Isooctene

Endpoint summary

Description of key information

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.  The male rat-specific kidney effects are indicative of alpha-2u-globulin nephropathy and are not considered relevant to humans. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Males and females were exposed to the test substance for 10 weeks prior to pairing.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

Males and females were paired for mating. Females were pregnant prior to termination.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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: Approximately 6 weeks
- Weight at study initiation: group mean range 161.7-166.6 g (males), 126.1-131.0 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 water 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

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: air

Remarks on MMAD:

MMAD / GSD: Not applicable

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).

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

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 count, 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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; a complete necropsy including gross pathological evaluation and weighing of selected organs (adrenals, brain, heart, kidneys, liver, lungs, ovaries, spleen, thymus, uterus, epididymides and testes) was performed.

HISTOPATHOLOGY: Yes; the following organs and tissues were examined histopathologically: all gross lesions, brain, spinal cord (cervical, thoracic and lumbar), sciatic nerve, pituitary gland, salivary glands, thyroid/parathyroid glands, adrenal glands, prostate gland, seminal vesicles, coagulation glands, uterus, oviducts, vagina, female mammary gland, thymus, lymph nodes, spleen, trachea, lungs, heart, aorta, liver, pancreas, kidneys, oesophagus, stomach, duodenum, jejunum, ileum, caecum, 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 lachrymal glands).
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).

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 15000 mg/m3 indicating marginal irritation and systemic toxicity of Isooctene.

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 was 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 15000 mg/m3.
The median of the prothrombin time was decreased in the male parents beginning in group 2 (5000 mg/m3), and in the female parents in the group exposed to 15000 mg/m3, only.

CLINICAL CHEMISTRY
In the female parents of the 15000 mg/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 15000 mg/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 5000 mg/m3 dose group. Regarding the medians of the protein fractions albumin and globulin the significant increase was only assessed in the 15000 mg/m3 dose group. The median of the globulin fraction was increased in the female rats of the 15000 mg/m3 dose group, too. The male parents showed increased medians of the bilirubin concentration in the 15000 mg/m3 dose group, and decreased medians of the glucose levels starting in the 5000 mg/m3 dose group. The medians of the cholesterol values were increased in the rats of both sexes beginning in the 5000 mg/m3 dose groups. The median of the triglyceride concentration was increased in the 15000 mg/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 5000 mg/m3, and in the females in the 15000 mg/m3 dose group; calcium: in the 15000 mg/m3 dose group of both sexes). The magnesium levels were increased only in the male parents beginning in the 1000 mg/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 5000 and 15000 mg 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 (5000 mg/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 1000 mg/m3 males is regarded to be adaptive and non-adverse in character.
In the respiratory tract no signs of toxicity were observed.

Dose descriptor:

NOAEC

Effect level:

1 000 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

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

Abs=absolute       rel=relative to final bodyweight

 

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 α2µ-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), 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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

1 000 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Males and females were exposed to the test substance for 10 weeks prior to pairing.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

Males and females were paired for mating. Females were pregnant prior to termination.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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: Approximately 6 weeks
- Weight at study initiation: group mean range 161.7-166.6 g (males), 126.1-131.0 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 water 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

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: air

Remarks on MMAD:

MMAD / GSD: Not applicable

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).

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

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 count, 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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; a complete necropsy including gross pathological evaluation and weighing of selected organs (adrenals, brain, heart, kidneys, liver, lungs, ovaries, spleen, thymus, uterus, epididymides and testes) was performed.

HISTOPATHOLOGY: Yes; the following organs and tissues were examined histopathologically: all gross lesions, brain, spinal cord (cervical, thoracic and lumbar), sciatic nerve, pituitary gland, salivary glands, thyroid/parathyroid glands, adrenal glands, prostate gland, seminal vesicles, coagulation glands, uterus, oviducts, vagina, female mammary gland, thymus, lymph nodes, spleen, trachea, lungs, heart, aorta, liver, pancreas, kidneys, oesophagus, stomach, duodenum, jejunum, ileum, caecum, 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 lachrymal glands).
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).

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 15000 mg/m3 indicating marginal irritation and systemic toxicity of Isooctene.

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 was 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 15000 mg/m3.
The median of the prothrombin time was decreased in the male parents beginning in group 2 (5000 mg/m3), and in the female parents in the group exposed to 15000 mg/m3, only.

CLINICAL CHEMISTRY
In the female parents of the 15000 mg/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 15000 mg/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 5000 mg/m3 dose group. Regarding the medians of the protein fractions albumin and globulin the significant increase was only assessed in the 15000 mg/m3 dose group. The median of the globulin fraction was increased in the female rats of the 15000 mg/m3 dose group, too. The male parents showed increased medians of the bilirubin concentration in the 15000 mg/m3 dose group, and decreased medians of the glucose levels starting in the 5000 mg/m3 dose group. The medians of the cholesterol values were increased in the rats of both sexes beginning in the 5000 mg/m3 dose groups. The median of the triglyceride concentration was increased in the 15000 mg/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 5000 mg/m3, and in the females in the 15000 mg/m3 dose group; calcium: in the 15000 mg/m3 dose group of both sexes). The magnesium levels were increased only in the male parents beginning in the 1000 mg/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 5000 and 15000 mg 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 (5000 mg/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 1000 mg/m3 males is regarded to be adaptive and non-adverse in character.
In the respiratory tract no signs of toxicity were observed.

Dose descriptor:

NOAEC

Effect level:

1 000 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

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

Abs=absolute       rel=relative to final bodyweight

 

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 α2µ-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), 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

15 000 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Non-human data

Oral and dermal

No studies available

Inhalation

Isooctene has been tested in a combined subchronic inhalation study with a reproduction/developmental toxicity screening test at vapor concentrations of 0, 1000, 5000, and 15,000 mg/m³ (BASF, 2007).  Male Wistar rats were exposed for approximately 13 weeks; females were exposed during premating (10 weeks), mating and gestation through day 4 after delivery (approximately 15 weeks). Absolute and relative liver and kidney weights were significantly increased in the mid- and high-exposure groups. The only histopathological effects were increased hyaline casts and basophilic cortical tubules, along with accumulation of alpha-2u-globulin in the cortical tubular cells of male rats at 5000 and 15,000 mg/m³. These male rat-specific kidney effects are indicative of alpha-2u-globulin nephropathy and are not considered relevant to humans. The NOAEC for this study is 1000 mg/m³.  

Justification for classification or non-classification

There are sufficient data available on isooctene to conclude that it is of low repeat dose toxicity by the inhalation route with no indication of serious functional or morphological effects. Classification is not warranted DSD or CLP.