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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
repeated dose toxicity: inhalation
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study. Minor restrictions: historical control data for clinical chemistry and haematology not available
Cross-reference
Reason / purpose:
reference to same study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Principles of method if other than guideline:
Combined Subchronic Vapor Inhaltion Study with Reproduction/Developmental Toxicity Screening Test.
GLP compliance:
yes (incl. certificate)
Remarks:
BASF AG, Dep. of Toxicology
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
isobutyl vinyl ether (IBVE), purity >99.5% as determined by GC.
The stability under storage conditions over the exposure
period was guaranteed.
Storage condition: refrigerator,
stored under nitrogen.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Wistar (CrlGlxBrlHan:WI) rats; female animals were nulliparous and non-pregnant
Source: Charles River, Germany
Housing: singly during the exposure period.
Free access to laboratory diet and tap water (analysed for contaminations) except during exposure and motor activity measurements.
Room: temperature 20-24°C
Relative air humidity 30-70%
Light/dark rhythm: 12 hrs.
Acclimatisation period: 6 or 13 days
Age st arrival: 4 or 5 weeks.

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
other: air
Remarks on MMAD:
MMAD / GSD: not applicable
Details on inhalation exposure:
Exposure to vapors of TS in an whole-body inhalation chamber (volume 1.4 m³) in individual cages in groups of 10 rats. For each concentration the test substance was supplied to a thermostated vapourizer at a constant rate by means of the metering pump; the vapour was generated with conditioned supply air (about 50% ± 20% relative humidity, 220 C ± 20 C) and passed into the inhalation system.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration measurement by gas chromatography. At the beginning of the study, daily means were calculated based on 2 measured samples per concentration. Constancy in each inhalation system was continuously monitored by means of a total hydrocarbon analyzer. Correctness of hydrocarbon analyzer checked by GC. In the control group one sample per week was analyzed
Duration of treatment / exposure:
13 weeks for males (93 days or 65 exposures and were sacrificed on the next workday after the last exposure)
15 weeks for females (67 to 72 exposures; sacrifice on study day 105)
13 weeks for satellite females (65 exposures in 92 days, sacrificed day 93)
Frequency of treatment:
5 days/week, 6 hrs/day
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 50, 500, 2000 ppm corresponding to 0, 208, 2080, 8300 mg/m³
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 208+-9.8, 2095+-57, 8301+-58 mg/m³ (means +- SD)
Basis:
analytical conc.
No. of animals per sex per dose:
10 male and 10 female rats per control and dose group
5 females in satellite groups (exposed 13 weeks, no mating, non-pregnant)
Control animals:
yes, concurrent vehicle
Details on study design:
The males were treated for appr. 13 weeks (10 weeks premating, 3 weeks mating and post mating); females were treated during premating (10 weeks), mating and gestation through day 4 after delivery (approx. 15 weeks). Additionally a satellite group of female rats (5 animals per concentration, nuliparous and non-pregnant) were exposed 13 weeks.
Mating pairs were formed from the same concentration group. The parental animals were examined for their mating and reproductive performances (see also Section 7.8.1).
Comment: in contrast to the documentation in the summary (page 18 of the report) it was mentioned that parenteral females were exposed "until including day 18 post coitum resulting in a total of 67 to 72 exposures" (page 31) suggesting that gestation day 18 was the last exposure day and no further exposure of dams and pups was performed.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
Rats controlled for clinical signs once daily (weekend) or twice daily (workdays); on exposure days clinical observation performed before, during and after
exposure.
Detailed clinical observations in an open field conducted prior to the start of the exposure period and weekly thereafter.
A functional observational battery (FOB) and measurements of motor activity (MA) were carried out on study days 56 and 57 (separate randomization) for males and females (n=5 each sex), respectively.
Body weight of the animals as well as food consumption was determined day0 and thereafter weekly; food efficiency calculated.

Hematology (leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count, reticulocytes, prothrombin time)
and clinical chemistry (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-y-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium)
performed towards the end of premating period.

After parturition the pups were sexed and weighed on the day after birth and on day 4 post partum. Their viability was recorded. Mating index, fertility index, and gestation index were calculated (see Section 7.8.1)
Sacrifice and pathology:
All F1 pups necropsied on day 4 post parturn and examined macroscopically for external and visceral findings (see Section 7.8.1).
Parenteral rats: A complete necropsy including gross pathological evaluation and weighing of selected organs (liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, thymus, spleen, brain, heart, lungs) performed.
Organs and tissues examined histopathologically in controls and high dose groups, except liver, nasal cavity, testis, epididymis, and ovaries (examined in all groups).

Histopathology of the following organs:
all gross lesions
brain
spinal cord (cervical, thoracic and lumbar cord)
sciatic nerve
pituitary gland
salivary glands (glandula mandibularis and glandula sublingualis)
thyroid glands/parathyroid glands
adrenal glands
prostate gland, seminal vesicles, coagulation glands
uterus, oviducts, vagina
female mammary gland
thymus
lymph nodes (mandibular and mesenteric)
spleen
trachea
lungs
heart
aorta
liver
pancreas
kidneys
esophagus
stomach (forestomach and glandular stomach)
duodenum, jejunum, ileum
cecum, colon, rectum
urinary bladder
sternum with marrow
bone marrow (femur)
head (with nasal cavities)
larynx
pharynx
eyes with optic nerve
femur with knee joint
skin
skeletal muscle
extraorbital lacrimal glands
Other examinations:
no
Statistics:
Clinical and neurofunctional examinations: DUNNETT's-test, Fisher's Exact test, Wilcoxon test, Kruskal-Wallis test.
Clinical pathology: Kruskal-Wallis test Pathology: Kruskal-Wallis test, Wilcoxon test
Level of significance selected was p=0.05

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
50 ppm (208 mg/m³): no adverse effects were detectec.

500 ppm (2080 mg/m³)
toxic effects in parenteral rats
- increased white blood cell counts in the parental females (+ 23%) and satellite females (+ 35%)
- increased lymphocytes in the parental females (+ 22%) and satellite females (+ 36%)
- increased triglyceride concentrations in the females (+ 77%) and satellite females (+59%)
- hyperplasia of the respiratory epithelium in the nasal cavity in 2 females
- mucous cell reduction in the nasal cavity in 6 males and 7 females
- degeneration of the olfactory epithelium in the nasal cavity in 9 males and 6 females
- inflammatory cell infiltration in the nasal cavity in one female
- increased absolute kidney weights in parental males and satellite females
- increased absolute and relative liver weights in satellite females
- increased cytoplasmic basophilia in the liver of one parental male
No test substance related adverse effects with respect to clinical examinations/reproductive performance of the parental animals during gestation and lactation period, and no test substance related effects on development of the offspring.

2000 ppm (8300 mg/m³)
Signs of toxicity in parental male and of female rats (excluding clinical signs of toxicity during gestation and lactation period)
- Unspecific clinical symptoms like visually increased respiration (day 0 - 105), alopecia (day30-105) and apathy (on study days 0 - 7)
- increased white blood cell counts in the parental females (+ 52%) and satellite females (+40%)
- increased polymorphonuclear neutrophils (absolute) in the parental females (+ 81 %) and satellite females (+ 88%)
- increased lymphocytes (absolute) in the parental females (+ 49%) and satellite females (+34%)
- increased alkaline phosphatase activities in the parental males (+ 42%)
- increased triglyceride concentrations in the parental females (+ 403%) and satellite females (+ 100%)
- increased cholesterol concentrations in the parental females (+ 129%) and satellite females (+ 43%)
- hyperplasia of the respiratory epithelium in the nasal cavity in 8 males
- mucous cell reduction in the nasal cavity in 10 males and 8 females
- degeneration of the olfactory epithelium in the nasal cavity in 8 males and 10 females
- inflammatory cell infiltration in the nasal cavity in 3 males and 3 females
- increased absolute and relative kidney weights in parental animals and in satellite females
- increased absolute liver weights in parental males and satellite females ; increased relative liver weights in animals and in satellite females
- increased cytoplasmic basophilia in the livers of 3 parental males and 1 parental female
Signs of toxicity of parental female animals during gestation and lactation
- Visually increased respiration before, during and after exposure in the course of pregnancy
- Visually increased respiration during lactation
- Statistically significantly lower mean body weights in the females on gestation day 20 (-12%; decrease during lactation not significant, -9%)
- Impairment of average female body weight gain during entire gestation (- 30 %)
- significantly decreased mean number of implantation sites per dam (8.9 vs. 11.8 in the control)
- higher resorption rate (% postimplantation loss 13 .1 vs. 2.1 in the control)
Pups
- Statistically significantly lower live birth index (7 % below control), only 69 pups liveborn vs. 115 pups in the control group, 5 pups stillborn and 2 pups cannibalized vs. 0 in the control

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
50 ppm
Sex:
male/female
Basis for effect level:
other: (corresponding to 208 mg/m³ air)
Dose descriptor:
LOAEL
Effect level:
500 ppm
Sex:
male/female
Basis for effect level:
other: (corresponding to 2080 mg/m³) local effects in the nasal cavity; adaptive effects in liver and kidney (increased metabolism of the test substance; no histopathological effects)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Food consumption and food efficiency were only temporary affected and not considered to be treatment related.

The open field observations, the functional observational battery, and the study on motor activity did not reveal any abnormalities.

Details on reproduction data are presented in Section 7.8.1.

Clinical chemistry: slightly increased alkaline phosphatase activities were seen in high dose males but no other enzyme parameters were altered; effects on triglyceride and cholesterol in mid and high dose females (see above) were isolated (no other blood chemistry parameters affected). Changes in white blood cell counts, polymorphonuclear neutrophils and lymphocytes were observed in the mid- and high-concentration females at the end of the exposure period; these findings were assessed as being treatment-related and were likely to be due to an inflammatory reaction in the upper respiratory tract. The increases in triglycerides and cholesterol levels in the sera of the mid- and highconcentration females were also considered to be test substance-related and may be caused by disturbances of the lipid metabolism in the liver. Interestingly, the lipid metabolism was more severely disturbed in parental females (triglycerides + 403 %, cholesterol +129 %) than in the non-pregnant satellite females (triglycerides + 100 %, cholesterol + 43 %).

The increase on absolute and relative spleen weight in mid and high dose group was found but these effects were not clearly dose dependent and considered by the authors not to be of toxicological relevance (no treatment-related histopathological findings were noted; the recorded weight increase is most likely due to variation in blood content of that organ).

Histopathology revealed no lesions in any organ except the nasal cavity (see above).

Table on incidences of changes in rat nasal cavity after repeated exposure to IBVE

Finding

0 ppm

50 ppm

500 ppm

2000 ppm

-

M

F

M

F

M

F

M

F

Respiratory epithelium hyperplasia

0/10

0/10

0/10

0/10

4/10

2/10

8/10

0/10

Mucous cell reduction

0/10

0/10

0/10

0/10

6/10

7/10

10/10

8/10

Olfactory epithelium degeneration

1/10

0/10

0/10

1/10

9/10

6/10

8/10

10/10

Inflammatory cell infiltration

0/10

0/10

0/10

0/10

0/10

1/10

3/10

3/10

M = males; F = females

Applicant's summary and conclusion

Conclusions:
After subchronic inhalation exposure in rats local effects in the nasal cavity were found as well as adapative effects in liver and kidney (increased metabolism of the test substance; no histopathological effects) at a concentration of >= 500 ppm (2080 mg/m³); the NOAEC was 50 ppm (208 mg/m³).
Executive summary:

In a GLP Guideline study according to OECD TG422 10 male and 10 female Wistar rats per test group were exposed to a vapor of isobutylvinylether (whole body, 6h/d, 5d/week) at dose levels of 0, 50, 500 or 2000 ppm (0, 208, 2080 and 8300 mg/m³). The males were treated for approx. 13 weeks (10 weeks premating, 3 weeks mating and post mating) and females during premating (10 weeks), mating and gestation through day 4 after delivery (approx. 15 weeks). Additionally a satellite group of female rats (5 animals per concentration group; nuliparous, non-pregnant) were exposed 13 weeks. The rats were sacrificed the day after the last exposure. The main target organ was the upper respiratory tract (hyperplasia of the respiratory epithelium, decrease of mucous cells, degeneration of the olfactory epithelium) at >=500 ppm. Furthermore, dose dependent changes in some blood parameters were seen in female animals at >=500 ppm (considered to be secondary to local effects). High serum triglyceride and cholesterol levels were noted in high-dose parental females (slight increase at mid dose level); the effect was less pronounced in non-pregnant females and absent in males (may be caused by disturbances of the lipid metabolism in the liver). Increased liver and kidney weight changes were noted in high-dose males and intermediate and high-dose non-pregnant females but no histopathological alterations. There was no treatment-related effect in the neurofunctional tests (Functional Observation Battery and Motor Activity Test) at any dose level.

Conclusion: After subchronic inhalation exposure in rats local effects in the nasal cavity were found as well as adapative effects in liver and kidney (increased metabolism of the test substance; no histopathological effects) at a concentration of >= 500 ppm (2080 mg/m³); the NOAEC was 50 ppm (208 mg/m³).