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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Isooctene, purity: 99.9% (gas chromatography); 99.84 area% (gas chromatography)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: about 4 weeks
- Housing: During the period when the rats were not exposed they were housed singly in wire cages (type DK III, Becker & Co., Castrop-Rauxel, FRG
(floor area about 800 cm²)). Underneath the cages, waste trays were fixed containing bedding material (type 3/4 dust free embedding, supplied by
SSNIFF, Soest, FRG). The following exceptions were made:
1. For the overnight mating, the females were put into the cages of the males.
2. From day 18 p.c. until sacrifice, the dams and their litters were housed in Makrolon type M III cages (floor area about 800 cm2). The M III cages
were also supplied by BECKER & CO. Pregnant females were provided with nesting material (cellulose wadding) towards the end of pregnancy. The
motor activity measurements were conducted in Polycarbonate cages with wire covers from Ehret, Emmendingen, FRG (floor area about 800 cm²) and bedding. The room was completely disinfected using a disinfector ("AUTEX", fully automatic, formalinammonia-based terminal disinfector) before
the start of the study. Usually, each week the floor and the walls were cleaned with water containing about 1 % Mikroquat®.
- Diet (e.g. ad libitum): The animals were maintained on milled mouse/rat laboratory diet “GLP” (Provimi Kliba SA, Kaiseraugst, Basel Switzerland) ad
libitum.
- Water (e.g. ad libitum): Tap water ad libitum.
During exposure and motor activity measurements food and water were withdrawn.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): relative humidity in the range of 30 - 70%
- Air changes (per hr): fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): A light/dark rhythm of 12 hours was maintained:
• 06.00 a.m. - 06.00 p.m. light
• 06.00 p.m. - 06.00 a.m. dark
Deviations from these ranges did not occur.

IN-LIFE DATES: From: 13.06.2006 To: 10.10.2006

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Duration of treatment / exposure:
The males were treated for approx. 13 weeks (10 weeks premating, 3 weeks mating and post mating). In females treatment was performed during
premating (10 weeks), mating and gestation through day 4 after delivery (approx. 15 weeks).
Frequency of treatment:
6 hours per day on 5 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
1, 5 and 15 g/m3
Basis:

No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: yes

Examinations

Observations and examinations performed and frequency:
The body weight of all parental animals was determined on day -5 (start preflow period), on day 0 (start exposure period) and then in weekly
intervals as well as prior to gross necropsy. As a rule, the animals were weighed at the same time of the day. Body weight change was calculated as
the difference between body weight on the respective exposure day and body weight on the day of the first exposure. Group means were derived
from the individual differences.
The following exceptions are notable for the female parental animals:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (day 0 p.c.) and on days 7, 14 and 20 post
coitum.
• Females with litter were weighed one day after parturition (day 1 p.p.) and on day 4 post partum.
• Females without litter were not weighed.
The pups were weighed on the day after birth (day 1 p.p.) and on day 4 after birth. Pups' body weight change was calculated from these results.
Furthermore the body weights on day 1 p.p. were used for the determination of "runts", i.e. pups, which weighed more than 25% less than the mean
weight of the respective control pups.

FOOD CONSUMPTION:
Food consumption was determined on day 0 (start of exposure period) and then in weekly intervals. It was calculated as mean food consumption in
grams per animal and day.
The following exceptions were notable:
• Food consumption was not determined during the mating period
• Food consumption of the parental females with evidence of sperm was determined for days 0 - 7, 7 - 14 and 14 - 20 p.c.
• Food consumption of parental females, which gave birth to a litter was determined for days 1 - 4 p.p.

Food efficiency (group means) was calculated based upon individual values for body weight and food consumption:
BWx - BWy
————— x 100 = Food efficiency for day x
FCy to x
BWx = body weight on day x (g)
BWy = body weight on day y (last weighing date before day x) (g)
FCy to x = mean food consumption from day y to day x; calculated as mean daily food consumption on day x, multiplied with the number of days
from day y to day x (g)

OPHTHALMOSCOPIC EXAMINATION: Before the start of the exposure period (day -3) the eyes of all animals, and at the end of the study (day 88 for
the parental male animals and day 102 for the parental female animals) the eyes of the animals of test group 0 (control group) and test group 3 (high concentration) were examined with an ophthalmoscope (HEINE Optotechnik, Herrsching, FRG)) for any changes in the refracting media.

HAEMATOLOGY: Parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Furthermore differential blood smears were prepared and stained according to Wright without being evaluated.

In the morning, blood was taken from the retro-orbital venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH Munich, Germany). The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a
randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with
commercial reference controls to assure reliable test results. The results of the clinical pathology examinations are expressed in units of the
International System (SI).
Sacrifice and pathology:
At termination of the study, the animals were sacrificed and underwent gross necropsy. The organs were weighed, preserved and processed
histotechnically. To examine whether there were treatment-related accumulation of alpha-2u-globulin in the kidneys of male animals, the slides were stained additionally according to Mallory-Heidenhain. Based on these results, the kidneys of one male animal of each concentration group was
stained immunohistochemically.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
No deaths were recorded throughout the study.
During the preflow period and the post-exposure observation days the animals showed no clinical signs and findings different from normal.
During the exposure period the animals of the control group, low concentration (1 g/m³), and intermediate concentration (5 g/m³) showed no clinical signs and findings different from normal.
Clinically, slight ataxia (from day 9 onward) and salivation (from day 8 onward) were observed at 15 g/m3 indicating marginal irritation and systemic toxicity of Isooctene. Corresponding to these observations, in the male animals the mean body weight, body weight change and food efficiency were found to be significantly decreased when compared with the controls. Moreover, a transient decrease of food consumption was observed in male and female animals on study day 7, which did not occure again after the animals adjusted to the test substance and the exposure procedure. All these clinical findings were considered to be exposure-related adverse effects.
Clinical Pathology: The increase of the g-glutamyltransferase activity in the male and female animals exposed to 15 g/m3 without any signs of hepatocellular damage in clinical pathology parameters is a sign of a microsomal enzyme induction in the liver. This hypothesis is supported by the increase of the haemostasis factor activity as well as by the increasing cholesterol and triglyceride values in the dosed rats of both sexes. The decrease of the aspartate aminotransferase activity in the treated female rats might be due to the mentioned microsomal enzyme induction, too. No adverse effects were observed, which could be associated with the fall of the aspartate aminotransferase activity. Therefore, the decrease of the aspartate aminotransferase is regarded as not toxicologically relevant. Moreover, in the males of this group the bilirubin levels as well as the alanine aminotransferase activity was increased. This might be an indication for a dysfunction of the hepatocytes as well as an intrahepatic affection of the bile flow. These findings correlate with the increased liver weights. There was no hint of a toxic damage of the hepatocytes. One reason for the higher serum albumin and globulin values in the rats of the 15 g/m3 dose groups might be the greater production of transport proteins by the liver. The increases of the serum potassium concentration in the female rats as well as the magnesium levels in the male rats and the calcium levels in the rats of both sexes might be the consequence of a slight acidotic metabolism. The decreased glucose levels in the male rats correlated with the retarded body weight development.

Pathology: In the pathology, the major organ weight changes in the treated groups concerned male liver and kidney. Absolute and relative liver and kidney weights of males were significantly increased in animals exposed to 5 and 15 g Isooctene/m3. Considering the findings in the clinico-pathology, the increased liver weight might be a results of adaptive enzyme induction, although there was no histopathological correlate. The treatment-related microscopic changes observed were increased hyaline cast(s) and basophilic cortical tubules in the kidneys of male animals from group 2 (5 g/m3) onward. In addition, in males there was suspected greater propensity for the development of large hyaline droplets related to the proximal tubules compared to controls. Mallory-Heidenhain stain of all male kidneys and immunhistochemistry of selected male kidneys with an alpha-2u-globulin antibody revealed a concentration-dependent accumulation of alpha 2u-globulin in cortical tubular cells in all treated groups. However, in the absence of any treatment-related morphological signs of cytotoxicity, the minimal to slight accumulation of alpha-2u-globulin in group 1 males is regarded to be adaptive and non-adverse in character.

In the respiratory tract no signs of toxicity were observed.

Reproductive/Developmental toxicity: The capability to cohabitate and to generate offspring was not affected in either genders. Furthermore, no adverse effects on the development of offspring until postnatal day 4 were noted in the litters of all exposed groups. Thus, under the conditions of this test, the test compound did not influence fertility and pre/postnatal development at dose levels as high as 15 g/m³.

Effect levels

Dose descriptor:
NOAEC
Effect level:
1 000 mg/m³ air
Sex:
male/female
Basis for effect level:
other: male kidney (α2µ)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion