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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
No data reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable with restrictions because it is an acceptable, well-documented study report following sound scientific principles.

Data source

Reference
Reference Type:
publication
Title:
Four-week inhalation exposures of rats to aerosols of three lubricant base oils.
Author:
Dalbey W, Osimitz T, Kommineni C, Roy T, Feuston M and Yang J
Year:
1991
Bibliographic source:
J Appl Toxicol, Vol 11(4), pp 297-302

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Principles of method if other than guideline:
The method did not strictly follow the guideline but is deemed appropriate as utilized in this report.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Oily liquid
Details on test material:
Two materials were examined in this study. The properties of the materials designated SRO and HBO are shown in the following table.

SRO Solvent refined oil, CAS # 64742-70-7

HBO Hydrotreated base oil, CAS #64742-54-7 [Severely
hydrotreated heavy paraffinic oil produced by treatment of the vacuum distillate with hydrogen at high temperature and pressure
(hydrotreating and hydrocracking)].

SRO HBO

Viscosity at 100 °F 106 161
Pour point (°F) 20 -5
API Gravity 32.8 33.6
Furfural (ppm) 1 <1
Nitrogen (ppm) 44 8
Sulfur (wt.%) 0.20 <0.06
Composition (wt.%)
Paraffins 36 29.7
Mononaphthenes 22.3 30.6
Polynaphthenes 22.3 37.3
Monoaromatics 12.8 0.6
Diaromatics 3.3 0.8
Polyaromatics 1.4 1.0
Unidentified aromatics 0.4 0
Aromatic sulfur types 1.1 0

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Age at study initiation: 11 to 12 weeks of age
- Weight at study initiation: not reported.
- Fasting period before study: overnight
- Housing: not reported
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2-week quarantine

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22 °C
- Humidity (%): 40 to 60%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 hours

IN-LIFE DATES: not reported

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
not specified
Vehicle:
other: no data
Remarks on MMAD:
MMAD / GSD: Mean ± SD aerosol concentration, Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard Deviation (GSD)

Material Aerosol MMAD (μm) GSD
Concentration
(mg m-3)
SRO 0 0 0
50±10 1.2±0.1 1.8±0.1
210±10 1.0±0.1 1.9±0.1
1020±60 1.2±0.1 1.8±0.1

HBO 0 0 0
47±2 1.3±0.1 1.8±0.2
220±10 1.2±0.1 1.8±0.1
980±50 1.2±0.1 1.7±0.1
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 400-litre inhalation chambers made of stainless steel and glass
- Method of holding animals in test chamber: not reported
- Source and rate of air: not reported
- Method of conditioning air: not reported
- System of generating particulates/aerosols: Nebulizers based on the design of Drew et al. were used to aerosolize the test substance; A glass impactor between the nebulizer and the main airstream to the chamber was used to maximize the delivery of small, respirable particles
- Temperature, humidity, pressure in air chamber: 20 to 23°C, 30 to 50% humidity, pressure not reported
- Air flow rate: not reported
- Air change rate: at least 12 air changes per hour
- Method of particle size determination: Aerosol concentration was measured hourly in the exposed groups by gravimetric analysis. Filter samples also were collected in the control chamber once per day. The aerodynamic size distribution of the aerosol was determined during most exposures with a cascade impactor (Sierra Series 220). In addition, selected glass-fiber filters were extracted with dichloromethane and analyzed with gas chromatography/flame ionization detection (GC/FID) to quantitate the amount of oil present on the filter and to measure any qualitative changes in the composition of the oil during an individual exposure and over the course of the 4-week exposure period. A J&W 30 metre x 0.32 millimetre DB-5 fused-silica capillary column was used (0.25 μm film thickness; oven temperature, 150°C for 30 minutes followed by 8°C min-1 to 300°C and a 60 minute hold; helium carrier gas).
- Treatment of exhaust air: not reported
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Measured aerosol concentrations were comparable among the three base stocks, as seen in the table below:

Mean ± SD aerosol concentration, Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard Deviation (GSD)
Material Aerosol concentration (mg m-3) MMAD (μm) GSD
SRO 0 0 0
50±10 1.2±0.1 1.8±0.1
210±10 1.0±0.1 1.9±0.1
1020±60 1.2±0.1 1.8±0.1

HBO 0 0 0
47±2 1.3±0.1 1.8±0.2
220±10 1.2±0.1 1.8±0.1
980±50 1.2±0.1 1.7±0.1

For the sake of simplicity, the respective aerosol concentrations will be referred to as 50, 220 and 1000 mg m-3. The mean mass median aerodynamic diameter was well under 2 μm for each base stock, with a geometric standard deviation of about ≤2. Analysis of dichloromethane extracts of selected glass-fiber filters by gas chromatography indicated that the composition of the aerosols remained constant throughout a given day of exposure, as well as over the four weeks of exposure. Qualitatively, the aerosols collected on the glass-fiber filters were virtually identical to each liquid base oil. Also, the analytically determined amount of oil recovered from a filter generally agreed closely with the gravimetrically measured values.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 220 or 1000 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
10 per sex per dose
Control animals:
yes, sham-exposed
Details on study design:
Exposures were conducted separately for each base stock. Sprague-Dawley rats (11 to 12 weeks of age, obtained from Charles River) were assigned randomly to treatment groups following a 2-week quarantine. Four experimental groups (10 animals per sex per group) were used for each separate study: sham-exposed controls and groups exposed to target concentrations of 50, 220 or 1000 mg/m3. Exposures were conducted for 6 hours per day, 5 days per week for approximately 4 weeks (17 days for SRO and 20 days for HBO). When not being exposed, animals were moved to a conventional animal room set to maintain 20 to 22°C, 40 to 60% relative humidity and a 12-hour day/night cycle. Animals had access to food and water ad libitum, except during exposure.
Positive control:
No data provided.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Conducted before and during exposures.

DETAILED CLINICAL OBSERVATIONS: No data reported.

BODY WEIGHT: No data reported

FOOD CONSUMPTION: No data reported.

FOOD EFFICIENCY: No data reported.

WATER CONSUMPTION: No data reported.

OPHTHALMOSCOPIC EXAMINATION: Not performed.

HAEMATOLOGY: Haematology was conducted for each animal, and parameters consisted of total white and red  cells, haemoglobin, haematocrit, MCV, MCH, and MCHC. A differential white cell count was also conducted.

CLINICAL CHEMISTRY: The following chemical parameters were measured in each animal: alanine transferase, albumin, albumin/globulin ratio, alkaline phosphatase, aspartate aminotransferase, total bilirubin, calcium, chloride, cholesterol, creatinine, globulin, glucose, iron, lactate dehydrogenase, inorganic phosphorus, potassium, total protein, sodium, triglycerides, urea nitrogen and uric acid.

URINALYSIS: Not performed.

NEUROBEHAVIOURAL EXAMINATION: Not performed.

OTHER: Lung tissue from three male and three female rats exposed to 980 mg/m3 WTO as well as one male and one female control rat were examined by transmission electron microscopy. Sperm morphology was used to assess toxicity in the male reproductive system. Sperm from the cauda epididymis of each control and high dose male was examined for an assessment of sperm morphology.
Sacrifice and pathology:
GROSS PATHOLOGY: No remarkable findings.
HISTOPATHOLOGY: No remarkable findings
Other examinations:
No data reported.
Statistics:
Data were analyzed by one-way analysis of variance. A probability of Type I error of < 5% (P<0.05) was considered to be statistically significant. Comparison of means was performed by Duncan's multiple range test or the Student-Neuman-Keuls multiple comparison. Data obtained from exposure to a given test article were analyzed together. No statistical procedures were carried out to compare the effects of different test articles with each other.

Results and discussion

Results of examinations

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):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Other than the occasional observation of loose stools in some of the exposed animals, there were no treatment-related clinical signs during the three series of exposures.

BODY WEIGHT AND WEIGHT GAIN
Body weight, measured weekly, was not affected by exposure.

HAEMATOLOGY and CLINICAL CHEMISTRY
At necropsy, no toxicologically significant effects were noted for any of the clinical chemistry or haematological parameters examined.

ORGAN WEIGHTS
No significant changes were noted in mean organ weights except in the lungs. Lung weight (wet and dry) increased in a concentration-related manner. Statistically significant increases were noted in wet weights of the right middle lung lobe in both sexes exposed to 1000 mg/m3 of each base stock. Moreover, statistically significant increases were seen in wet lung weights of females exposed to 210 mg/m3 of HBO and in males exposed to 210 mg/m3 of SRO. In addition, the ratio of dry lung weight to wet lung weight was increased (P<0.05) over controls in both sexes exposed to 1000 mg/m3 of both base oils. Similar statistically significant increases were noted among groups exposed to 210 mg/m3, but the values were within the upper range of data for historical control groups and their biological significance is highly doubtful.

Some minor morphological differences were noted in the responses to the three aerosols. SRO caused accumulation of foamy macrophages in alveoli at the lowest concentration and a mild dose-related infiltration of PMNs into the alveoli; HBO caused neither. However, slight thickening of some alveolar walls and foamy macrophages in the alveolar interstitium were noted only with HBO. The accumulation of macrophages in the lymph nodes may have resulted from a general increase in macrophages populations in the lung as a result of exposure and subsequent lymphatic drainage. The foamy macrophages noted in the tracheobronchial lymph nodes of animals exposed to SRO may have either entered already vacuolized from the lung or developed in response to the presence of SRO in the lymph nodes. The accumulations of alveolar macrophages were associated with a concentration-dependent increase in lung weights. Each base stock produced increases in wet weight in both sexes exposed to 1000 mg/m3 and in one sex exposed to 210 mg/m3. The increases with 1000 mg/m3 were generally 30 to 40% above control values. The reasons for the weight gain are unclear but may be related to increased numbers of alveolar macrophages and other cells, residual oil or, possibly, more subtle changes that were not apparent by light microscopy. The ratio of dry to wet weight also was increased significantly with exposure to 1000 mg/m3, indicating a disproportionate accumulation of non-volatile material. Presumably, residual oil may have added to the dry weight; but the amount of retained oil was unknown.

MICROSCOPIC PATHOLOGY
Morphologically, treatment-related effects were seen only in the lung and tracheobronchial lymph nodes. In the lung, foamy macrophages with numerous vacuoles of varying size were present in the alveolar spaces of many of the exposed animals. The number of foamy macrophages was dose-related. No foamy macrophages were seen in any of the control animals or in animals exposed to 50 mg/m3 of HBO. The number of foamy macrophages increased with aerosol concentration until there were 3 to 6 foamy macrophages in many alveoli of animals exposed to 1000 mg/m3 of each of the oils. These foamy macrophages were most prevalent in the alveoli close to the alveolar ducts. Generally, the accumulations of macrophages were similar among the three base stocks, although both SRO and WTO caused accumulation of a few foamy macrophages at the lowest concentration while HBO did not. Exposure to 1000 mg/m3 of HBO did produce mild thickening of some of the alveolar walls and occasional foamy macrophages in the alveolar interstitium in scattered areas. The macrophages probably contributed to the thickening of alveolar walls. A few relatively mild changes were noted in addition to the accumulation of foamy macrophages in the alveolar lumen. With SRO, a very mild infiltration of polymorphonuclear leukocytes (PMNs) into the alveoli was noted in some animals exposed to the higher concentrations. Examined by transmission electron microscopy; PMNs were not observed in rats treated with HBO. The large alveolar macrophages observed by transmission electron microscopy (TEM) contained vacuoles that appeared to be membrane-bound and probably represented phagosomes that had lost their contents during processing for electron microscopy. The tracheobronchial lymph nodes of animals exposed to the high concentration of HBO (to a lesser extent) contained numerous normally appearing macrophages (histiocytosis). With SRO, these same lymph nodes had accumulations of foamy macrophages. The anterior mediastinal lymph nodes were normal, except for histiocytosis in nine of twenty animals in the high concentration of SRO.

OTHER FINDINGS
The percent of sperm with aberrant morphology, including breakage, was not affected by exposure to any of the materials.

Effect levels

open allclose all
Dose descriptor:
NOEC
Effect level:
ca. 220 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: based on mild accumulation of alveolar macrophages in lungs consistent with oil deposition
Dose descriptor:
NOAEC
Effect level:
> 980 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: based on lack of systemic toxicity

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Chamber concentrations
The aerosol concentrations were comparable among the two base stocks. Qualitatively, the aerosols were virtually identical to each liquid base
  oil. The actual concentrations (in mg/m
3) for each of the aerosols was as follows:
  

Nominal

Actual

SRO

0

0

50

50 ±10

220

210 ±10

1000

1020 ±60

WTO

0

0

50

50 ±10

220

210 ±10

1000

980 ±20

HBO

0

0

50

47 ±2

220

220 ±10

1000

980 ±50


The mass median diameter was well under 2µm for each base stock

Toxicity assessment

Apart from occasional loose stool, there were no treatment-related
 clinical observations, and body weights were unaffected by exposure. No treatment-related effects were found in any of the haematological or clinical chemical parameters that were measured. The percent sperm with aberrant morphology, including breakage, was unaffected by exposure to any of the three base oils. There were no treatment-related observations at necropsy, and, with the exception of the lungs, there were no significant changes in organ weights. Wet and dry lung weights increased in a dose-related manner. The percentage increases in wet weight are shown in the following table.  For simplicity, increases are shown to nearest whole numbers.                 
                              

% Increase in wet lung weight

Sex

Dose

(mg/m3)

SRO

WTO

HBO

Female

50

3

8

2

220

4

23*

34*

1000

38*

64*

36*

Male

50

5

-

1

220

12*

1

6

1000

33*

31*

32*

*  denotes differences that are statistically significant (P<0.05) compared to controls.
The ratios of wet to dry lung weights were significantly increased for
 both sexes at the highest dose concentration for all three base oils.

Morphologically, treatment-related changes were only observed in the
lungs and tracheobronchial lymph nodes. Foamy macrophages with numerous vacuoles of varying size were present in the alveolar spaces of the lungs of many of the exposed animals. The histological changes are summarized in the following table:

No. of animals in each group with a given
        histopathological change

Tissue/change

Dose group (mg/m3)

SRO

50

220

1000

Lung

1 to 2 Foamy macrophages (FM)

20

20

20

3 to 6 FM

0

0

20

Thickened alveolar wall

0

0

0

FM in alveolar interstitium

0

0

0

Mild alveolar PMN infiltrate

0

5

20

Lymph nodes

Anterior mediastinal

Macrophage accumulation

NE

NE

9

Tracheobronchial

FM accumulation

NE

NE

19

Macrophage accumulation

NE

NE

0

HBO

Lung

1 to 2 Foamy macrophages (FM)

0

16

16

3 to 6 FM

0

0

16

Thickened alveolar wall

0

0

16

FM in alveolar interstitium

0

0

16

Mild alveolar PMN infiltrate               

0

0

0

Lymph nodes

Anterior mediastinal

Macrophage accumulation

NE

NE

2

Tracheobronchial

FM accumulation

NE

NE

2

Macrophage accumulation

NE

NE

2

NE denotes Not Evaluated
Only 16 animals in the HBO high dose group were examined

Applicant's summary and conclusion

Conclusions:
No systemic effects were observed. The NOAEL for lung changes associated with oil deposition in the lungs was 220 mg/m3. As no systemic toxicity was observed, the overall NOAEL for systemic effects was > 980 mg/m3.
Executive summary:

In a subchronic, repeated-dose inhalation toxicity study, one of two lubricant base oils [solvent-refined oil (SRO); and severely hydrotreated, hydrocracked oil (HBO)] was administered to Sprague-Dawley rats (10/sex/dose) by dynamic inhalation exposure at nominal concentrations of 0, 50, 220, or 1000 mg/m3 for 6 hours per day, 5 days per week for approximately 4 weeks (18 days exposure for WTO, 17 days for SRO, and 20 days for HBO).  The mass median aerodynamic diameter was approximately 1μm.

 

Foamy macrophages accumulated in the lungs of exposed animals with each material in a concentration-related manner, especially in alveoli close to alveolar ducts. Mild infiltration of PMNs into alveoli was noted also with high aerosol concentrations. Increased numbers of alveolar macrophages are expected following deposition of a significant number of particles in the alveoli. The alveolar macrophages and the associated increase in neutrophilic leukocytes are part of the normal mechanism for removal of an increased particle load. The presence of neutrophils, therefore, is not necessarily a pathological occurrence.

Therefore, the NOEL is 220 mg/m3 based on accumulation of alveolar macrophages in lung and the NOAEL is >980 mg/m3 based on lack of systemic toxicity in males and females.

This study received a Klimisch score of 2 and is classified as reliable with restrictions because it is an acceptable, well-documented study report following sound scientific principles.