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Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
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.
Objective of study:
distribution
toxicokinetics
Principles of method if other than guideline:
3 day toxicokinetic study in rats.
GLP compliance:
not specified
Radiolabelling:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Møllegaard A/S, L1, Skensved, Denmark
- Age at study initiation: 40-50 days
- Weight at study initiation: 150 - 200 g
- Individual metabolism cages: no
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 4-6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±1 during exposure
- Humidity (%): 70 ± 20 during exposure
- Photoperiod (hrs dark / hrs light): 10/14
Route of administration:
inhalation: vapour
Vehicle:
unchanged (no vehicle)
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMPER DESCRIPTION
- Exposure apparatus: conical 0.7 m³ inhalation chambers with a glass front door and walls, accommodating 4 cages each containing 4 rats each.
TYPE OF INHALATION EXPOSURE: whole body

Dynamic exposure of anomals was performed in conically shaped 0.7 m3 steel chambers with glass front door and walls as described elsewhere (Walseth & Nilsen 1984). The concentration of hydrocarbons in the inhalation chambers was monitored automatically by on-line gas chromatography, Concentrations were measured in 15 min intervals. Altogehter 44 measurements at steady state each day.
Duration and frequency of treatment / exposure:
1, 2, and 3 days, 12 hours/day
Remarks:
Doses / Concentrations:
0.52 mg/L (corresponding to 100 ppm)
No. of animals per sex per dose / concentration:
4 per exposure duration
Control animals:
no
Positive control reference chemical:
not applicable
Details on study design:
The aimed concentration was 1000 ppm. All exposures were performed at daytime for 12 hr (8 a.m. - 8 p.m.). Measurements were done on days 1, 2, and 3 after 12 hr exposure. Animals were one by one removed, killed, and blood and organs obtained within 3 min after removal from exposure chamber.
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: blood, brain, liver, kidneys, perirenal fat
- Time and frequency of sampling: day 1, 2, and 3 within 3 min of removal from inhalation chamber
Preliminary studies:
Not performed
Details on absorption:
Not addressed.
Details on distribution in tissues:
Normal-Heptane demonstrated moderate concentrations in kidneys. In perirenal fat, concentration are highest, however decreasing with lasting exposure. This is in contrast to other n-alkanes, which showed increasing concentrations.
Key result
Test no.:
#1
Transfer type:
blood/brain barrier
Observation:
distinct transfer
Details on excretion:
Not addressed.
Metabolites identified:
not measured

Blood and tissue values in µmol/kg (with SD):

day

1

2

3

blood

2.4 ± 0.8

2.9 ± 0.9

2.1 ± 0.2

Brain

5.2 ± 0.8

6.9 ± 0.6

6.2 ± 1.0

liver

1.6 ± 0.6

2.3 ± 0.1

1.5 ± 0.1

kidney

15.7 ± 4.2

15.2 ± 2.6

17.1 ± 3.0

fat

140 ± 14

127 ± 28

121 ± 10

Conclusion:

n-Heptane was found in moderate concentrations in the kidneys and only in marginal concentrations in blood, brain and liver. In perirenal fat, concentrations were the highest, however, decreasing with lasting exposure.

Conclusions:
Interpretation of results: other: see conclusions below
Normal-Heptane was found in moderate concentrations in the kidneys and only in marginal concentrations in blood, brain and liver. In perirenal fat, concentrations were the highest, however, decreasing with lasting exposure.
Executive summary:

Normal-Heptane was found in moderate concentrations in the kidneys and only in marginal concentrations in blood, brain and liver. In perirenal fat, concentrations were the highest, however, decreasing with lasting exposure.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given.
Objective of study:
absorption
Principles of method if other than guideline:
The comparative rates of uptake of 19 hydrocarbon (including heptane) vapours by rats were determined by a dual-column gas chromatography method.
GLP compliance:
not specified
Radiolabelling:
no
Species:
rat
Strain:
other: F344/N
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Lovelace ITRI colony
- Age at study initiation: 12 to 15 weeks
- Weight at study initiation: mean 298 g
- Housing: Before exposure, animals were housed in polycarbonate cages (2 animals/cage) with hardwood chip bedding and filter caps.
- Individual metabolism cages: yes/no
- Diet (e.g. ad libitum): AM. Food (Lab Blox, Allied Mills, Chicago, IL, USA); ad libitum
- Water (e.g. ad libitum): water from bottles with sipper tubes; ad libitium


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22.2
- Humidity (%): 20 to 50
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
inhalation: vapour
Vehicle:
unchanged (no vehicle)
Details on exposure:
TYPE OF INHALATION EXPOSURE: nose only

The exposure apparatus, exposure procedures, and method for handling data were described in detail by Dahl et al., 1987 (Amer Ind Hyg Assoc J 48:505-510)

The vapour was pumped at 400 mL/min from a Teflon supply bag through one sampling loop of a dual-column gas chromatograph, past the nose of a rat confined in a nose-only exposure tube, through the second sampling loop of the dual column gas chromatograph and, finally, into an exhaust bag.
The amount of hydrocarbon vapour absorbed was calculated from the output of the gas chromatograph and the flow rate past the rat´s nose. Rat exposures were preceded by a 10-15 min pre-exposure equilibration/calibration period without a rat in the system.


Duration and frequency of treatment / exposure:
80 min for 5 consecutive days (totally 450 min)
Remarks:
Doses / Concentrations:
on day 1: 1 ppm
on day 2: 10 ppm
on day 3: 100 ppm
on day 4: 1000 ppm
on day 5: 5000 ppm
See also "any other information on materials and methods".
No. of animals per sex per dose / concentration:
at 100 ppm: 10 male rats (not further specified)
Control animals:
not specified
Positive control reference chemical:
no data
Details on study design:
All animals were exposed for 80 min/day for 5 consecutive days with escalation of vapour concentration daily.
Details on dosing and sampling:
During the exposures (80 min/day), respiratory and gas chromatographic data were collected at 1 min intervals.
Statistics:
The calculation of vapour uptake from gas chromatography data see attached document.
Details on absorption:
Only data from one exposure at 100 ppm were available. Uptake of inhaled heptane vapour was 4.5 ± 0.3 nmol/kg/min/ppm (N=10). The value is given for uptake during minutes 60 to 70 from start of exposure.
Conclusions:
Interpretation of results: bioaccumulation potential cannot be judged based on study results
Taking into account all data of the report, a number of trends relating uptake to chemicals properties were observed. Among these, highly volatile hydrocarbons are less well-absorbed than less volatile hydrocarbons; unsaturated compounds are better absorbed than saturated ones; and branched hydrocarbons are less well-absorbed than unbranched ones. These trends can be used to predict relative uptake rates within classes of hydrocarbons.
Executive summary:

Taking into account all data of the report, a number of trends relating uptake to chemicals properties were observed. Among these, highly volatile hydrocarbons are less well-absorbed than less volatile hydrocarbons; unsaturated compounds are better absorbed than saturated ones; and branched hydrocarbons are less well-absorbed than unbranched ones. These trends can be used to predict relative uptake rates within classes of hydrocarbons.

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
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.
Principles of method if other than guideline:
Guidance for conduct of the in vitro dermal kinetic experiments was posted in the United States FR, April 26, 2004 (Volume 69, Number 80), pages 22402-22441, "In vitro dermal absorption rate testing of certain chemicals of interest to the occupational safety and health administration".
GLP compliance:
not specified
Radiolabelling:
yes
Species:
other: in vitro human skin model
Strain:
other: in vitro human skin model
Sex:
not specified
Details on test animals or test system and environmental conditions:
not applicable
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Duration of exposure:
up to 60 min
Doses:
infinite dose: 1200 µL/cm2
10 min: 20 µL
60 min: 20 µL
No. of animals per group:
in vitro human skin model
Control animals:
no
Details on in vitro test system (if applicable):
see "any other information on materials and methods"
Signs and symptoms of toxicity:
not examined
Dermal irritation:
yes

The flux values for Normal-Heptane and the 10 and 60 min short-term absorption values (the quantity of chemical remaining in the skin plus that portion that had penetrated the skin was detected in the receptor fluid) were 63.2 µg/cm2/h, 113 µg/cm2/h (for the 10 min flux) and 22.1 µg/cm2/h (for the 60 min flux). Therefore, 10 min flux value for Normal-Heptane (based on both the amount in the skin and the receptor solution) was greater than the flux measured in a similar manner over 60 min.

Skin integrity measurements were taken before and after each experiment. All reporting laboratories (Normal-Heptane: Hask, DuPont Haskell Laboratory, USA) either used tritiated water permeability or electrical resistance (impedance) to confirm skin integrity; for consistency and to ease comparisons, all tritiated Kp values were converted to electrical impedance values expressed in kilo-ohms (k-ohms). A ratio of post- to pre-test impedance of "1" indicates that the skin barrier did not change over the course of the experiment. In the Kp experiments, skin exposed to Normal-Heptane had a damage ratio of 0.57, confirming that approx. 43% of the skin barrier function was lost due to exposure to Normal-Heptane. The barrier properties for the skin in the short-term experiments were given as the ratios of 0.90 for 10 min and 0.88 for 60 min.

Recovery of the applied dose, based on liquid scintillation count data when the radioactive chemical form was spiked into the non-radiolabeled chemical, was 95.5% (for the Kp experiment), 54.0% (for the 10 min experiment) and 110.0% (for the 60 min experiment).

At the end of the Kp experiment, the portion of Normal-Heptane in the skin (0.01%) was less than the portion in the receptor solution (0.12%). The portion of Normal-Heptane in the donor solution (wash) was 95.4%. In contrast to the Kp experiment, the skin (0.14%) retained a larger percentage of Normal-Heptane following a 10 min exposure. The portion of Normal-Heptane in the donor solution (wash) was 6.84% at 10 min. The greater portion of the applied dose remaining in the skin at 10 min suggests that partitioning into the skin from the donor solution is the driver of penetration with this brief exposure. After the 60 min experiments, there was also a larger percentage of n-heptane in the receptor solution (0.12%) than in the skin (0.06%). The increased proportion of Normal-Heptane detected in the receptor solution illustrates and confirms the movement of the chemical from the skin into the receptor solution.

Conclusions:
Under the test conditions, Normal-Heptane was able to penetrate the skin. During prolonged exposure, the penetration of the skin was aggravated, since the exposure to n-heptane simultaneously reduced skin barrier function.
Executive summary:

Under the test conditions, Normal-Heptane was able to penetrate the skin. During prolonged exposure, the penetration of the skin was aggravated, since the exposure to Normal-Heptane simultaneously reduced skin barrier function.

Description of key information

Short description of key information on bioaccumulation potential result:

See toxicokinetics, metabolism and distribution.

Short description of key information on absorption rate:

Under dermal in vitro test conditions, heptane was able to penetrate the skin. During prolonged exposure, the penetration of the skin was aggravated, since the exposure to heptane simultaneously reduced skin barrier function.

Due to the experimental setup, e. g. undepletable reservoir of test substance and therefore absence of any evaporation, the dermal penetration factors reported by Fasano and McDougal (2008) are very conservative. In contrast, when using a diffusion cell, which is a more realistic setup for volatile subsances like hydrocarbon solvents, dermal penetration rates of 0.1 µg/cm2/h and 0.0005 µg/cm2/h were obtained for heptane and octane, respectively (Tsuruta et al., 1982).

Key value for chemical safety assessment

Additional information

The uptake of inhaled Normal-Heptane vapors was explored by Dahl et al. (1988) in male rats exposed for 5 consecutive days, 80 min/day with escalation of vapor concentration daily (from 1 ppm up to 5000 ppm). During the exposures, respiratory and gas chromatographic data were collected at 1 min intervals. For Normal-Heptane, only data from one exposure at 100 ppm were available. Uptake of inhaled heptane vapor was 4.5 ± 0.3 nmol/kg/min/ppm (n = 10). The value is given for uptake during minutes 60 to 70 from the start of exposure of the experiment.

Toxicokinetic properties of Normal-Heptane were investigated in rats during inhalation of 100 ppm of the hydrocarbon for 3 days, 12 hours/day (Zahlsen et al., 1992). The concentration of Normal-Heptane was measured by head space gas chromatography in blood, brain, liver, kidneys and perirenal fat. Normal-Heptane was found in moderate concentrations in the kidneys and only in marginal concentrations in blood, brain and liver. In perirenal fat, concentrations were the highest, however, decreasing with lasting exposure. This is in contrast to other n-alkanes, which showed increasing concentrations.

Partition coefficients of Normal-Heptane were determined in human blood and tissues by Perbellini et al. (1985). The solubility of heptane was tested in blood, saline, olive oil and in the most important human tissues (lung, kidney, liver, brain, muscle, heart, and fat). The solubility of Normal-Heptane in saline was low and very high in olive oil, displaying a partition coefficient of 452 (20.0 SD). The partition coefficients were therefore high in fat and fatty tissues compared to the other examined tissues.

Alkanes that are metabolized to gamma diketones can produce peripheral neuropathy in experimental animals and man. Perbellini et al. (1986) have demonstrated that Normal-Heptane does not generate neurotoxic metabolites or produce them in quantities too low to produce neurotoxic effects. Exposure of male rats to 1800 ppm Normal-Heptane for 6 hours only produced the major urinary metabolites 2-heptanol and 3-heptanol. 2,5-Heptanedione, a potentially neurotoxic metabolite was only present in a low concentration (4.4 µg/24 hours) in the urine, approximately 0.8% of urinary metabolites. Other metabolites included 2- or 3-heptanone and gamma valerolactone. At the end of exposure, the amount of 2-heptanol in blood and tissue (liver, muscle, kidney, and nervous tissue) was between 0.2 and 2.0 mg/L. The concentration of the parent compound Normal-Heptane was 5.7 mg/L in blood and 10 to 25.6 mg/L in tissues. 24 hours after the end of exposure, no quantifiable amounts were present in blood or tissue, demonstrating rapid clearance of Normal-Heptane and its metabolites.

 

Discussion on bioaccumulation potential result:

See toxicokinetics, metabolism and distribution.

Discussion on absorption rate:

Fasano and McDougal (2008) described the procedures for determination of a permeability coefficient (Kp) and two short-term dermal absorption rates at 10 and 60 min. The flux values for heptane and the 10 and 60 min short-term absorption values (the quantity of chemical remaining in the skin plus that portion that had penetrated the skin was detected in the receptor fluid) were 63.2 µg/cm2/h, 113 µg/cm2/h (for the 10 min flux) and 22.1 µg/cm2/h (for the 60 min flux). Therefore, the 10 min flux value for heptane (based on both the amount in the skin and the receptor solution) was greater than the flux measured in a similar manner over 60 min.

Skin integrity measurements were taken before and after each experiment. A ratio of post- to pre-test impedance of "1" indicates that the skin barrier did not change over the course of the experiment. In the Kp experiments, skin exposed to Normal-Heptane had a damage ratio of 0.57, confirming that approx. 43% of the skin barrier function was lost due to exposure to heptane. The barrier properties for the skin in the short-term experiments were given as the ratios of 0.90 for 10 min and 0.88 for 60 min. At the end of the Kp experiment, the portion of Normal-Heptane in the skin (0.01%) was less than the portion in the receptor solution (0.12%). The portion of Normal-Heptane in the donor solution (wash) was 95.4%. In contrast to the Kp experiment, the skin (0.14%) retained a larger percentage of Normal-Heptane following a 10 min exposure. The portion of Normal-Heptane in the donor solution (wash) was 6.84% at 10 min. The greater portion of the applied dose remaining in the skin at 10 min suggests that partitioning into the skin from the donor solution is the driver of penetration with this brief exposure. After the 60 min experiments, there was also a larger percentage of heptane in the receptor solution (0.12%) than in the skin (0.06%). The increased proportion of Normal-Heptane detected in the receptor solution illustrates and confirms the movement of the chemical from the skin into the receptor solution. Under the test conditions, Normal-Heptane was able to penetrate the skin. During prolonged exposure, the penetration of the skin was aggravated, since the exposure to heptane simultaneously reduced skin barrier function.