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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
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

Constituent 1
Reference substance name:
Diesel fuel
IUPAC Name:
Diesel fuel
Details on test material:
Substance is a possible end product of the substances being registered (i.e., substances being produced in a refinery as petrochemical intermediates).

- Name of test material (as cited in study report): Diesel fuel
- 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 degrees Celcius

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:
other: unchanged (no vehicle)
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; 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:
13 weeks
Frequency of treatment:
Twice a week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 0.25, 0.75, or 1.50 mg/L
Basis:
nominal conc.
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

open allclose all
Dose descriptor:
NOAEC
Remarks:
Systemic effects
Effect level:
> 1.71 mg/L air (analytical)
Sex:
male/female
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/m3

750 mg/m3

1500 mg/m3

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/m3

750 mg/m3

1500 mg/m3

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:
The sub-chronic inhalation study of diesel fuel (read-across to cracked gas oils) resulted in a conservative sub-chronic NOAEC of 750 mg/m3 determined for local effects on the lung (increased relative wet weight in the absence of histopathological change). A NOAEC of >1710 mg/m3 was established for systemic effects, based on no significant findings at this level.
Executive summary:

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 250, 750 or 1500 mg/m3aerosol (MMAD 0.43-0.75 microns) 4 hour per day, two days per week for 13 weeks (total of 26 exposures). 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 14th and 26th 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, 14th and 26th exposures (to evaluate acute/short term effects), and in both sexes (8/sex/dose) prior to the 14th and 26th 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 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.

 

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. No treatment -related effects were noted in examination of the weight and histopathology of the testes.

 

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 1710 mg/m3 (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 750 mg/m3 is determined for local effects on the lung (increased relative wet weight in the absence of histopathological change). A NOAEC of >1710 mg/m3 is established for systemic effects, based on no significant findings at this level.