment for 6 hours of males with concentrations up to and including 40 mg/m3 (target concentration) no biologically relevant indications of a clastogenic effect of Isophorone Diisocyanate were found in male NMRI mice under the experimental conditions of this study.