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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2003-10-14 to 2004-11-09
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: According to ECHA Practical Guide 6 the maximum score for read across is rel. 2

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
isophorone diisocyanate of Bayer Polymers, batch no. LL48/3-55, purity 99.8 %, sampled 07 Aug 2003

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ORGANISMS
- Source: Hsd Cpb:WU from Harlan-Winkelmann GmbH, Borchen (Germany)
- Age: between 14 and 17 weeks (females)
- Weight at study initiation: 201 - 244 g (females)
- Number of animals: 27 per dose / control group
Housing conditions:
- Room temperature: 22°C+/-2°C;
- humidity: appr. 50%;
- light: 12hours light/dark;
- air change: 10 times per hour;
- animal room was cleaned daily
- nutrition: standard diet Provimi Kliba Maus/Ratte-Haltung-GLP, Art.-No 3883.0.15 (Provimi Kliba SA, 4303 Kaiseraugst, Switzerland) ad libitum
- water: tap water ad libitum

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure (if applicable):
nose only
Vehicle:
other: air
Details on exposure:
Females:
- exposure by inhalation (nose only) daily from day 6 to 19 post coitum for 6 hours between approximately 08:00 and 14:00 CET.

Males:
- not treated (used only for mating)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Doses used in this study were verified by analysis. The analyses of the test atmospheres are described in the analytical report (study no. T 7072620) which is part of the main study. The test atmosphere was determined by high performance liquid chromatography (HPLC) after derivatization of the
isocyanate functionality. Samples were taken by using glass powder filled tubes containing nitro-reagent as scavenging agent. For
reference/calibration purposes the test compund was used. The precision, accuracy and stability in solvents as well as on the adsorbent used were
checked prior to the study. Chamber samples were taken in the vicinity of the breathing zone.
Details on mating procedure:
MATING PROCEDURES: 
Two females and one male were placed in a cage  overnight. If sperm was detected in the vaginal smear taken in the  morning, this day was 
regarded as day 0 of gestation.
Duration of treatment / exposure:
days 6 through 19 post coitum
Frequency of treatment:
6 hours/day, daily
Duration of test:
The femals were subjected to gross pathological examination at the time of cesarian section on day 20 post coitum. The females were sacrificed using cardiotomy under deep carbon dioxide anesthesia.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 (air control); 0.25; 1.0; 4.0 mg/m3
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
--; 1.1; 5.0; 23.6 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
--; 0.206; 0.929; 4.536 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
27 inseminated females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Sex: female
Duration of test: cesarean section on day 20

Examinations

Maternal examinations:
PARAMETERS ASSESSED DURING STUDY: 
- Mortality: daily on days 0 through 5 and 20, twice daily on days 6  through 19
- Clinical signs: daily on days 0 through 5 and 20, twice daily on days 6  through 19
- Body weight gain: Weighing on days 0 and 6 through 20, correction for  weight of uterus on day 20
- Food consumption: Cumulative on days 3, 6, 9, 12, 15, 18, and 20; water  consumption 3 times/week

Ovaries and uterine content:
- Examination of uterine content: 
Number of corpora lutea, number of  implantations (in females without visible implantation sites after  staining with 10 % ammonium sulfide 
solution), uterine weights,  individual weight and appearance of the placentas, number of early  resorptions (only implantation sites visible), 
number of late resorptions  (fetal or placental remnant visible), number of dead fetuses (i.e.  without signs of life, without maceration), number and sex of live fetuses
Fetal examinations:
- Examination of fetuses: sex, individual weight, external malformations  or other findings deviating from normal, visceral malformations and other   
findings deviating from normal (Wilson technique), findings in abdominal,  pelvic, and thoracic organs as well as skeletal and cartilage findings  
(modified Dawson technique) with the addition of cartilage staining:  evisceration, cartilage staining with alcian blue GX, clearing of the  fetuses 
with diluted potassium hydroxide solution, staining of the  skeletal system with alizarin red S and evaluation of the skeletal system  including 
cartilaginous findings. Every other fetus within a litter was  prepared for either skeletal or visceral evaluation with generally the  first fetus of 
each litter assigned to skeletal analysis. 
Statistics:
STATISTICAL METHODS: 
- Females without implanatation sites were excluded. Skeletal  localizatins with mechanical damage in single fetuses were excluded from  the 
calculation of percentages of affected localizations but reported in  the tables of individual skeletal findings.
- Analysis of variance, and in case of significant results Dunnett's test  for: feed consumption; body weights (incl. gains and corrections);  
uterine weights; number of corpora lutea, of implantations, of live  fetuses (incl. percentages) per female; placental and fetal weights per  female.
- 2 by N Chi(square) test, and in case of significant differences  Fisher's exact test with Bonferroni correction for: fertility and  gestation rate; 
number of implantations per group; number of  preimplantation losses per group; number of postimplantation losses,  early resorptions, late 
resorptions, or dead fetuses per group; number of  live fetuses per group in percent of implantations; number of male or  female fetuses or fetuses  with undeterminable sex per group; number of  fetuses or litters with external, visceral, and skeletal findings; number  of fetuses or litters with 
malformations.
- Kruskal-Wallis test, and in case of significant differences Dunn's test  for: number of preimplantation losses, postimplantation losses, early  
resorptions, late resorptions or dead fetuses per female; number of male  or female fetuses or fetuses with undeterminable sex per female;  
proportin of placental, fetal external, and fetal visceral findings per  female.
- Chi(square) test (correction according to Yates) for: number of 
fetuses  or litters with cartilaginous tissue observations.
Indices:
For external, skeletal and visceral malformations a scheme for classification and an incidence table is described within the study.
Historical control data:
Description of historical control data is part of the study report (annex) for e.g.: vehicles, clinical findings, feed consumption, body weight gain,
gross pathological findings, fertility and gestation index, total resorptions, Cesarean section data, placental findings, number of malformations,
spontaneous malformations, external and visceral deviations, skeletal findings in fetuses and litters (including cartilaginous findings), incidence of
eye malformations in different historical dose groups

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
No mortalities were reported. Treatment with the test substance Isophorondiisocyanat (IPDI) at the 4 mg/m3 exposure level affected the respiratory tract and the fur and comprised decreased respiratory rate (bradypnea), laboured breathing, breathing sounds, reddish encrusted nostrils, serous nasal discharge and rough fur. Furthermore, decreased feed intake, body weight loss for 2 days and impaired body weight gain was evident in the 4 mg/m3 exposure group. Necropsy revealed no treatment related gross pathological findings at an exposure level up to and including 4 mg/m3.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEC
Effect level:
1 mg/m³ air
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
1 mg/m³ air
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEC
Effect level:
0.929 mg/m³ air (analytical)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
0.929 mg/m³ air (analytical)
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Reduction of fetal weight was evident in the 4 mg/m3 group and treatment relationship could not be completely excluded for marginally impaired
placental weight at the 4 mg/m3 exposure level. A treatment related effect on the incidence and type of fetal malformations was not assumed at an
exposure level up to and including 4 mg/m3. Slightly retarded ossification of few localizations (phalanges, sternebrae, sacral and caudal vertebrae)
was assumed and slightly impaired descensus testi could not be completely excluded at the 4 mg/m3 exposure level in relation to decreased fetal
weights and dose dependency. All signs of developmental toxicity observed at the 4 mg/m3 exposure level i.e. reduced fetal weight, delayed
descensus testis and slightly retarded ossification were indicative of delayed fetal development and were only seen in the presence of maternal
toxicity and thus considered a secondary effect.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

For developmental toxicity a read across to HDI oligomers, isocyanurate type (EC 931 -274 -8) is applied. This substance is a close structural analogue to HDI oligomers, iminooxadiazindione type, also derived from catalytic oligomerisation of 1,6 -hexamethylene diisocyanate (HDI; CAS 822 -06 -0) and also belonging to the CAS number 28182-81-2 (Hexane, 1,6 - diisocyanato-, homopolymer).The read across is based on physicochemical and toxicological similarity. In fact, comparison of the toxicological endpoints, that are available for both of the two substances (Acute oral toxicity, Acute inhalation toxicity, Skin and Eye Irritation/Corrosion, Skin Sensitisation, Bacterial mutagenicity (Ames)) reveal good correlation. With respect to Inhalation Toxicity an expert statement is available justifying the read across (Pauluhn, Comparison of pulmonary irritation potency..., Bayer HealthCare AG, 2008).

Therefore, all available toxicological data for HDI oligomers, isocyanurate type can be used for the toxicological evaluation of HDI oligomers, iminooxadiazindione type and all waiving conclusions drawn for HDI oligomers, isocyanurate type are also valid for HDI oligomers, iminooxadiazindione type. This approach is in accordance with Annex XI, section 1.5 of the REACH Regulation (Regulation (EC) No 1907/2006).

Comments for IPDI:

Result: not teratogenic

ACTUAL DOSE RECEIVED BY DOSE LEVEL BY SEX: Chemical analyses demonstrated  satisfactory stability and agreement 

between nominal and actual  concentrations of the test material.
MATERNAL TOXIC EFFECTS BY DOSE LEVEL: 
- Mortality and day of death: No mortalities were reported.
- Description, severity, time of onset and duration of clinical signs:  decreased respiratory rate (bradypnea), labored breathing, 

breathing  sounds, reddish encrusted nostrils, serous nasal discharge, rough fur (4  mg/m3 group). Effects on breathing as well as 

nasal discharge were not  observed in the other groups, while effects on nose and nostrils were  rare (maximum 2 females/group) 

and likely related to restraint. Rough fur  occurred in some females of all study groups, including the control  group, but showed a 

sharp increase in incidence with the 4 mg/m3 group. 
- Food/water consumption: Decreased feed intake throughout the exposure  period was observed in the 4 mg/m3 group  (14.7 % 

below control; p < 0.01)  and during the last interval (days 18-20) in the 0.25 mg/m3 group (p <  0.05, not dose related).  After 

start of inhalation, reduced feed intake  was observed in all study groups including control, most probably due to  the inhalation 

procedure. Beyond this, feed intake was normal. No effects  on water intake and on excretion of urine and feces were observed in 

any  group.
- Body weight: The body weight in the 4 mg/m3 group from day 0 to day 20  was lower by 7.9 %, the corrected body weight 

(body weight minus unterine  weight) was lower by 9.2 %. The body weight gain in the 4 mg/m3 group  from day 0 to day 20 was 

lower by 23.5 % (absolute; relative to initial  weight: -21.7 %; corrected: -91.3 %; compared to control; p < 0.01).   After start of 

inhalation, body weight loss was observed in all study  groups including control, most probably due to the inhalation  procedure.  

Beyond this, body weight development was normal.
- Gross pathology incidence and severity: There were no treatment related  gross pathological findings in any group.
- Number pregnant per dose level: control: 24/27; 0.25 mg/m3: 23/27; 1  mg/m3: 24/27; 4 mg/m3: 26/27
- Number of implantations: control 11.9; 0.25 mg/m3: 11.9; 1 mg/m3: 13.0,  4 mg/m3: 12.0 (mean per female with implantation sites) 

= no significant  differences
- Pre and post implantation loss: control: 1.7 / 0.8; 0.25 mg/m3: 2.1 /  0.7; 1 mg/m3: 1.2 / 0.7; 4 mg/m3: 1.4 / 0.5 

(mean pre- / postimplantation  loss per female with implantation sites) = no significant differences
- Number of corpora lutea: control: 13.6; 0.25 mg/m3: 14.0; 1 mg/m3:  14.1, 4 mg/m3: 13.4 (mean per female with implantation sites)

 = no  significant differences
- Number aborting: No abortion in any group
- Number of resorptions: There were no females with total resorption in  any group.
- Duration of pregnancy: determined by cesarean section on day 20
- Other findings: Placental weights were marginally decreased at the 4  mg/m3 level (-6.6 %, not statistically significant but slightly 

below  historical control data range).
FETAL DATA: 
- Litter size and weights: mean fetal weight in control: 3.51 g; 0.25  mg/m3: 3.49 g; 1 mg/m3: 3.46 g; 4 mg/m3: 3.27 g, i.e. reduction 

of fetal  weight in 4 mg/m3 group (-6.8 %; p < 0.01)
- Number viable: control: 11.1; 0.25 mg/m3: 11.2; 1 mg/m3: 12.3; 4 mg/m3:  11.5, i.e. no treatment related findings
- Sex ratio: control: 49.3; 0.25 mg/m3: 48.3; 1 mg/m3: 50.0; 4 mg/m3:  52.0 % males, i.e. no treatment related findings
- Grossly visible abnormalities: There is no evidence for treatment  relation. A marginally higher number of common eye 

malformations in the 4  mg/m3 group (1 % of the fetuses and 7.7 % of litters affected vs. 0.4 %  of fetuses and 4.2 % of litters in 

control), which is well within the  range of historical control data (up to 1.8 % of fetuses and 20 % of  litters affected), is 

considered to be either incidental or secondary  (reduced oxygen supply to offspring by maternal bradypnea).
  control:    1.1 % of fetuses / 12.5 % of litters showed malformations
  0.25 mg/m3: 1.6 % of fetuses / 13.0 % of litters showed malformations
  1.0  mg/m3: 2.0 % of fetuses / 20.8 % of litters showed malformations
  4.0  mg/m3: 1.7 % of fetuses / 11.5 % of litters showed malformations
- External abnormalities: External deviations were not observed in this  study.
- Soft tissue abnormalities: Statistical significance was only evident  for reduced number of tracheal findings (membraneous part of 

trachea  slightly folded, lying in tracheal lumen; possibly of artefactual origin)  in the 1 mg/m3 and 4 mg/m3 groups and for reduced 

total number of fetuses  with deviations in the 1 mg/kg exposure group. Based on lack of dose  relationship (control: 23; 0.25 mg/m3: 

11; 1 mg/m3: 6; 4 mg/m3: 10 %),  highest incidence of the tracheal finding in the actual control group and  lack of pathological 

significance per se for a reduced number, these  findings were considered incidental. Other deviations observed during  visceral 

evalution were either common or without dose relationship. In  conclusion, an effect on incidence and type of external and visceral  

deviations was not evident at an exposure level up to and including 1  mg/m3, while slightly retarded descensus testis (4 % of fetuses 

and 38.5  % of litters vs. 2.2 % of fetuses and 25 % of litters in the control  group) was observed at the maternally toxic 4 mg/m3 

exposure level. In  relation to decreased fetal weights treatment relationships could not be  totally excluded, although the individual 

alterations lay well in the  range of historical data and statistical significance was not evident.
  control:    27.0 % of fetuses / 95.8 % of litters showed deviations
  0.25 mg/m3: 24.4 % of fetuses / 87.0 % of litters showed deviations
  1.0  mg/m3: 17.0 % of fetuses / 87.5 % of litters showed deviations
  4.0  mg/m3: 23.0 % of fetuses / 96.2 % of litters showed deviations
- Skeletal abnormalities: Fetal skeletal including cartilaginous tissue  evaluation for degree of ossification and 

incidence of variations  relealed no toxicologically relevant effects at an exposure level up to  and including 1 mg/m3. Dose relation 

was missing for all findings at 0.25  and 1 mg/m3, and statistical significance was absent either on a fetus  base, or on a litter base, 

or for both. Statistically significant fetal  skeletal findings at the 4 mg/m3 exposure level included retarded  ossification of distal and 

proximal phalanges of digits and toes, of  metacarpal bones, 6th sternal segment, 7th cervical vertebral body,  sacral and caudal 

vertebral arches and caudal vertebral bodies. Incidence  of findings at the proximal phalanges of digits and distal phalanges of  toes, 

of metacarpals, and sacral and caudal vertebrae lay outside the  range of recent historical control data on a fetal basis, and although  

statistical significance on a litter basis was restricted to delayed  ossification of proximal phalanges of digits, treatment relationship 

was  assumed for retarded ossification of these localizations in relation to  as well impaired fetal weight. Other findings in the 

4 mg/m3 group were  considered to be of no toxicological relevance.

Applicant's summary and conclusion

Conclusions:
Animals treated with the test substance Isophorone diisocyanate (IPDI) at the 4 mg/m3 exposure level showed signs of maternal toxicity (respiratory tract affected, decrased respiratory rate (bradypnea), laboured breathing, breathing sounds, reddish encrusted nostrils, serous nasal discharge,
rough fur, decreased feed intake, body weight loss). The incidence and type of fetal malformations were unaffected by treatment at an exposure level up to and including 4 mg/m3. Even if a treatment related effect was taken into consideration, it would be considered secondary to maternal toxicity.
Thus under the conditions of this study the test substance Isophorone diisocanate is considered to be not teratogenic. All signs of developmental
toxicity observed at the 4 mg/m3 exposure level (i.e. reduced fetal weights, delayed descensus testis and slightly retarded ossification) were
indicative of delayed fetal development and were only seen in the presence of maternal toxicity and thus considered a secondary effect. Animals
treated with the test substance at the 4 mg/m3 exposure level showed signs of maternal toxicity (respiratory tract affected, decrased respiratory
rate (bradypnea), laboured breathing, breathing sounds, reddish encrusted nostrils, serous nasal discharge, rough fur, decreased feed intake, body weight loss). Summarizing and evaluating all data investigated the following no-observed-adverse-effect concentrations (NOAECs) were determined: Maternal toxicity: 1 mg/m3
Developmental toxicity: 1 mg/m3
Executive summary:

Female inseminated Wistar rats were treated daily for 6 hours/day by inhalation (nose-only) with Isophorone diisocyanate (IPDI) from day 6 to day 19 post coitum to examine potential developmental toxicity effects of the test substance. Dosages used were 0 (air control); 0.25; 1 and 4 mg/m3, respectively.

Animals treated with the test substance Isophorone diisocyanate (IPDI) at the 4 mg/m3 exposure level showed signs of maternal toxicity (respiratory tract affected, decrased respiratory rate (bradypnea), laboured breathing, breathing sounds, reddish encrusted nostrils, serous nasal discharge, rough fur, decreased feed intake, body weight loss). No mortalities were reported. Necropsy revealed no treatment releated gross pathological findings at an exposure level up to and including 4 mg/m3. With respect to the parameters of intrauterine development, gestation rate, postimplantation loss, mean litter size, fetal sex distribution and placental appearance were not affected by treatment with the test substance at an exposure level up to and including 4 mg/m3. Reduction of fetal weight was evident in the 4 mg/m3 group and treatment relationship could not be completely excluded for marginally impaired placental weight at the 4 mg/m3 exposure level.

The incidence and type of fetal malformations were unaffected by treatment at an exposure level up to and including 4 mg/m3. Slightly retarded ossification of few localizations (phalanges, sternebrae, sacral and caudal vertebrae) was assumed and slightly impaired descensus testis could not be completely excluded at the 4 mg/m3 exposure level in relation to decreased fetal weights and dose dependency. All signs of developmental toxicity observed at the 4 mg/m3 exposure level (i.e. reduced fetal weights, delayed descensus testis and slightly retarded ossification) were indicative of delayed fetal development and were only seen in the presence of maternal toxicity and thus considered a secondary effect. Summarizing and evaluating all data investigated the following no-observed-adverse-effect concentrations (NOAECs) were determined: Maternal toxicity: 1 mg/m3

Developmental toxicity: 1 mg/m3