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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment, not according to guideline, only lung and trachea examined at necropsy

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990
Reference Type:
study report
Title:
Unnamed
Year:
1991
Reference Type:
publication
Title:
Altered lung function in rats after subacute exposure to n-Butylisocyanate
Author:
Pauluhn J, Eben A
Year:
1992
Bibliographic source:
Arch. Toxicol. 66, 118-125.

Materials and methods

Principles of method if other than guideline:
TEST ORGANISMS (rat) - Age: 2 - 3 months  - Number of animals: 20/concentration ADMINISTRATION / EXPOSURE  - Type of exposure: dynamic head-nose only vapor exposure Test material was filled into a glass bubbler. Dry nitrogen from a pressurized glas cylinder was metered through the bubbler using a gas pump and was subsequently diluted with dry air. The test atmosphere was passed into a stainless steel head-nose inhalation chamber (7l). The chamber was operated in a fume hood. chamber concentration: Analysis of the test atmosphere:  The stability of the test atmosphere was monitored continuously. Analytical concentration of the chamber atmosphere was determined by taking samples near the animal breathing zone; The exposure atmosphere was sampled using an impinger containing a nitroreagent and the reaction product was determined quantitatively bei HPLC.  - Doses:  target concentrations: 1, 5, 15, and 25 mg/m³ nominal concentrations:  5, 13, 30, and 48 mg/m³ mean actual analytical concentrations: 1.1, 6.2 14.8, and 26 mg/m³ Due to mortality in the 25 mg/m³ group another group (15 mg/m³) was exposed 2 months later. CLINICAL OBSERVATIONS AND FREQUENCY:  - Clinical signs: twice daily throughout the exposure period and once daily thereafter - Mortality: twice daily - Body weight: before the first exposure, at the 4. and 7. exposure and then weekly; additionally the individual body weights were determined before pulmonary function test and bronchoalveolar lavage. - Rectal Temperature: days 0, 4 and 7 - Reflex: day 3 of exposure CLINICAL PATHOLOGY during follow-up week 2 and 5 - Hematology (due to mortality 25 mg/m³ animals were excluded from the 5 week post exposure examination) ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC):  - Gross Pathology:  after the lung lavage the animals were examined macroscopically Fixed organs: lung, trachea Organ Weights: Lungs (relative wet weight of arterially  exsanguinated lungs, except the animals of the high dose group) - Microscopic: trachea and  lungs from 10 (6, respectively from the high concentration group) animals evaluated from the control and all concentration groups. ADDITIONAL MEASUREMENT during follow-up week 2 and 5: -Pulmonary function tests (due to mortality 25 mg/m³ animals were excluded from the 5 week post exposure examination) cover the following parameters:  Respiratory Rate  Dynamic Compliance (Cdyn) Static Compliance (Cstat) Specific Compliance (Cspec) Lung Resistance (Res) Delta-Res after bronchial challenge (DRes) Peak Expiratory Flow (PEF) Mean Mid Expiratory Flow (MMEF) Forced Expiratory Vol. after 0.05 sec (FEVO.05) Forced Expiratory Vol. after 0.1 sec (FEVO.1) Forced Expiratory Vol. after 0.2 sec (FEVO.2) Forced Expiratory Vol. after 0.4 sec (FEVO.4) Forced Exp. Flow at 75% FVC (FEF75) Forced Exp. Flow at 50% (FEF50) Forced Exp. Flow at 25% (FEF25)  Forced Exp. Flow at 10% (FEF10) Tidal Volume (TV) Inspiratory Volume (IV) Inspiratory Capacity (IC) Vital Capacity (VC) Forced Vital Capacity (FVC) Residual Volume (RV) Functional Residual Capacity (FRC) Total Lung Capacity (TLC) VC % of TLC (VC/TLC) RV % of TLC (RV/TLC) FRC % of TLC (FRC/TLC) Fraction-Neon alveolar/inspired (F - Ne) Fraction-CO alveolar/inspired (F - CO) Diffusing Capacity (DLco) Krogh Diffusing Cap. (DLcoKr) Diffusing Capacity/kg b.w. (DLco/kg) - Arterial blood gas and acid-base measurement (due to mortality 25 mg/m³ animals were excluded from the 5 week post exposure examination) cover the following parameters: Hemoglobin (Hb) O2-Halfsaturation pressure (p50) Oxyhemoglobin (HbO2) Carboxyhemoglobin (HbCO) Met-Haemoglobin (MetHb) a-pO2 - Concentration (pO2) a-pCO2-Concentration (pCO2) O2-Saturation (SAETT%) Blood pH (pH) a-Bicarbonat-Concentration (HCO3) Total a-C02-Concentration (TCO2) Actual Base Excess (ABE) Standard Base Excess (SBE) Standard bicarbonate (SBIC) Alveolar O2-concentration (ApO2) Arterioalveolar 02-difference (A-apO2) Venous admixture (Qs/Qt) - Analysis of bronchoalveolar lavage fluid (BALF) (due to mortality 25 mg/m³ animals were excluded) cover the following parameters: cells/g lung total number of cells in BALF Lactate dehydrogenase (LDH) gamma-Glutamyltranspeptidase (gGT) Protein Sialic acid Lymphocytes Granulocytes Macrophages
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
IUCLID4 Test substance: other TS: n-butyl isocyanate, purity: 99.5 %

Test animals

Species:
rat
Strain:
Wistar
Sex:
male

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
nose/head only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: no data
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
5 d; 6 h/day
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1, 5, 15, and 25 mg/m³ (target concentrations)
Basis:

No. of animals per sex per dose:
20
Control animals:
other: conditioned air
Details on study design:
Post-exposure period: 5 weeks

Examinations

Statistics:
STATISTICAL METHODS: one way analysis of variances (FORTRAN); a modification of the Tukey-Kramer post hoc test ; the Mann-Whitney U test; the Fisher´s Exact test

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):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 mg/m³ air
Sex:
male
Dose descriptor:
LOAEL
Effect level:
5 mg/m³ air
Sex:
male

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

RS-Freetext:
MORTALITY: 
12/20 of the high concentration group (group 5) died during
recovery period (day 10 - day 15)

CLINICAL SIGNS:
Group 1, 2 and 3 (controls, 1 mg/m³ and 5 mg/m³): 
all rats tolerated the exposures without any clinical signs.
Group 4 (15 mg/m³): 20/20 with signs (unkempt appareance,
labored breathing pattern, reduced motility, increased
serous discharge) which started at the beginning of the
exposure (day 0) and lasted up to day 13 
Group 5 (25 mg/m³): 20/20 with signs which started at the
beginning of the exposure (day 0) and lasted up to day 15.
Severe respiratory distress (during the exposure period),
reduced motility, lethargy, unkempt fur, serous discharge
from nose, perinasal wetness, and cyanosis; in the post
observation period tachypnoea was observed

REFLEX EXAMINATION:
no substance induced effects

RECTAL TEMPERATURE:
The body (rectal) temperatures of rats of the 25 and 15
mg/m³ group were significantly dose dependent reduced after
the first exposure. At the last exposure day a significant
decrease was only observed for the high dose animals and
returned to normal during the post observation period (day 7
of the study).

BODY WEIGHTS: 
A decrease of body weight gain during the exposure period
was observed in the 15 and 25 mg/m³ groups; recovery of body
weight loss was observed during the post observation period 

HEMATOLOGY and CLINICAL PATHOLOGY: 

At follow-up week 2 erythrocytes, hematokrit and hemoglobin
was elevated in the 25 mg/m³ animals. At follow-up week 5 no
deviations from control animals were observed at any
concentration examined (1-15 mg/m³).

GROSS PATHOLOGY: 

rats died intercurrent (= 25 mg/m³):
pale liver, spleen, and kidneys, gastrointestinal tract was
filled with a yellow mucous or bloody mucous mass, reddening
of the intestinal mucosa, lobular pattern of the liver,
pulmonary edema, and hydrothorax; liver like changes in the
lung; distended lungs.

rats sacrificed at the end of the post observation period
(1-15 mg/m³): no lung lesions 
rats sacrificed at the end of the post observation period
(25 mg/m³): liver like changes in the lung; distended lungs


LUNG WEIGHTS (1-15 mg/m³):
Significant increase in rats exposed to 15 mg/m³ during week
2, but not during week 5.

HISTOPATHOLOGICAL FINDINGS: 

TRACHEA:
1/10 animal(0, 1 and 5 mg/m³ group) and 2/10 animals (25
mg/m³) showed hyperaemia; round-cell infiltration was only
observed in 1/10 animals of the 15 mg/m³ group.
Histopathological lesions were restricted to the high dose
animals (25 mg/m³).

LUNG:
Significant histopathological lesions were restricted to the
high dose animals (25 mg/m³). The lesions in the region of
central acinus were predominant and were characterized by
inflammatory responses; emphysema (5/6), thickening of septa
(4/6) and intraalveolar edema (6/6).

ADDITIONAL MEASUREMENT: 

- PULMONARY FUNCTION TESTS

During follow-up week 2 (1-25 mg/m³):

Effects were restricted to the high dose group (25 mg/m³). 

The following parameters were elevated compared to control
animals (although the changes did not reach in every case
statistical significance they were considered to be
toxicologically relevant):

Residual Volume (RV)
Functional Residual Capacity (FRC)
Total Lung Capacity (TLC)
Lung Resistance (Res)
Delta-Res after bronchial challenge (DRes)

The following parameters were decreased compared to control
animals (although the changes did not reach in every case
statistical significance they were considered to be
toxicologically relevant):

Mean Mid Expiratory flow (MMEF)
Peak Expiratory Flow (PEF)
Dynamic Compliance (Cdyn)
Static Compliance (Cstat)
Specific Compliance (Cspec)

During follow-up week 5 (1-15 mg/m³)

The following parameters were elevated compared to control
animals (although the changes did not reach in every case
statistical significance they were considered to be
toxicologically relevant):

5 and 15 mg/m³: Delta-Res after bronchial challenge (DRes)
15 mg/m³: Dynamic Compliance (Cdyn), Residual Volume (RV),
Functional Residual Capacity (FRC), Total Lung Capacity
(TLC)

The following parameters were decreased compared to control
animals (although the changes did not reach in every case
statistical significance they were considered to be
toxicologically relevant):

5-15 mg/m³: Static Compliance (Cstat), 
1-15 mg/m³: Diffusing Capacity (DLco)** 
15 mg/m³: Lung Resistance (Res), Specific Compliance (Cspec)

** the physiological relevance of this change is not
apparent, since no clear correlation with changes of
arteriolar oxygen difference or venous admixture could be
ascertained

- ARTERIAL BLOOD GAS and ACID-BASE MEASUREMENT

During follow-up week 2 (1-25 mg/m³):

Effects were restricted to the high dose group (25 mg/m³). 

The following parameters were elevated compared to control
animals (although the changes did not reach in every case
statistical significance they were considered to be
toxicologically relevant):

Hemoglobin (Hb)
a-pCO2-Concentration (pCO2)
a-Bicarbonat-Concentration (HCO3)
Arterioalveolar O2-difference (A-apO2)
Venous admixture (Qs/Qt)

The following parameters were decreased compared to control
animals (although the changes did not reach in every case
statistical significance they were considered to be
toxicologically relevant):

Oxyhemoglobin (HbO2)
Standard bicarbonate (SBIC)
a-pO2 - Concentration (pO2)

During follow-up week 5 (1-15 mg/m³)

Effects were restricted to the high dose group (15 mg/m³)

The following parameters were elevated compared to control
animals (although the changes did not reach in every case
statistical significance they were considered to be
toxicologically relevant):

a-pCO2-Concentration (pCO2)
a-Bicarbonat-Concentration (HCO3)

- BRONCHOALVEOLAR LAVAGE FLUID (BALF) 

During follow-up week 2 (1-15 mg/m³):

The following parameters were elevated compared to control
animals:

5 mg/m³: gamma-Glutamyltranspeptidase (gGT)
15 mg/m³: 
Lactate dehydrogenase (LDH)
gamma-Glutamyltranspeptidase (gGT)
Protein
Sialic acid
Granulocytes

The following parameters were decreased compared to control
animals: 

15 mg/m³: 
Lymphocytes
Macrophages

During follow-up week 5 (1-15 mg/m³)

The following parameters were elevated compared to control
animals:

5 mg/m³: 
Granulocytes

15 mg/m³: 
Lactate dehydrogenase (LDH)
gamma-Glutamyltranspeptidase (gGT)
Protein
Sialic acid
Granulocytes

The following parameters were decreased compared to control
animals: 

15 mg/m³: 
Lymphocytes
Macrophages

Applicant's summary and conclusion

Conclusions:
CL-Freetext:
These results demonstrate obstructive and progressive lung disease with associated gas trapping and severe disturbance
of the ventilation perfusion relationship which is considered to be the cause of delayed mortality. In terms of
variability and sensitivity the increase in bronchoalveolar lavage fluid (BALF) parameters was most sensitive in
indicating the diseased state of the lung.
Executive summary:

TEST ORGANISMS (rat) - Age: 2 - 3 months  - Number of animals: 20/concentration

ADMINISTRATION / EXPOSURE  - 5 d/6 hours per day Type of exposure: dynamic head-nose only vapor exposure

DOSES:  mean actual analytical concentrations: 1.1, 6.2 14.8, and 26 mg/m³

RESULT: NOAEC = 1 mg/m³ air (male rats); LOAEC = 5 mg/m³ air (male rats)

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