Isoheptane

Endpoint summary

Description of key information

Inhalation

Inhalation systemic NOAEC (Rat): 12470 mg/m³

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration

Reference 2

Endpoint:

sub-chronic toxicity: oral

Data waiving:

other justification

Justification for data waiving:

other:

Justification for type of information:

The 'Justification for the read across' is provided in the 'Attached justification' section below.

Species:

rat

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Male and nulliparous, nonpregnant female Sprague-Dawley rats (VAF/ Plus Crl:CD BR)
- Source: Charles River Laboratories, Kingston, NY
- Age at study initiation: approximately 5 wk old when purchased, acclimated for approximately 2 wk prior to the initiation of the study
- Housing: Animals were housed individually in suspended stainless steel wire mesh cages in air-conditioned rooms
- Diet (e.g. ad libitum): Certified rodent diet 5002 (PMI Feeds, Inc., St. Louis, MO) ad libitum
- Water (e.g. ad libitum): water from an automated watering system was available ad libitum
- Acclimation period: 2 weeks. All animals were assigned a temporary identification number at receipt and examined by the staff veterinarian during the acclimation period.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26°C
- Humidity (%): 40-70% relative humidity
- Photoperiod (hrs dark / hrs light): 12/12 during the acclimation and all nonexposure periods

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: nitrogen

Details on inhalation exposure:

Rats were exposed to wholly vaporized LAND-2 generated in nitrogen, by inhalation in whole-body exposure cages 6 h/d, 5 d/wk for 13 wk at analytical concentrations of 668, 2220, and 6646 ppm (2.4, 8.1, and 24.3 g/m3). Exposure levels were determined three times daily by gas chromatography. The highest concentration was approximately 75% of the lower explosive limit. Animals’ positions in the cages were rotated for each exposure to ensure uniform exposure of every animal.

Chamber Operation: Animals were housed individually in wire mesh, stainless steel cages within a 1000-L glass and stainless steel exposure chamber. Chamber temperature and humidity were monitored every half hour during exposure and maintained, to the extent possible, within the ranges of 20–24°C temperature and 40–60% relative humidity. Animals did not receive food or water during the exposure period. Exposure chambers were operated dynamically at a calibrated airflow rate of 200 L/min (lpm). Recordings of airflow and static pressure were made every half hour. All animals remained in the chamber for a minimum of 30 min at the end of exposure while the chamber was operated using clean air only. Chambers were exhausted through a system of coarse filter, HEPA filter, and charcoal filter.

Atmosphere Generation: LAND-2 was pumped directly from the 5-gal container, housed within a freezer constantly flushed with nitrogen, using a laboratory pump, equipped with a piston, that was insulated within a styrofoam container. An ice bag was placed on top of the piston to keep the piston chamber cold to inhibit volatilization of LAND-2 in the pump and delivery lines. LAND-2 was delivered onto the central glass helix of a countercurrent volatilization chamber (one generator per chamber). The glass helix was heated by an internal nichrome wire inserted in the center of the glass tube that supported the helix (external to the volatilization chamber) and was controlled by a variable autotransformer. House-line nitrogen delivered from a regulator with a backpressure gauge was divided with a stainless steel T into the generation flow system and a purge flow system. Purge nitrogen was delivered to the bottom of the tube containing the nichrome wire to continuously purge the area surrounding the wire, protecting it from oxidation. Nitrogen for the generation system was directed through a flowmeter to the ball-and-socket joint at the bottom of the volatilization chamber, flowed up the chamber, passed over the coil, and volatilized the test material. The LAND-2-laden nitrogen flowed through a T tube at the top of the volatilization chamber into the turret of a 1-m3 glass and stainless steel exposure chamber, where it mixed with room air to appropriate exposure concentrations as it was drawn into the chamber (flow rate of 200 lpm). Control animals were sham-exposed to nitrogen alone introduced into the turret and mixed with air in the chamber.

Exposure Chamber Monitoring: Samples for determination of analytical exposure levels and the major components of LAND-2 vapor were withdrawn by vacuum pump from the breathing zone in the exposure chambers three times per exposure for treated groups and once per exposure for controls. Samples were pulled through Teflon lines into the multipositional control module and directed to a Hewlett Packard 5890II gas chromatograph, equipped with a flame ionization detector for analysis by ASTM method D5134-92 (ASTM, 1992). Composition and stability of the test material were evaluated by characterizing neat LAND-2 and comparing the major components in the neat and generated atmospheres at the beginning and end of the study. Particle size distribution measurements of any background aerosol were performed once during each exposure for chambers and room air using a TSI Aerodynamic Particle Sizer. Samples were drawn for 20 s at a rate of 5 L/min. Mean mass aerodynamic diameter (MMAD), geometric standard deviation (GSD), and total mass concentration (TMC) were calculated. Nominal concentrations (mg/m3) were calculated from the loss of weight from the generation apparatus divided by the total air flow through the chamber during exposure. This value was converted to parts per million (ppm) using an average molecular weight for this mixture of 89.2.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Determined three times daily by gas chromatography.
Composition and Uniformity Chamber Gas Chromatographic Results (% Weight): n-Butane: 3.21; iso-Pentane: 34.343; 2,3-Dimethylbutane: 12.977, 2-Methylpentane: 4.096; 2,4-Dimethylpentane: 5.663; 2,3-Dimethylpentane: 2.680; 2,2,4-Trimethylpentane: 16.885; 2,3,4-Trimethylpentane: 3.578; 2,3,3 –Trimethylpentane: 4.505; 2,2,5-Trimethylhexane: 2.499.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
0, 668, 2220, and 6646 ppm (0, 2.4, 8.1, and 24.3 g/m3)
Basis:
analytical conc.

No. of animals per sex per dose:

12

Control animals:

yes, sham-exposed

Details on study design:

Neurobehavioral evaluations of motor activity (MA) and functional operational battery (FOB) were performed pretest and during wk 5, 9, 14, and 1 8 (recovery groups). Animals were not exposed to LAND-2 on the days of neurobehavioral testing. Exposure days were added to ensure that each animal received at least 65 exposures.

Following 13 wk of exposure, 12 animals/sex/group were necropsied and microscopic examination was performed on selected tissues. Nervous tissue from 6 rats/sex/group was also examined microscopically. At the end of the 4-wk recovery period, 12 animals/sex from the high and control groups were necropsied and selected tissues examined microscopically.

Observations and examinations performed and frequency:

Animals were evaluated twice daily for mortality and gross signs of toxicological or pharmacological effects. During each exposure, rats were observed as a group once for abnormal behavior. Detailed physical examinations were performed twice pretest and weekly during the study. Ophthalmoscopic evaluations were performed pretest and just prior to the scheduled sacrifices at wk 14 (terminal) and 18 (recovery).

Body Weights and Food Consumption. All animals were weighed twice pretest, weekly during the study period, and prior to scheduled sacrifice. Food consumption was measured once during the week prior to treatment initiation and over a 6-d interval each week during the study period.

Animals were transported from the room in which they were housed during non-exposure hours to the neurotoxicity laboratories. Behaviors were evaluated pretest and during wk 5, 9, 14, and 18 (recovery groups). Temperature, humidity, and illumination were measured and recorded to minimize variation in environmental conditions during evaluations. Noise levels were not recorded. Testing was staggered over 8 sessions within a 4-d period, each session consisting of approximately 2 rats/sex/treatment group.

Motor Activity. Locomotor activity was monitored as a function of the number of beam breaks in an activity box, using an automated photo- beam activity system. Sessions were 60 mm in length divided into twelve 5-mm intervals. Rats were evaluated at pretest, at 3 time points during the treatment period (wk 5, 9, and 1 4), and at the end of the recovery period. Treatment groups were counter balanced across test times. Three sets of analyses were performed. The first analysis was conducted using the pre-dose data with animals nested within the interaction effect of sex crossed with treatment group, with a profile measure across the 12 time intervals. The second analysis used data from the 3 treatment time points, and animals were nested within the interaction effect of sex crossed with treatment group, with a profile measure across the 12 time intervals nested within the 3 time periods. The third analysis was similar to the first, using the recovery data. All three analyses tested for treatment effects, sex differences, treatment group by sex interactions, and these effects crossed with periods (second analysis only) and intervals. Analyses were repeated using transformed rank data to achieve a normal distribution of the residuals. Residuals from the models were tested for normality by the Shapiro-Wilk W-test or the Kolomogorov D-test.

Functional Operational Battery. The battery was comprised of home- cage evaluations (posture, vocalizations, and palpebral closure), handling evaluations (reactivity to general stimuli, signs of autonomic function), open-field behavior (arousal level and gait, urination and defecation frequency, convulsions, tremor, abnormal behaviors, piloerection, and exophthalmos), and reflex assessments (response to visual and auditory stimuli, tail pinch, pupillary function). Animals were also evaluated for forelimb and hindlimb grip strength, landing foot splay, and air righting ability. For landing foot splay, a small dot of paint was applied to each hindpaw. The rat was dropped from a height of 2 ft above a flat surface and the distance between the marks left by the hindpaws was measured in centimeters. To evaluate air righting reflex ability, the rat was held upside down, dropped from a height of 2 ft above a container of bedding, and the landing position was observed. Treatment groups were counterbalanced across test times. The observer performing the evaluation did not know the identity of the animal’s dose group.

Sacrifice and pathology:

All animals were sacrificed by intraperitoneal injection of sodium pentobarbital, and tissues were preserved in situ by transcardial perfusion with phosphate-buffered saline (pH 7.4) followed by 4% paraformaldehyde/1 % glutaraldehyde in the same buffer. Animals were killed at termination of 13 wk of exposure (week 13 terminal sacrifices performed during week 14) or at the completion of the 4-wk recovery period (control and high-dose groups only). A complete macroscopic examination was performed on all animals and 12 organs were weighed: adrenals, brain, heart, kidneys, liver, lung, ovaries, prostate, spleen, testes (with epididymides), thymus, and uterus. The length and width of the brain of each rat was measured.

Thirty-nine tissues were preserved from all animals in all dose groups. Tissues from all animals in the control and high-dose groups were processed, embedded in paraffin, mounted on glass slides, and stained with hematoxylin and eosin for histopathological examination. The kidneys of selected animals were also stained with Mallory-Heidenhain stain. In addition, tissues of the nervous system were fixed for all animals. Brain, spinal cord, ganglia, and spinal nerve roots were processed, embedded in paraffin, mounted on glass slides, and stained with hematoxylin-eosin, Luxol fast blue, and Sevier-Munger stains. Peripheral nerve sections (sciatic, tibial, sural, and optic) were embedded in glycol methacrylate and stained with toluidine blue. Slides of nervous system tissues were examined from animals (6/sex/group) designated through random selection for neuropathology, in the control and high-dose groups sacrificed at the end of 13 weeks of exposure. Specific brain regions examined were forebrain, cerebral cortex, hippocampus, basal ganglia, midbrain cerebellum and pons, and medulla.

Statistics:

Statistical evaluations were performed on the following parameters: body weights, body weight change from wk 0, and food consumption; hematology and clinical chemistry; and organ weights, organ/terminal body weight ratio, and organ/brain weight ratio. Barlett’s test at 1 % significance, two-sided risk level, was used to determine if groups had equal variance. All other tests were conducted at 5% and 1 % significance, two- sided risk level. Parametric procedures were standard one-way analysis of variance (ANOVA) using F distribution for significance. If significant differences among means were indicated, Dunnett’s test was used to determine significant differences from controls. The Kruskal-Wallis test was the nonparametric procedure for testing equality of means, and if differences were indicated, Dunn’s summed rank test was used to determine differences from controls.

A statistical test for trend in the dose levels was also performed, using standard regression techniques with a test for trend and lack of fit where variances were equal orJonckheere’s test for monotonic trend in nonparametric cases.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

All animals survived the treatment period and were sacrificed according to study design at the end of 13 wk or at 18 wk (recovery groups). No test-related observations were noted in the exposure chambers during any exposure period for any treatment groups or during non-exposure periods. From weekly clinical observations, the only apparent treatment-related finding was an increased incidence of red facial staining in both male and female rats in the high dose group. No LAND-2-related ocular disease was observed. All groups showed similar mean body weights, body weight gains, and food consumption.

At wk 13 terminal sacrifice, there was a statistically significant decrease, relative to control values, in hemoglobin (5%), hematocrit (5%), and erythrocytes (7%) in blood of high dose males (data not shown). The hemoglobin was still decreased (4%) after the 28-d recovery period. However, because these differences were small and within the historical range for control animals in this laboratory, they are not considered toxicologically relevant. Clinical chemistry results showed a statistically significant decrease in aspartate aminotransferase (AST 32%) and alanine aminotransferase (ALT 46%) in females of the high-dose group (data not shown) compared to controls. However, several control female rats had elevated AST and ALT relative to the other nine female rats in the group and historical controls. Comparison of ALT and AST values from high-dose females with these elevated concurrent control values produced a statistical significance that is not toxicologically relevant. These results are not considered LAND-2 related.

NEUROBEHAVIORAL STUDIES

Motor Activity. Shapiro-Wilk analysis of data from the predose period indicated that the only statistically significant effects on response pattern occurred in the low-dose group due to inactivity among females for intervals 6—8, and increased activity of males during interval 10 (data not shown). Data from the treatment intervals and the recovery period were analyzed based on the Blom transformed data because the residuals from the model were not normally distributed by the Shapiro—Wilk statistic at the .01 level. There were statistically significant differences in the number (ct < .04) and relative pattern (cx. < .02) of beam breaks among the dose groups over the treatment testing periods. There were expected differences between sexes, and pattern differences across the 1 2 measuring intervals. In the recovery period in which only the room air controls and the high-dose animals were evaluated, there were no dose-group-related differences in response. Overall, dose-group differences did not occur in a dose-related pattern. Although statistically significant, the magnitudes of the differences were not large, and none of the treatment-group differences were larger than differences seen during the pre-dose period.

Functional Operational Battery. No differences were detected in the distance between foot splay for male or female rats in any dose group over any time interval evaluated. Grip strength of both fore- and hindlimbs in general increased from pretest through wk 14 for both sexes in all treatment groups. Values for control and high-dose recovery animals were lower at wk 18 than in previous treatment weeks but similar between the groups. There was no test-material-related effect on any endpoint evaluated within the functional operational battery of tests.

Pathology. At the wk 13 terminal sacrifice there were statistically significant dose-related increases in absolute and relative kidney weights in males of all 3 treatment groups. The kidney weights of high-dose males remained elevated after the recovery period. These increases correlated with microscopic observations of hyaline droplet formation in the proximal convoluted tubules considered to contain an alpha2-microglobulin-hydrocarbon complex, based on positive staining reaction by the Mallory-Heidenhain method, and increase in incidence and severity of nephropathy and dilated tubules at the corticomedullary junction. These microscopic finding are characteristic of ‘light hydrocarbon nephropathy” also known as hyaline droplet nephropathy and are male rat specific and are not considered relevant to humans. Statistically significant increases in absolute and relative liver weights were observed in high-dose male and female rats at wk 13 sacrifice. Differences were not present after the recovery period and had no microscopic correlate. Lung and brain weights were comparable to controls. Lungs were macroscopically and microscopically comparable to controls. Brain length and width measurements showed no test-material-related effects. There were no microscopic findings in the brain, spinal cord, or peripheral nerves that could be attributable to exposure to LAND-2.

Key result

Dose descriptor:

NOAEC

Effect level:

24 300 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: no effects except adaptive response of liver weight to exposure equivalent to 6646 ppm

Critical effects observed:

not specified

Conclusions:

The NOAEC of LAND-2 for subchronic toxicity and neurotoxicity is 6646 ppm.

Executive summary:

The NOAEC of LAND-2 for subchronic toxicity and neurotoxicity is 6646 ppm.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study meets generally accepted scientific principles, acceptable for assessment. Only one dose employed; statistical methods not identified.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Principles of method if other than guideline:

Single concentration repeated dose study for peripheral nerve toxicity.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: not specified
- Weight at study initiation: 308 ± 18 g

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: room air

Remarks on MMAD:

MMAD / GSD: not applicable, vapour

Details on inhalation exposure:

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography and Kitagawa gas detection

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Animals were actually exposed to 2960 ± 200 ppm of Normal-Heptane.

Duration of treatment / exposure:

16 weeks

Frequency of treatment:

12 hours/day, 7 days/week

Remarks:

Doses / Concentrations:
12.47 mg/L (re-calculated; corresponding to 3000 ppm)
Basis:
nominal conc.

No. of animals per sex per dose:

7 males

Control animals:

yes, sham-exposed

Details on study design:

- Post-exposure recovery period: none

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: no data
- Cage side observations included: behaviour


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before and after 4, 5, 12, 16 weeks of exposure


BODY WEIGHT: Yes
- Time schedule for examinations: not further specified, very likely weekly


OTHER:
Neurophysiology: motor nerve conductivity velocity (MCV), distal latency (DL), mixed nerve conduction velocity (MNCV)
- Time schedule: before and at 4, 5, 12, 16 weeks of exposure

Sacrifice and pathology:

GROSS PATHOLOGY: No data
HISTOPATHOLOGY: Yes (one rat): gastrocnemeius and soleus muscles, the dorsal trunk of the tail nerve and the tibial nerve were examined by light and electron microscopy

Statistics:

Employed but not identified by test name.

Clinical signs:

no effects observed

Mortality:

no mortality observed

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:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
No abnormal behavioural changes were observed.

BODY WEIGHT AND WEIGHT GAIN
Body weight gain was statistically significantly depressed (p<0.01) after 8 weeks of exposure compared to controls but gradually increased throughout the experiment, albeit to body weight levels below control values, but not statistically significantly lower.


HISTOPATHOLOGY: NON-NEOPLASTIC
Peripheral nerves, muscles and neuromass junctions, examined microscopically, were normal.

OTHER FINDINGS
Neurophysiology: There were no statistically significant differences in motor nerve conduction velocity, distal latency or mixed nerve conduction velocity in any region of the tail.

Key result

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

12 470 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no effects except reversible body weight changes

Key result

Dose descriptor:

NOAEC

Remarks:

neurotoxicity

Effect level:

12 470 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on lack of adverse treatment-related effects

Critical effects observed:

not specified

Normal-Heptane is not a neurotoxicant in this assay system.

Conclusions:

Normal-heptane is not a neurotoxicant in this assay system.

Executive summary:

Normal-heptane is not a neurotoxicant in this assay system.

Reference 3

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

17 April 1978 - 30 March 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions. Limited documentation on animal housing, only 2 concentrations tested, exposure duration 84 days, no ophthalmological examination.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass. 01887
- Age at study initiation: males 6 wks, females 7 wks
- Weight at study initiation: males 185 g mean (range 165-217 g); females 162 g mean (range 138-189)
- Fasting period before study: no
- Housing: paired in chamber, individual out of chamber
- Diet (e.g. ad libitum): Standard laboratory pellet diet (Purina Laboratory Chow) ad libitum (out of chamber only)
- Water (e.g. ad libitum): ad libitum (out of chamber only)
- Acclimation period: 13 days

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: not applicable, vapour

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel and glass chambers with 1 cubic meter total volume (760 L effective volume)
- Source and rate of air:
- Method of conditioning air:
- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber:
- Air flow rate: 134 L/min
- Air change rate: 8 per hour
- Method of particle size determination: not applicable, vapour


TEST ATMOSPHERE
- Brief description of analytical method used: Atmospheric sampling was performed using a Wilks Scientific Corp., Miran 1A Ambient Air Analyzer (long pathlength infrared). A calibration curve relating the absorption to the airborne concentration of the test material was prepared. On each exposure day, three samples were drawn from each exposure chamber (at about 1, 3, and 5 hours) and the exposure concentration calculated by comparing the absorption of this sample to the standard curve.
In addition, the composition of the test atmosphere was analyzed for homogeneity by gas chromatographic analysis of several charcoal-trapped vapour samples collected from each chamber during the 12-wk exposure period

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test atmosphere was analysed for concentration and homogeneity by measurement of the infrared spectrum and by gas chromatographic analysis, respectively. Based on the infrared analysis the animals were exposed to cumulative mean concentrations of 385 and 1200 ppm, respectively. Gas chromatographic analysis of the chamber atmosphere demonstrated that the test material composition was representative of the initial sample.

Duration of treatment / exposure:

12 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
400, 1200 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

35

Control animals:

yes, sham-exposed

Details on study design:

- Rationale for animal assignment (if not random): assigned to group by weight

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: incidence of abnormal signs


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly (full recorded physical assessment)


BODY WEIGHT: Yes
- Time schedule for examinations: weekly, from 5 days prior to exposure through termination


WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes (retro-orbital sinus)
- Time schedule for collection of blood: 4, 8, 12 weeks
- Anaesthetic used for blood collection: Yes (exsanguination under ether anesthesia)
- Animals fasted: Yes (fasted overnight prior to bleeding)
- How many animals: 10/sex/group (4 and 8 weeks), 15/sex/group (12 weeks, all survivors)
- Parameters examined: hemoblobin, hematocrit, erythrocyte count, clotting time, total and differential leukocytes


CLINICAL CHEMISTRY: Yes (retro-orbital sinus)
- Time schedule for collection of blood: 4, 8, 12 weeks
- Animals fasted: Yes (exsanguination under ether anesthesia)
- How many animals: 10/sex/group (4 and 8 weeks), 15/sex/group (12 weeks, all survivors)
- Parameters examined: blood urea nitrogen, serum glutamic pyruvic transaminase (SGPT), glucose, alkaline phosphatase


OTHER:
Organ weights and organ/body weight ratios determined in animals sacrificed at 4, 8 and 12 weeks (adrenals, brain (sans pituitary), gonads, kidneys, liver, lungs)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes: adrenals, brain (without pituitary), gonads, kidneys, liver, lungs
HISTOPATHOLOGY: Yes (control and 1200 ppm group): adrenals (2), bone marrow (sternal), brain (2 sections), eye, gonad, heart (with coronary vessels) intestine, colon, duodenum, ileum, kidneys (2), liver (2 sections), lung (2 sections), lymph node (mesenteric), mammary gland, pancreas, pituitary, salivary gland, skeletal muscle, skin, spinal cord (cervical), spleen, stomach, thyroid, trachea, urinary bladder, uterus/prostate, gross lesions, tissue masses

Statistics:

Body weight, hematology and clinical chemistry parameters, organ weights and organ/body weight ratios were statistically evaluated. Mean values for all treatment groups were compared to the control group at each time interval (4, 8, and 12 weeks). Hematology and clinical chemistry parameters were compared by the F-test and Student's t-test. When variances differed significantly (F-test), Student's t-test was appropriately modified using Cochran's approximation (t'). Body weight, organ weight and organ/body weight ratios were compared to control according to Dunnett.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

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:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

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:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No treatment-related mortality occured (1 male of the 1200 ppm group was accidentally killed).
Several animals in all groups exhibited dry rales and red and mucoid nasal discharge (more numerous in the treated groups, but not clearly treatment-related), moist rales, excessive lacrimation, hair loss and chromodacryorrhea were found in a limited number of animals in all groups (not treatment-related)
1200 ppm: singular occurrences of excessive salivation, laboured, irregular breathing; yellow staining of the anogenital fur in 6 males and 35 females from wk 3 through 12
400 ppm: yellow staining of the anogenital fur in 2 females
Control: singular occurrences of excessive salivation and bleeding inside the ear; a limited number of animals with brown staining of the ano-genital region and soft stool; three observations (in one animal) of an abnormally dark red or red and yellow eye

BODY WEIGHT AND WEIGHT GAIN
1200 ppm: mean body weights in males significantly higher at wk 2 and significantly lower (p?0.05) from wk 8 through 11 than in controls
400 ppm: mean body weight and weight gains in males similar to control throughout the study, except wk 2 (significantly higher, p?0.01), in females mean body weights significantly depressed (p?0.01 and p?0.05) at wk 5 through 8.

HAEMATOLOGY
Several statistically significant (p < 0.05 and p < 0.01) decreases in mean hematocrit values of males and females of both treated groups at wk 4 and 8, statistically significant decreases (p?0.05) in mean hemoglobin values at wk 8 in the males of both treated groups and the females of the 400 ppm group at wk 4. Mean red blood cell values were significantly decreased in 1200 ppm males at wk 8 and 400 ppm females at wk 12. Since all values were within normal biological limits, these findings were not considered to be treatment-related.

CLINICAL CHEMISTRY
Mean SGPT levels were significantly (p?0.01) depressed in 1200 ppm males at wk 4, 400 and 1200 ppm males at wk 8, and in 1200 ppm females at wk 12. Mean blood urea nitrogen levels were significantly increased in the males of both treated groups at wk 8. Mean glucose levels were significantly (p?0.01 or p?0.05) increased in 400 ppm males at wk 8, decreased in 1200 ppm males at wk 12, and decreased in 1200 ppm females at wk 4 and 12. The observed effects were not considered to be treatment-related.

ORGAN WEIGHTS
Mean kidney weights and kidney/body weight ratios were significantly (p?0.05) higher in the 1200 ppm males at wk 8. In the 400 ppm males these values were also elevated, but not statistically significant. At wk 12, mean kidney weights and kidney/body weight ratios for 400 and 1200 ppm males were significantly (p?0.01) elevated, indicating a treatment-related response. The only other statistically significant (p?0.05) findings were elevated mean adrenal/body weight ratios for the 1200 ppm males at wk 4 and the 400 ppm females at wk 12.

GROSS PATHOLOGY
Microscopic evaluation of organs and tissues from the control and high level exposure groups revealed a mild tubular injury in the kidneys of some exposed male rats sacrificed after exposure for 8 and 12 wk. Other changes were unrelated to group or sex and were considered to be spontaneous.

HISTOPATHOLOGY: NON-NEOPLASTIC
See Gross Pathology

Key result

Dose descriptor:

NOAEC

Effect level:

1 200 ppm (nominal)

Sex:

male

Basis for effect level:

other: overall effects

Critical effects observed:

not specified

Significantly increased mean kidney weights and kidney/body weight ratios were observed in males at 400 ppm, which were considered to be treatment-related by the authors of the study.

The kidney was confirmed as potential target organ for the test material-induced toxicity by the observation of mild tubular injury found in the histopathological examination of high dose males.

The fact, that these effects were strictly limited to male rats and that the test substance belongs to a category of substances which are known for their ability to induce nephropathy in male rats due to their exclusive expression of alpha-2u-globulin, the protein known to play the crucial role in the onset of this disease, the observed effects in the kidney have to be regarded as species-specific and therefore not relevant for risk assessment in humans. Therefore, these effects were not considered for the determination of the NOAEC.

Conclusions:

In a 12 -week inhalation study with rats the test substance hydrocarbons, C7 -C9, isoalkanes was tested. Significantly increased mean kidney weights and kidney/body weight ratios were observed in males at 400 ppm, which were considered to be treatment-related by the authors of the study.

The kidney was confirmed as potential target organ for the test material-induced toxicity by the observation of mild tubular injury found in the histopathological examination of high dose males.

The fact, that these effects were strictly limited to male rats and that the test substance belongs to a category of substances which are known for their ability to induce nephropathy in male rats due to their exclusive expression of ?2u-globulin, the protein known to play the crucial role in the onset of this disease, the observed effects in the kidney have to be regarded as species-specific and therefore not relevant for risk assessment in humans. Therefore, these effects were not considered for the determination of the NOAEC.

Renal effects were strictly limited to males, therefore the authors concluded an ?2u-globulin-related mechanism for the observed nephropathy. The observation was not considered for determination of the NOAEC.

Executive summary:

In a 12 -week inhalation study with rats the test substance hydrocarbons, C7-C9, isoalkanes was tested. Significantly increased mean kidney weights and kidney/body weight ratios were observed in males at 400 ppm, which were considered to be treatment-related by the authors of the study. The kidney was confirmed as potential target organ for the test material-induced toxicity by the observation of mild tubular injury found in the histopathological examination of high dose males. The fact, that these effects were strictly limited to male rats and that the test substance belongs to a category of substances which are known for their ability to induce nephropathy in male rats due to their exclusive expression of ?2u-globulin, the protein known to play the crucial role in the onset of this disease, the observed effects in the kidney have to be regarded as species-specific and therefore not relevant for risk assessment in humans. Therefore, these effects were not considered for the determination of the NOAEC. Renal effects were strictly limited to males, therefore the authors concluded an ?2u-globulin-related mechanism for the observed nephropathy. The observation was not considered for determination of the NOAEC.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

12 470 mg/m³

Study duration:

subchronic

Experimental exposure time per week (hours/week):

84

Species:

rat

Quality of whole database:

Three key read across and one supporting read across sub-chronic toxicity studies from structrual analogues available for assessment.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration

Critical effects observed:

not specified

Reference 2

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There is no data available for Isoheptane. However, data is available for a structural analogues, Heptane; Hydrocarbons, C7 -C9, isoalkanes; and Light Alkyl Naphtha Distillate and presented in the dossier. This data is read across to Isoheptane based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

Oral

There is no data available for Isoheptane. However, in order to comply with standard information requirements for Annex X substances, an OECD Guideline 90-day sub-chronic (OECD 408) toxicity test is proposed for structural analogue Hydrocarbons, C7-C9, isoalkanes, (EC# 921 -728 -3). The testing proposal for the same has been presented in the lead registrant dossier for this substance already submitted to ECHA. This study will be conducted subsequent to ECHA's approval and this endpoint will be updated upon completion of the above study.

Inhalation

Heptane

In a comparative study designed to evaluate peripheral nerve toxicity of n-pentane, n-hexane and n-heptane male rats were exposed to 0 or 3000 ppm 12 hours/day, 7 days/week, for 16 weeks. The conduction velocity of tail nerves was measured to determine the functional status of the peripheral nerves. For animals exposed to n-heptane, the body weight gain was statistically significantly depressed (p<0.01) after 8 weeks of exposure compared to control animals but gradually increased throughout the experiment to body weight levels below control values but not statistically significantly lower. No abnormal behavioral changes were observed. There were no statistically significant differences in motor nerve conduction velocity, distal latency or mixed nerve conduction velocity in any region of the tail for n-heptane exposed rats. Peripheral nerves, muscles and neuromass junctions, examined microscopically were normal. Only n-hexane induced neuropathy. The NOAEC was determined to be > 3000 ppm corresponding to 12470 mg/m³ (Ono et al., 1979 and Takeuchi et al., 1980, 1981).

Rats were exposed to n-heptane via whole body inhalation at 0, 398 and 2970 ppm for 26 weeks with a subsequent 2-week recovery period conducted similar to OECD 413 (Shell, 1980). There were no treatment-related deaths during the study. The only treatment-related observations were labored breathing or rapid breathing and slight prostration during the first week of exposure and anogenital fur and dry rales during weekly observations. The in chamber signs were generally more numerous and severe at the higher concentration and appeared to abate by the second week of the study. No treatment-related effects were observed for body weight, hematology or urinalysis. Serum alkaline phosphatase was significantly elevated in high-concentration females and slightly elevated in low-concentration females. All other clinical chemistry values appeared normal with the exception of one high-concentration male whose serum glutamic pyruvic transaminase and serum alkaline phosphatase levels were markedly elevated when compared to all other exposed male rats. Proteinuria, elevated specific gravity and ketones were observed but were not considered related to treatment. Clinical pathology results had no correlate in histopathology. The NOAEC was 2970 ppm corresponding to 12200 mg/m³.

Light alkylate naphtha distillate

 

A 13-week inhalation toxicity study was conducted using wholly vaporized light alkylate naphtha distillate (LAND-2) generated in nitrogen (Schreiner et al., 1998). Male and female rats were exposed by inhalation in whole-body exposure cages 6 hours/day, 5 days/week for 13 weeks at analytical concentrations of 0, 668, 2220, and 6646 ppm. All animals survived the treatment period and were sacrificed according to study design at the end of week 13 or 18 (recovery group). No test-related observations were noted in the exposure chambers during any exposure period for any treatment groups or during non-exposure periods. From weekly clinical observations, the only apparent treatment-related finding was an increased incidence of red facial staining in both male and female rats in the high dose group. At week 13, there were statistically significant dose-related increases in absolute and relative kidney weights in males of all 3 treatment groups. The kidney weights of high-dose males remained elevated after the recovery period. These increases correlated with microscopic observations of hyaline droplet formation in the proximal convoluted tubules considered to contain an alpha2-microglobulin-hydrocarbon complex as well as an increase in incidence and severity of nephropathy and dilated tubules at the corticomedullary junction. These microscopic finding are characteristic of "light hydrocarbon nephropathy" also known as hyaline droplet nephropathy and are male rat specific. Therefore these effects are not considered to be relevant to humans. Statistically significant increases in absolute and relative liver weights were observed in high-dose male and female rats at week 13 after sacrifice. Differences were not present after the recovery period and had no microscopic correlate. Thus, the NOAEC for systemic toxicity was 8117 mg/m³ corresponding to 2200 ppm.

 

Hydrocarbons C7-C9, isoalkanes

 

Systemic toxicity of hydrocarbons, C7-C9, iso-alkanes was assessed in a 12-week inhalation toxicity study in rats (ExxonMobil Chemical,1979). In this study, repeated exposure to 400 or 1200 ppm of the test substance for 6 hours/day, 5 days/week, for 12 weeks resulted in male rat kidney effects consistent with the alpha-2µ-globulin-induced nephropathy in male rats. There was no treatment-related mortality and clinical findings were unremarkable. Under the test conditions, the NOAEC (excluding male rat nephropathy) was determined to be >1200ppm.

 

The fact, that alpha-2µ-globulin-induced nephropathy was strictly limited to male rats and that the test substance belongs to a category of substances which are known for their ability to induce nephropathy in male rats due to their exclusive expression of alpha-2µ -globulin, the protein known to play the crucial role in the onset of this disease, the observed effects in the kidney have to be regarded as species-specific and are not relevant for risk assessment in humans. Therefore, additional experimental data were used to evaluate repeated dose toxicity via inhalation.

Justification for classification or non-classification

Based on available substance specific and read across data, Isoheptane does not meet the criteria for classification for repeated dose toxicity (STOT-RE) under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).