Gas oils (petroleum), straight-run

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

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

Adequacy of study:

key study

Study period:

1982-08-09 to 1983-01-24

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study is classified as reliable with restrictions because there is no GLP statement, but the study was generally conducted in accordance with OECD 413 guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Diesel Fuel

IUPAC Name:

Diesel Fuel

Constituent 2

Reference substance name:

most likely 68334-30-5

IUPAC Name:

most likely 68334-30-5

Test material form:

aerosol dispenser: not specified

Remarks:

migrated information: aerosol

Details on test material:

Substance represents the marketed fuel product containing straight-run gas oil substances being registered (i.e., substances being produced in a refinery as petrochemical intermediates).

- Name of test material (as cited in study report): Diesel obscurant aerosol
- Test substance: Diesel fuel
- Physical state: Liquid
- Analytical purity: Not reported
- Lot/batch No.: Not reported
- Expiration date of the lot/batch: Not reported
- Stability under test conditions: Not reported
- Storage condition of test material: In the Oak Ridge Laboratories respository at 5 °C

Test animals

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, Massachussetts
- Age at study initiation: 18 to 21 weeks old
- Weight at study initiation: Approximately 500 grams for males and 260 to 290 grams for females
- Housing: Individaully in hanging, stainless steel, wire mesh cages
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 10 to 13 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not reported
- Humidity (%): Not reported
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

IN-LIFE DATES: From: 1982-08-09 To: 1983-01-24

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: Ranged from 0.43 microns to 0.75 microns with a standard geometric deviation of 1.4 to 1.7

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Method of holding animals in test chamber: 1.5 m3 New York University style inhalation chambers
- Source and rate of air: Not reported
- Method of conditioning air: Not reported
- System of generating particulates/aerosols: Vycor heater
- Temperature, humidity, pressure in air chamber: 20 to 24 degrees Celcius, 70% humidity, and air pressure not reported
- Air flow rate: Only specified as constant
- Air change rate: Not reported
- Method of particle size determination: Cascade impactation at various times during the experiment
- Treatment of exhaust air: Not reported

TEST ATMOSPHERE
- Brief description of analytical method used: Infrared backscatter probes and periodic filter samples taken for gravimetric determination, which were also analyzed by high performance liquid chromatography and gas chromatography
- Samples taken from breathing zone: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The gravimetric data indicate that all concentrations were greater than the target concentrations by 40%, 17%, and 14% for the 250, 750, and 1500 mg/m3 target concentrations respectively; however, the backscatter probes found the concentration of the 250 mg/m3 group to be lower than the target concentration. This was considered a result of the vapour concentration and aerosol concentrations were considered to be close enough to the target to be acceptable.

Duration of treatment / exposure:

4hrs/day for 13 weeks

Frequency of treatment:

Twice a week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 0.25, 0.75, or 1.50 mg/L
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 0.35, 0.88, 1.71 mg/L
Basis:
analytical conc.

No. of animals per sex per dose:

24 for sham control and treatment groups and 12 for untreated controls

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: This was phase 3 of the testing protocol. Phase 1 and 2 were acute and short-term testing. Doses were based on the previous tests.
- Rationale for animal assignment (if not random): Not reported
- Rationale for selecting satellite groups: Not reported
- Post-exposure recovery period in satellite groups: 2 months
- Section schedule rationale (if not random): Not reported

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During exposure and on removal from the chamber
- Cage side observations were not reported.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At end of exposure and after 2 month recovery
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: Six animals per sex per treatment
- Parameters checked included red blood cell counts, white blood cell counts, and haematocrit

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Terminal sacrifice (immediately after exposure and after 2-month recovery)
- Animals fasted: No data
- How many animals: Six animals per sex per treatment
- Parameters checked in table 1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to study initiation, at various time points over the 13 week exposure period, and once a month during the 2-month recovery
- Dose groups that were examined: All groups
- Battery of functions tested: other: Breathing frequency and startle response

OTHER: The number of alveolar macrophages in pulmonary lavage fluid and pulmonary function were measured.

Sacrifice and pathology:

GROSS PATHOLOGY: No data
HISTOPATHOLOGY: Yes (see table 2)

Other examinations:

Select organs were weighed (Table 2).

Statistics:

Analysis of variance

Results and discussion

Results of examinations

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):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: There were no deaths during the exposure phase or during the 2-month recovery period. Animals were described as inactive during treatment but no overt clinical signs were present.

BODY WEIGHT AND WEIGHT GAIN: Body weight was decreased in both the sham control and the diesel-exposed groups relative to animal room controls at the start of exposure (that is, when the animals were first introduced into the chambers). Exposed animals exhibited a reduced weight gain (relative to the sham control group) until the start of the fourth week of treatment (statistically significant for mid- and high dose males and all exposed females), after which male body weight increased while body weights for females remained relatively static. Terminal body weights (after 25 exposures) were significantly decreased by 7%, 13% and 11% in low-, mid- and high-dose males and by 11%, 17% and 16% in the corresponding groups of females, relative to the sham controls (Table 1). Body weights for exposed males were comparable to the sham control group by the third week of the recovery period, whereas statistically significant decreases remained in mid- and high-dose females until recovery weeks 7 and 5, respectively.

FOOD CONSUMPTION: Food intake was significantly decreased by approximately 10-15% in mid- and high dose animals (no difference between the sexes) during study weeks 4-12, but did not differ from that of the sham control group thereafter.

HAEMATOLOGY: There were no treatment-related effects on red blood cell counts, white blood cell counts, or haematocrit.

CLINICAL CHEMISTRY: Some clinical chemistry parameters were apparently altered in high-dose animals (LDH, cholesterol and creatinine in females at study termination; LDH in males 2 month post-exposure), however the report discounts the biological relevance of the findings and the data are not reported.

NEUROBEHAVIOUR: Breathing frequency was indistinguishable between sham control and exposed animals during the test and recovery periods. Results from the startle reflex tests showed that reaction time was statistically significantly increased in high- and mid-dose males immediately after exposure, and in mid-dose females immediately before exposure, however the magnitude of the alteration (2 msec) was small and considered of doubtful toxicological relevance by the study authors. In contrast, statistically significant increases in peak time were present in high dose males (2-4 msec greater than sham controls) at all of the time points investigated, intermittently in mid-dose males (after the 14th exposure, before the 26th exposure, at one month post-exposure; 2-5 msec) and intermittently in low dose males (after the 14th exposure and at the one month into the recovery phase; 2-3 msec). Similar statistically significant increases in peak time (increased 2-4 msec) were also present in high- and mid-dose females and persisted for up to one month post-exposure. The magnitude of these differences (up to 5 msec) lead the authors to conclude that treatment-related decrement in performance was probably present. There was no obvious treatment-related change in the maximum force exerted by the animals following exposure to the noise stimulus.

ORGAN WEIGHTS: Relative liver weight (as a proportion of body weight) was statistically significantly increased in high-dose males (+29%) and females (+14%) at study termination, but comparable to the sham controls by the end of the two month recovery period. Similarly the relative wet weight of the right middle lobe of the lung was increased 18-19% (significant) in high-dose males and females immediately post-exposure, with smaller (non-significant) increases of 7-9% present in the mid-dose groups (Table 2). Kidney, spleen, adrenal and testis weights were comparable for the sham control and the treated groups.

HISTOPATHOLOGY: Histopathological examination of lung revealed no treatment-related lesions, while findings in the kidney (glomerulosclerosis), adrenal (small cortical adenomas) and heart (degeneration of single cardiac fibres) occurred at a similar frequency in control and treated animals and were considered spontaneous phenomena, unrelated to treatment. No treatment-related lesions were present in 15-20 other tissues that were sampled and subjected to microscopic evaluation. This included the nasal turbinates, where no adverse changes were present even in high-dose animals.

OTHER FINDINGS: The number of alveolar macrophages in pulmonary lavage fluid was increased 8-19% in exposed animals of both sexes (significant for low- and high-dose groups), but had resolved by the end of the 2-month recovery period (numbers of other cells unaffected).Pulmonary function tests showed no obvious dose-related difference or trend in lung resistance, multibreath nitrogen washout, single breath carbon monoxide diffusing capacity, maximal forced exhalation, peak expiratory flow, vital capacity, inspiratory capacity, functional residual capacity or specific compliance. Only total lung capacity and residual volume were altered by treatment, with statistically significantly decreases (-7% and -13%, respectively) noted in high-dose animals (both sexes combined) when compared with the sham controls.

Effect levels

1

Dose descriptor:

NOAEC

Remarks:

Systemic effects

Effect level:

>= 1.71 mg/L air (analytical)

Sex:

male/female

Basis for effect level:

other: see Details on results

2

Dose descriptor:

NOAEC

Remarks:

Local effects

Effect level:

0.88 mg/L air (analytical)

Sex:

male/female

Basis for effect level:

other: Lung weight

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

 

Table 1. Body weight
	Sham Control	250 mg/m^3	750 mg/m^3	1500 mg/m^3
Males
Initial	508.7±9.2	512.6±9.4	510.3±11.7	507.6±7.1
End of exposure	580.3±12.2	543.7±10.9 *	525.6±13.6 *	528.1±8.8 *
End of recovery	606.1±22.9	608.4±13.9	570.0±20.7	588.4±17.4
Females
Initial	284.6±3.5	287.7±4.0	266.0±3.7 *	267.4±3.7 *
End of exposure	331.5±6.1	301.3±5.1 *	279.4±4.2 *	283.5±4.3 *
End of recovery	336.5±9.9	336.3±12.1	311.2±9.4	321.4±9.7

Significantly different from the control * p<0.05

   

Table 2. Select Relative Organ Weights
	Sham Control	250 mg/m^3	750 mg/m^3	1500 mg/m^3
Males
Liver After exposure 2 month post exposure	10.24±0.55 9.67±0.60	10.82±0.49 11.56±6.3 *	10.72±0.48 9.63±0.55	13.18±0.51 * 10.61±0.57
Lung (wet weight right middle lobe) After exposure 2 month post exposure	162±8 143±9	160±7 148±9	173±7 149±8	193±7 * 148±8
Lung (wet/dry ratio) After exposure 2 month post exposure	5.64±0.19 5.04±0.20	5.55±0.17 5.05±0.22	5.36±0.16 5.31±0.19	5.52±0.17 6.95±0.20 **
Females
Liver After exposure 2 month post exposure	10.49±0.72 11.63±0.65	11.12±0.89 11.38±0.67	10.83±0.47  11.17±0.78	11.98±0.92 * 11.41±0.74
Lung (wet weight right middle lobe) After exposure 2 month post exposure	157±10 149±9	161±12 157±9	171±14 144±11	185±13 * 161±11
Lung (wet/dry ratio) After exposure 2 month post exposure	5.53±0.25 4.99±0.23	5.35±0.31 4.91±0.23	5.15±0.0.33 5.01±0.27	5.31±0.32 7.67±0.26 **

Significantly different from the control * p<0.05; ** p< 0.0001

Applicant's summary and conclusion

Conclusions:

In a read-across, sub-chronic inhalation study of diesel fuel, findings resulted in a conservative sub-chronic NOAEC of 0.88 mg/L determined for local effects on the lung (increased relative wet weight in the absence of histopathological change). A NOAEC of greater than or equal to 1.71 mg/L was established for systemic effects, based on no significant findings at this level.

Executive summary:

Justification for Read Across

Detailed compositional analyses are available for SRGO substances, generated using GCxGC (see Section 2 in the CJD). Comparison of these data with with substances in the VHGO category, including samples of diesel fuel (see Section 3 of the CJD) indicate that the type and range of structures present are similar for substances in these two categories. Therefore read-across of experimental data from diesel fuel to the SRGO category is considered to be appropriate.

 

The sub-chronic inhalation toxicity of diesel fuel, including potential effects on neurological and pulmonary function, has been investigated in a well conducted, well reported study in which groups of male and female Sprague-Dawley rats were exposed whole body to nominal concentrations of 0, 0.25, 0.75 or 1.5 mg/L aerosol (MMAD 0.43-0.75 microns) 4 hour per day, two days per week for 13 weeks (total of 26 exposures) (analytical concentrations:0.35, 0.88, and 1.71 mg/L). The study also included a sham (chamber) control group and an untreated (animal room) control group, with animals in all groups aged 18-21 weeks at the start of the study. Body weight (all animals) and food consumption (6/sex/dose level) were recorded weekly. Breathing frequency was measured using a barometric method (groups of 12 rats/sex/dose level, temporarily housed in sealed chambers) prior to the first exposure (baseline), before the 14^th and 26^th exposures (i.e., study weeks 7 and 13), and after one or two months recovery. Startle reflex (assessed by measuring reaction time, peak time and peak height following exposure to five 10 msec pulses of 110 dB noise) was quantified in males only (8/dose) immediately after the first, 14^th and 26^th exposures (to evaluate acute/short term effects), and in both sexes (8/sex/dose) prior to the 14^th and 26^th exposures and after one and two months recovery (to assess chronic/cumulative changes). The type and number of free cells present in pulmonary lavage fluid and serum chemistry (alkaline phosphatase, aspartate aminotransferase, cholesterol, triglyceride, uric acid, urea nitrogen, glucose, bilirubin, creatinine, sodium, potassium) were determined in groups of animals (6/sex/dose) at study termination and following a two-month recovery period. Lung function tests (pulmonary resistance, nitrogen washout, carbon monoxide diffusion, functional reserve capacity, peak expiratory flow, total lung capacity, vital capacity, inspiratory capacity, functional residual capacity, residual volume, specific compliance) were performed on groups of anaesthetised animals (8/sex/dose level, fitted with a tracheal cannula) after 13 weeks exposure and also following a 2-month recovery period using whole body plethysmography. After completion of the lung function tests, animals were subject to necropsy (including gross examination and collection of organ weight data), followed by histological examination of around 15-20 tissues.

 

There were no deaths during the exposure phase or during the 2-month recovery period. Animals were described as inactive during treatment but no overt clinical signs were present. Body weight was decreased in both the sham control and the diesel-exposed groups relative to animal room controls at the start of exposure (that is, when the animals were first introduced into the chambers). Exposed animals exhibited a reduced weight gain (relative to the sham control group) until the start of the fourth week of treatment (statistically significant for mid- and high dose males and all exposed females), after which male body weight increased while body weights for females remained relatively static. Terminal body weights (after 25 exposures) were significantly decreased by 7%, 13% and 11% in low-, mid- and high-dose males and by 11%, 17% and 16% in the corresponding groups of females, relative to the sham controls. Body weights for exposed males were comparable to the sham control group by the third week of the recovery period, whereas statistically significant decreases remained in mid- and high-dose females until recovery weeks 7 and 5, respectively. Food intake was significantly decreased by approximately 10-15% in mid- and high dose animals (no difference between the sexes) during study weeks 4-12, but did not differ from that of the sham control group thereafter.

 

Breathing frequency was indistinguishable between sham control and exposed animals during the test and recovery periods. Results from the startle reflex tests showed that reaction time was statistically significantly increased in high- and mid-dose males immediately after exposure, and in mid-dose females immediately before exposure, however the magnitude of the alteration (2 msec) was small and considered of doubtful toxicological relevance by the study authors. In contrast, statistically significant increases in peak time were present in high dose males (2-4 msec greater than sham controls) at all of the time points investigated, intermittently in mid-dose males (after the 14^th exposure, before the 26^th exposure, at one month post-exposure; 2-5 msec) and intermittently in low dose males (after the 14^th exposure and at the one month into the recovery phase; 2-3 msec). Similar statistically significant increases in peak time (increased 2-4 msec) were also present in high- and mid-dose females and persisted for up to one month post-exposure. The magnitude of these differences (up to 5 msec) lead the authors to conclude that treatment-related decrement in performance was probably present. There was no obvious treatment-related change in the maximum force exerted by the animals following exposure to the noise stimulus.

 

The number of alveolar macrophages in pulmonary lavage fluid was increased 8-19% in exposed animals of both sexes (significant for low- and high-dose groups), but had resolved by the end of the 2-month recovery period (numbers of other cells unaffected). Pulmonary function tests showed no obvious dose-related difference or trend in lung resistance, multibreath nitrogen washout, single breath carbon monoxide diffusing capacity, maximal forced exhalation, peak expiratory flow, vital capacity, inspiratory capacity, functional residual capacity or specific compliance. Only total lung capacity and residual volume were altered by treatment, with statistically significantly decreases (-7% and -13%, respectively) noted in high-dose animals (both sexes combined) when compared with the sham controls.

 

Relative liver weight (as a proportion of body weight) was statistically significantly increased in high-dose males (+29%) and females (+14%) at study termination, but comparable to the sham controls by the end of the two month recovery period. Similarly the relative wet weight of the right middle lobe of the lung was increased 18-19% (significant) in high-dose males and females immediately post-exposure, with smaller (non-significant) increases of 7-9% present in the mid-dose groups. Kidney, spleen, adrenal and testis weights were comparable for the sham control and the treated groups. Some clinical chemistry parameters were apparently altered in high-dose animals (LDH, cholesterol and creatinine in females at study termination; LDH in males 2 month post-exposure), however the report discounts the biological relevance of the findings and the data are not reported. Red cell and white cell counts were unaffected.

 

Histopathological examination of lung revealed no treatment-related lesions, while findings in the kidney (glomerulosclerosis), adrenal (small cortical adenomas) and heart (degeneration of single cardiac fibres) occurred at a similar frequency in control and treated animals and were considered spontaneous phenomena, unrelated to treatment. No treatment-related lesions were present in 15-20 other tissues that were sampled and subjected to microscopic evaluation. This included the nasal turbinates, where no adverse changes were present even in high-dose animals.

 

These results demonstrate statistically significant alterations in a number of parameters (body weight, food consumption, startle reflex, certain lung function parameters) in rats following sub-chronic inhalation exposure to diesel aerosol, however the magnitude of these changes was small suggesting that they are of doubtful biological relevance. Statistically significant increases in relative liver weight and relative wet lung weight were observed in animals exposed to 1.71 mg/L (actual concentration) diesel aerosol for 13 weeks, however there was no histopathological involvement, again making the relevance of these findings unclear.  It is noted that the use of whole body exposure probably resulted in ingestion of the test sample during grooming, and may account for the systemic findings that were observed. All of the changes present following 13 weeks exposure were reversed after a 2-month recovery period. A conservative sub-chronic NOAEC of 0.88 mg/L is determined for local effects on the lung (increased relative wet weight in the absence of histopathological change). A NOAEC of greater than or equal to 1.71 mg/L is established for systemic effects, based on no significant findings at this level.