Registration Dossier

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 compliant, guideline study, available as published report, no restrictions, fully adequate for assessment
Cross-reference
Reason / purpose:
reference to same 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:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): isooctene
- Synonym: Di-n-butene
- Physical state: Clear, clourless liquid
- Analytical purity: 99.9%, 99.84 area% (gas chromatography)
- Lot/batch No.: K402 destillate
- Expiration date of the lot/batch: Unlimited
- Stability under test conditions: Unlimited
- Storage condition of test material: room temperature; air sensitive, under N2

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: group mean range 161.7 - 166.6 g (males), 126.1 - 131 g (females)
- Housing: Individually in wire cages
- Diet: Milled mouse/rat laboratory diet (Provimi Kliba SA, Kaiseraugst, Basel Switzerland) ad libitum except during exposure and motor activity measurements
- Water: tap ad libitum except during exposure and motor activity measurements
- Acclimation period: Approximately two weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity: 30 - 70%
- Air changes (per hr): Not reported; air-conditioned
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 13 June 2006 To: 26 April 2007

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: glass-steel inhalation chamber, volume of about 1.4 m³ (BASF Aktiengesellschaft).
- Air flow: Approximately 20 per hour
- Air conditions: Test group 0: A positive pressure was maintained by adjusting the airflow of the exhaust air system. This ensured that no laboratory air reached the control animals.
Test groups 1 - 3: A negative pressure was maintained by adjusting the airflow of the exhaust air system. This ensured that the laboratory was not contaminated as the result of any leakage from the inhalation chamber.
- System of generating particulates/aerosols: For each concentration the test substance was supplied to a thermostated vaporizer at a constant rate by means of the metering pump.
- Temperature, humidity, pressure in air chamber: The vapour was generated with conditioned supply air (actual range 54.6-61.1% relative humidity, temperature 20.9 - 22.7°C) and passed into the inhalation system.

TEST ATMOSPHERE
- Brief description of analytical method used: The nominal concentration was calculated from the study means of the test pump rates and the supply airflows used during exposure to generate the respective concentrations.
The concentrations of the inhalation atmospheres were analyzed by gas chromatography in all test groups including control.
- Total hydrocarbon analyzers were used to continuously monitor the constancy of concentrations of test substance vapours in the inhalation systems.
- No surveillance of the oxygen content in the inhalation system was performed. The air change within the inhalation systems was judged to be sufficient to prevent oxygen depletion by the breathing of the animals and the concentrations of the test substance used could not have a substantial influence on oxygen partial pressure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The vapour generation effectiveness was as expected for these high concentrations (range of 83.9 - 88.8%). Real time surveillance of the inhalation atmospheres with total hydrocarbon analyzers generally proved the constancy of each concentration throughout the daily exposures.
Duration of treatment / exposure:
6 hours per day / 5 days per week
Frequency of treatment:
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).
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1000, 5000 and 15000 mg/m3
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
1200, 5600 and 16900 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1000±100 , 50000±200 and 14900±700 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
10
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: as requested by the sponsor

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The clinical conditions of the test animals were recorded at least 3 times (before, during and after exposure) on exposure days and once during the preflow period, on the day of neurofunctional test and prior to gross necropsy. During exposure only a group wise examination was possible.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed in all animals prior to the exposure period and thereafter in weekly intervals.

BODY WEIGHT: Yes
- Time schedule for examinations: 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.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, with the following exceptions; Food consumption was not determined during the mating period
Food consumption of the parental females with evidence of sperm (p.c.) 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 post partum (p.p.)

FOOD EFFICIENCY:
- Food efficiency (group means) was calculated based upon individual values for body weight and food consumption.

OPHTHALMOSCOPIC EXAMINATION: Yes
- 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: Yes
- Time schedule for collection of blood: Towards the end of the premating period, blood was taken from the retro-orbital venous plexus
- Anaesthetic used for blood collection: Yes (Isoba®, Essex GmbH Munich, Germany)
- Animals fasted: Yes
- How many animals: 10 animals per test group and sex.
- Parameters examined: leukocyte count, erythrocyte counr, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count, differential blood count and reticulocyte count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Towards the end of the premating period, blood was taken from the retro-orbital venous plexus
- Animals fasted: Yes
- How many animals: 10 animals per test group and sex.
- Parameters examined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and γ-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol and magnesium.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: A functional observational battery (FOB) and measurements of motor activity (MA) were carried out on study days 50 and 51 for males and females, respectively.
- Dose groups that were examined: From each concentration group, neurofunctional tests were performed in five male and five female animals.
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; a complete necropsy including gross pathological evaluation and weighing of selected organs was performed.
HISTOPATHOLOGY: Yes; organs and tissues were examined histopathologically as required by the test guidelines.
In addition, to examine whether there were treatment-related accumulation of alpha-2u-globulin in the kidneys of male animals, the slides were stained according to Mallory-Heidenhain. Based on these results, the kidneys of one male animal of each concentration group was stained immunohistochemically.
Other examinations:
After ten weeks of exposure, the parental animals were mated to produce a litter. Mating pairs were formed from the same concentration group. The parental animals were examined for their mating and reproductive performances. The pups were sexed and were weighed on the day after birth and on day 4 post partum. Their viability was recorded. All pups were underwent necropsy on day 4 post partum and were examined macroscopically for external and visceral findings.
Statistics:
Means and standard deviations of each test group were calculated for parameters measured. Further statistical analyses were performed as appropriate, according to the methods of Dunnett’s t-test (two-sided), Fisher’s Exact test, Wilcoxon test (one-sided), and/or Kruskal-Wallis test (two-sided).

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:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
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:
CLINICAL SIGNS AND MORTALITY
There was no mortality. 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. Ataxia affected all animals at 15000 mg/m3 only during exposure and persisted throughout the study. Animals were normal before and after exposure. In the FOB observations on day 50 there were no abnormalities of gait recorded in either males or females.

BODY WEIGHT AND WEIGHT GAIN
In male animals the mean body weight and body weight change were found to be significantly decreased when compared with the controls.

FOOD CONSUMPTION
A transient decrease of food consumption was observed in male and female animals on study day 7, which did not occur again after the animals adjusted to the test substance and the exposure procedure.

FOOD EFFICIENCY
In male animals, food efficiency were found to be significantly decreased when compared with the controls.

HAEMATOLOGY
After inhalation of the substance for about 2 months there was noticed a slight increase of the median of the platelet counts in the parent males exposed to 15 g/m3.
The median of the prothrombin time was decreased in the male parents beginning in group 2 (5 g/m3), and in the female parents in the group exposed to 15 g/m3, only.

CLINICAL CHEMISTRY
In the female parents of the 15 g/m3 group the median of the aspartate aminotransferase activity was decreased after 2 months of substance inhalation, in the male parents of the same dose group there was assessed an increased median of the alanine aminotransferase activity. The median of the γ-glutamyltransferase activity was slightly increased in the 15 g/m3 group of the rats of both sexes, but this was only statistically significant in the males.
In the male rats the median of the total protein levels was increased beginning in the 5 g/m3 dose group. Regarding the medians of the protein fractions albumin and globulin the significant increase was only assessed in the 15 g/m3 dose group. The median of the globulin fraction was increased in the female rats of the 15 g/m3 dose group, too. The male parents showed increased medians of the bilirubin concentration in the 15 g/m3 dose group, and decreased medians of the glucose levels starting in the 5 g/m3 dose group. The medians of the cholesterol values were increased in the rats of both sexes beginning in the 5 g/m3 dose groups. The median of the triglyceride concentration was increased in the 15 g/m3 dose group of the females, only. In this group, there was found a decrease of the urea and the creatinine concentration, too.
The medians of the potassium and calcium serum levels were increased in the rats of both sexes (potassium: males beginning in the 5 g/m3, and in the females in the 15 g/m3 dose group; calcium: in the 15 g/m3 dose group of both sexes). The magnesium levels were increased only in the male parents beginning in the 1 g/m3 dose group. The increase in the low-concentration group was still within the range of biological variations. Therefore, this is not regarded as a toxicologically relevant effect.

ORGAN WEIGHTS
Absolute- and relative liver and kidney weights of males were significantly increased in animals exposed to 5 and 15 g Isooctene/m3

HISTOPATHOLOGY: NON-NEOPLASTIC
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 the group 1g/m3 males is regarded to be adaptive and non-adverse in character.
In the respiratory tract no signs of toxicity were observed.

OTHER FINDINGS
The fertility indices for F0 males were 90, 100, 90 and 100% for the control, 1 g/m3, 5g/m3 and 15 g/m3 groups respectively. the female mating index was 100% for the control, 1 g/m3 and 15 g/m3 groups and 90% for the 5 g/m3 group.
The gestation index was 100% in the 5 and 15 g/m³ groups, 89% in the control group and 90% in the 1 g/m³ group. The number of liveborn and stillborn pups was comparable between all test groups. Thus, the live birth index amounted to 100% in the 5 g/m³ group, 99% in 15 g/m³ group, 98% in the control group and 96% in the 1 g/m³ group.
The mean number of delivered F1 pups/dam and the rate of live- and stillborn F1 pups was not affected by the test substance. The total average of delivered F1 pups/dam was 10.7, 11.8, 11.1 and 11.3 pups/dam in the control, 1, 5 and 15 g/m³ groups.
No adverse effects on the development of offspring until postnatal day 4 were noted in the litters of all exposed groups.

Effect levels

Dose descriptor:
NOAEC
Effect level:
1 000 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Intergroup comparison of bodyweights (g) selected timepoints

 

males

females

 

Dose level of isooctane (mg/m3)

Day

0

1000

5000

15000

0

1000

5000

15000

0

166.6

163.3

162.0

161.7

131.1

126.1

129.1

129.3

21

289.8

276.5

277.9

261.0**

185.0

182.8

188.3

183.2

49

373.3

355.0

355.2

328.2**

219.1

219.1

224.9

217.8

91

422.0

406.1

403.0

371.3**

-

-

-

-

105

-

-

-

-

270.3

258.8

273.7

263.4

*p<=0.05  **p<=0.01

Intergroup comparison of food efficiency, selected timepoints

 

males

females

 

Dose level of isooctane (mg/m3)

Day

0

1000

5000

15000

0

1000

5000

15000

7

27.1

27.8

28.5

24.2*

16.5

19.4

17.3

17.6

28

15.9

15.9

14.3

11.8**

9.5

7.1

8.6

9.2

63

7.4

6.4

7.7

5.6

3.9

6.4

7.4

3.3

*p<=0.05  **p<=0.01

Intergroup comparison of selected organ weights (g)

 

males

females

 

Dose level of isooctane (mg/m3)

 

0

1000

5000

15000

0

1000

5000

15000

Kidney, abs

2.377

2.411

2.717*

2.837**

1.751

1.758

1.814

1.826

Kidney, rel

0.61

0.644

0.725**

0.825**

0.698

0.741*

0.713

0.754*

Liver, abs

9.804

9.873

10.948*

12.201**

8.709

7.751**

7.701**

8.317

Liver, rel

2.5

2.638

2.924**

3.54**

3.475

3.253

3.03**

3.436

*p<=0.05  **p<=0.01

 

Applicant's summary and conclusion

Conclusions:
The NOAEC in this study was 1000 mg/m3 for male and female rats. The male kidney was identified as the target organ as increased hyaline casts and basophilic cortical tubules were observed in the kidneys of male animals exposed to 5000 mg/m3 onwards. These changes were associated with accumulation of alpha 2u globulin in cortical tubular cells. The presence of increased hyaline casts and basophilic cortical tubules are an indication of proximal tubular cell damage and a classical effect of α2u-nephropathy which is male rat-specific effect not considered relevant human hazard assessment.
Executive summary:

The 90-day inhalation exposure of rats to Isooctene at 1000, 5000 and 15000 mg/m3 induced toxicity in the male kidney (target organ), effects in the male liver, but no effects in the respiratory tract. Under the current test conditions, the no-observed adverse effect level (NOAEC) in this study is 1000 mg/m3 for the male and female rats.