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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
Study period:
1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted according to GLP. The histopathological findings were re-evaluated in 2001; the re-evaluation has confirmed the conclusions of the initial report.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1985
Report Date:
1985
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001
Reference Type:
publication
Title:
Inhalation Toxicity of 1,6-Hexamethylene Diisocyanate Homopolymers (HDI-IC and HDI-BT): Results of Subacute and Subchronic Repeated Inhalation Exposure Studies
Author:
Pauluhn, J. and Mohr, U.
Year:
2001
Bibliographic source:
Inhalation Toxicology 13: 513 - 532

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes
Remarks:
Bayer AG
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain as given in the study report: Wistar, Bor:WISW (SPF-Cpb)
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: approx. 160 g for the males and 130 g for the females
- Housing: 5 animals per cage in Makrolon Type III cages
- Diet: Altromin 1324 maintenance diet for rats and mice (Altromin GmbH, Lage, Germany), ad libitum except during exposure
- Water: tap water, ad libitum except during exposure
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 40 - 70%
- Air changes: 10 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose/head only
Vehicle:
other: acetone
Remarks on MMAD:
MMAD / GSD: MMAD (µm)/GSD:
Low test concentration: 1.4/1.3
Mid test concentration: 1.4/1.3
High test concentration: 1.7/1.6

Alveolar mass
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE
The test substance was tested as a test item in 10% acetone solution; the mixture was injected into the exposure chamber as a well-inhalable aerosol under dynamic conditions. For generation of the test aerosol, a volume of 100 µL/minute of the solution mentioned above was mixed to the air stream (10 L air/minute) and was conducted with a dispersion pressure of 600 kPa into a 3 L three-necked flask. Particles with an aerodymic diameter > 5 µm were separated by means of a pre-separator.

CHAMBER DESCRIPTION
The inhalation chamber consisted of a prechamber (volume: 20 L) and the main exposure chamber (volume: 20 L).

TEST ATMOSPHERE
About 60 to 80% of the air supplied were extracted from the exposure chamber to create slight over pressure conditions therein. The air change was about 15 times per hour. The extracted air was conducted through an activated carbon filter for cleaning.
The nominal concentrations of test item in the test atmosphere was calculated on the basis of the whole test item conducted into the exposure chamber and the air supply. The analytical method for determination of the actual test item concentrations in the breathing zone of the animals was based on the nitro-reagent principle, and the measurements were based on HLPC. Samples of test atmosphere were taken from the breathing zone 3 times/exposure day on several days per week. The sampling volume was 150 L for the low test concentration level (3 mg test item/m³ air), 100 L for the mid test concentration level (15 mg test item/m³ air), and 50 L for the high test concentration (75 mg test item/m³ air).
In addition, a gravimetric monitoring of the test item concentration was done for the high test concentration. For this purpose, 50 L of air were extracted via a glass fiber filter. The absolute amount of test item measured gravimetrically was about 3 to 5 mg per filter (i.e., ca. 3% of the absolute filter mass).

PARTICLE SIZE DETERMINATION
Sampling was done close to the noses of the test animals. The measurement of the particle distribution was done by means of a TSI-Laser-Velocimeter.
For the high test concentration level, the aerosol atmosphere was examined using a cascade impactor. The mass median aerodynamic diameter (MMAD), the number median aerodynamic diameter (NMAD) and the mean geometric standard deviation (GSD) were calculated. The particle mass with a MMAD < 5 µm was considered as the respirable (alveolar) fraction of the aerosol.

VEHICLE
Acetone was selected as vehicle for nebulisation since the test item was of high viscosity. The concentration of the test item in the vehicle was about 90%. The nominal acetone concentration did not exceed 405 mg acetone/m³ and thus was below the MAK value (2400 mg/m³; MAK-Liste 1983). Nevertheless, a vehicle (acetone) control group was added. For this group, the acetone test atmosphere was generated by dynamic evaporation of acetone. For this purpose, air (0.2 L) was conducted throughout an acetone-containing flask and was then further diluted with 20 L of air. The nominal concentration of acetone was calculated taking into account the flask weight and the air flow through the exposure chamber per exposure day.

REMARK
The tendency of the test compound to polymerize in the ducting system to the inhalation chamber, including nozzles, made it necessary to dismantle, clean, and reassemble all of the equipment on a day-to-day basis.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
3 weeks
Frequency of treatment:
6 hours/day, 5 days/week (15x6h in total)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
3, 15, 75 mg/m³
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
3.7, 17.5, 76.6 mg/m³
Basis:
analytical conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
yes, sham-exposed

Examinations

Observations and examinations performed and frequency:
CLINICAL SIGNS AND MORTALITY
The animals were observed several times on exposure day for clinical symptoms.

BODY WEIGHT
The body weights of the animals were recorded prior to the first exposure and weekly thereafter.

FOOD AND WATER CONSUMPTION
Not examined.

OPHTHALMOSCOPIC EXAMINATION
Not done.

HAEMATOLOGY AND CLINICAL CHEMISTRY
Blood sampling was done at necropsy by cardiac puncture.The blood samples needed for glucose measurement were collected from the fasted animals during the last treatment week from the tail.
The following haematological parameters were examined: haematocrit (HCT), haemoglobin (HGB), erythrocyte count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), thrombocyte count (THROM), and differential blood count. Clotting analysis was performed and the prothrombin time (Hepato Quick´s Test; HQT) was determined.
For clinical chemistry, following parameters were examined: glucose, urea, bilirubin, creatinine, total protein, aspartate aminotransferase, alanine aminotransferase, glutamate dehydrogenase, lactate dehydrogenase, alkaline phosphatase, triglycerides, cholesterol, albumin, sodium, potassium, calcium, chloride.

URINALYSIS
Urine was sampled from all animals during the last week of treatment. For this purpose, the animals were held individually in metabolic cages overnight and did not receive drinking water. Prior to placing into the metabolic cages, each animal received 5 mL water by gavage. For urinalysis, the following parameters were examined: volume, specific gravity, pH, presence of blood, protein and glucose, presence of ketones, sediment composition.

NEUROBEHAVIOURAL EXAMINATION
Not done.
Sacrifice and pathology:
GROSS PATHOLOGY
All animals were sacrificed at test ending for the purpose of necropsy and were subjected to gross pathological examination. Animals found dead were necropsied and examined for gross pathological changes.

ORGAN WEIGHTS
The weights of following organs were recorded: heart, gonads, liver, lung, spleen, adrenals, kidneys, thyroid gland (a lateral half of the gland was weighed whereas the remaining half was left close to the larynx for a better histological examination of the organ).

HISTOPATHOLOGY
Particular attention was paid to the examination of the entire respiratory tract, which included the nasal cavities, lung-associated lymph nodes, larynx, lung and trachea.
The following organs and tissues were fixed in 10% buffered formalin: eye, duodenum, brain, lung-Hilus-Lymph nodes, heart, gonads, head (nose-pharynx), liver, lung, stomach, spleen, adrenals, kidneys, oesophagus, trachea, larynx, thyroid gland.
The fixed tissues were embedded in paraplast and sectioned; the sections were then haematoxylin-eosin stained for microscopy. The olfactory epithelium sections were subjected to iron hematoxylin staining according to M. Heidenhain for a better tissue examination.

RE-EVALUATION OF THE HISTO(PATHO)LOGICAL FINDINGS
Slides from the entire respiratory tract of the animals from the present study have been re-examined 2001 (BAYER AG, PH 30632) as a part of a retrospective and comparative analysis of inhalation effects induced by different substances (HDI oligomers, biuret type and isocyanurate type), for the purpose of publication (see Pauluhn et al., Inhalation Toxicol 13: 513 - 532, 2001). The re-evaluation of the findings did not reveal any other effects or lead to any other conclusions as those stated in the inital study report.
Statistics:
Data of the different groups were compared with the pooled sham control (air) and for analysis, a Mann-Whitney-Wilcoxon U-test was basically used.
With respect to histopathological data of the respiratory tract, findings were compiled and reanalyzed using Fisher's exact test. For all tests, the criterion for statistical significance was set at p < 0.05.

Results and discussion

Results of examinations

Details on results:
ANALYTICAL MONITORING OF TEST CONCENTRATIONS
The analytically measured test concentrations for the substance were 3.7 mg/m³ for the low test concentration group (nominal 3 mg/m³), 17.5 mg/m³ for the mid test concentration group (nominal 15 mg/m³) and 76.6 mg/m³ for the high test concentration group (nominal 75 mg/m³).
According to the particle size determination the alveolar mass
CLINICAL SIGNS AND MORTALITY
The sham exposed animals (air), the vehicle control animals (acetone) and the animals of the low test concentration group (3.7 mg/m³ test item) did not exhibit any clinically significant effects.
At a test concentration of 17.5 mg/m³, the animals exhibited ruffled fur, reduced motility, nasal bleeding and tachypnea during the first week of treatment; thereafter, they only exhibited ruffled fur. On the day following treatment day as well as on treatment-free weekends, the animals were inconspicuous.
At a test concentration of 76.6 mg/m³, similar symptoms as described above were noticed, and additionally, the animals also exhibited signs of respiratory distress and breathing sounds. In this group, 3 cases of mortality occurred; two males died on day 7 and 21 of testing, respectively, and one female was sacrificed in extremis on day 16 because of moribund state.

BODY WEIGHT AND WEIGHT GAIN
The body weights were significantly decreased in the high test concentration group (76.6 mg/m³) for both, males and females.
For the males, the group mean body weights at test ending were 201, 208, 197, 197 and 175 g for the sham control, the vehicle control, the low concentration group (3.7 mg/m³), the mid concentration group (17.5 mg/m³) and the high concentration group (76.6 mg/m³), respectively.
For the females, the group mean body weights at test ending were 147, 147, 147, 148 and 143 g for the sham control, the vehicle control, the low concentration group (3.7 mg/m³), the mid concentration group (17.5 mg/m³) and the high concentration group (76.6 mg/m³), respectively.

HAEMATOLOGY
For all considered parameters, no treatment-related differences between control groups and treated groups were noticed. Thus, the treatment with the substance did not affect the haematology of the test animals.

CLINICAL CHEMISTRY
For the males of the high test concentration group (76.6 mg/m³), a slight increase in albumin level and a slight increase in globulin were evidenced; for the females a similar tendency was also noticed. For all remaining parameters, no treatment-related differences between control groups and treated groups were noticed; some incidental statistically significant differences were found, which were without toxicological relevance because, e.g., of absence of a dose-response relationship.

URINALYSIS
For all considered parameters, no treatment-related differences between control groups and treated groups were noticed.

GROSS PATHOLOGY
The pathological examination of the animals sacrificed at test ending revealed some cases of dark red, distended lungs in the mid and high test concentration groups (17.5 and 76.6 mg/m³); in one male of the mid and one of the high test concentration group, foamy content in the trachea further was seen. Moreover, the findings were more severe in the high test concentration group than in the mid one, and in the high test concentration group, the findings were more severe in males than in females. No further gross pathological abnormalities were found.
The pathological examination of the intercurrently died 3 animals was only possible in one case, because of cannibalism. In the one case, examination revealed distended lung, dark content of the stomach and intestines, ulcer-like spots in the glandular stomach, enlarged and dark red adrenals (right side), brain edema and hyperaemia in all relevant organs.

ORGAN WEIGHTS
The absolute and relative lung weights were significantly increased in a dose-related manner in the mid and the high test concentration groups (17.5 and 76.6 mg/m³). For the remaining organ weights, no conclusive changes were evident when compared to controls; in fact, some changes in liver weight were reported which were incidental and not treatment-related.

HISTOPATHOLOGY
The findings observed by gross pathology examinations coincided with round-cell infiltrations and focal hyperplasia in the larynx and trachea, increased incidence of proliferative responses in the bronchio-alveolar region, inflammation, thickening of septa, fibrosis, and increased influx of alveolar macrophages.These findings were more pronounced and severe in the high test concentration group than in the mid group, indicating a dose-response relationship. Histopathological changes in the nasal passages consisted of a goblet-cell hypertrophy and hyperplasia with no conclusive difference to the acetone control group.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Effect level:
3.7 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects were seen at the lowest tested concentration level.
Dose descriptor:
LOAEC
Effect level:
17.5 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: effects at 17.5 mg/m³: clinical symptoms, pathological and histopathological lesions in respiratory tract. effects at 76.6 mg/m³: mortalities, clinical symptoms, reduced body weights, pathological and histopathological lesions in respiratory tract.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Incidence of exposure-related histopathologic lesions after 3 weeks (range-finding study)

Tissue and lesion

Air

control

Acetone control

Low-concentration group

Mid-concentration group

High-concentration group

Larynx

(20)

(20)

(20)

(20)

(20)

Round cell infiltration

2

1

1

5

0

Hyperplasia

0

0

0

3

8**

Trachea

(20)

(20)

(20)

(20)

(20)

Round-cell infiltration

0

1

0

2

2

Hyperplasia

0

0

0

5*

6**

Lungs

(20)

(20)

(20)

(20)

(20)

Increased number of AM

0

3

1

6*

18**

Thickening of septa

0

1

0

3

18**

Fibrosis

0

1

0

0

18**

Bronchio-alveolar proliferation

1

3

0

1

17**

Values in parentheses are nuber of animals with the tissue examined

AM: alveolar macrophages

* p < 0.054; ** p < 0.01

 

Table 2: Characterization of chamber aerosol atmosphere

3 week range-finding study

Unit

Air

control

Acetone control

Low-concentration group

Mid-concentration group

High-concentration group

Target concentration

mg/m³

-

-

3

15

75

Acetone nominal concentration

mg/m³

-

9292

58

180

405

Analytical concentraition

mg/m³

-

-

3.7 ± 1.7

17.5 ± 5.0

76.6 ± 17.1

Gravimetric concentration

mg/m³

-

-

-

-

70.1 ± 9.1

MMAD

µm

-

-

1.4 ± 0.1

1.4 ± 0.1

1.7 ± 0.1

GSD

-

-

-

1.3 ± 0.0

1.3 ± 0.0

1.6 ± 0.1

Applicant's summary and conclusion