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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
chronic toxicity: inhalation
Remarks:
other: Chronic combined repeated dose and carcinogenicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2003-03-19 to 2006-04-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restrictions because it is an acceptable study that is well documented.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 451
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: mix of aerosol and vapour
Details on test material:
- Name of test material (as cited in study report): Bitumen condensate
Sample API 06-01
- Substance type: Bitumen
- Physical state: Not reported
- Analytical purity: Not reported
- Composition of test material, percentage of components: Polycyclic aromatic hydrocarbon content in µg/g (results from two separate laboratories): Naphthalene 81.8-110, Acenaphthylene 0.58-1.09, Acenaphthene 74.3-78.4, Fluorene 137-216, Phenanthrene 71.2-85.0, Anthracene 22.4-32.7, Fluoranthene 7.23-7.4, Pyrene 7.3, Benz(a)anthracene 1.37-1.5, Triphenylene 1.8, Chrysene 1.8, Benzo(b)fluoranthene 0.42-0.53, Benzo(k)fluoranthene 0.07-0.08, Benzo(e)pyrene 0.73, Benzo(a)pyrene 0.18-0.23, Indeno(1,2,3-cd)pyrene 0.02-0.03, Dibenz(ah)anthracene 0.03-0.05, Benzo(ghi)perylene 0.16- 0.17, total 441-512
- Stability under test conditions: Not reported
- Storage condition of test material: Sealed glass bottles protected from the light
- PAH and Total hydrocarbon content in exposure atmoshere in attachment

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: Approximately 9 weeks old
- Weight at study initiation: Not reported
- Housing: Two per polycarbonate cage
- Diet (e.g. ad libitum): Ad libitum, except during exposure period
- Water (e.g. ad libitum): Ad libitum, except during exposure period
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24
- Humidity (%): 30% to 70%
- Air changes (per hr): 12 to 15
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

IN-LIFE DATES: From: 2003-03-31 To: 2005-11-11

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
nose only
Remarks:
aerosol and vapour
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: The median mobility diameter was 106, 85, and 83 nanometres for the low-, mid-, and high-dose groups, respectively.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Direct flow nose only inhalation system
- Method of holding animals in test chamber: Tapered acrylic glass tubes with adjustable backstops
- Source and rate of air: Slowly flowing cool air stream
- System of generating particulates/aerosols: Fumes were generated using an evaporation condensation generator.
- Temperature, humidity, pressure in air chamber: Temperature ranged from 20.3 to 23.9 degrees Celsius, humidity ranged from 42.7% to 58.5% in a slight overpressure in the exposure system
- Air flow rate: 100 litres per minute
- Method of particle size determination: Scanning mobility particle sizer
- Treatment of exhaust air: Into a laboratory hood

TEST ATMOSPHERE
- Brief description of analytical method used: Samples were collected on a combination glass fibre filter and a XAD absorption tube with a sample flow rate of approximately 2 litres per minute. Samples were then extracted and analyzed by IR spectroscopy and presented as mg of total hydrocarbons per cubic metre.
- Samples taken from breathing zone: Yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The mean total hydrocarbon concentrations for combined vapour and aerosol were 4.1 +/1 0.3, 20.7 +/- 1.8, and 103.9 +/- 9.7; therefore were well within the acceptable range.
Duration of treatment / exposure:
24 months
Frequency of treatment:
6 hours a day 5 days a week (except during holidays)
Doses / concentrations
Remarks:
Doses / Concentrations:
4, 20, or 100 mg/m3
Basis:
nominal conc.
No. of animals per sex per dose:
Fifty to eighty-six animals per sex per treatment group
Control animals:
yes
Details on study design:
- Dose selection rationale: Doses were selected based on a previous 14-day dose range finding study and a 90-day study.
- Rationale for animal assignment (if not random): Animals were stratified by weight via computer generated randomized program
- Rationale for selecting satellite groups: Not reported
- Section schedule rationale (if not random): Random
Positive control:
None reported

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations for clinical symptoms.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly for the first 13 weeks, then monthly until study termination

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/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: Terminal sacrifice
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: Fifty animals per group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: A bronchoalveolar lavage (BAL) was conducted on control and high-dose animals (6 per sex per dose per time) after 7 days, 3 months, and 12 months of exposure. The following parameters were measured: cell number, related to recruitment of leukocytes in lungs; differential cell count, related to inflammatory reactions (PMNs, lymphocytes); total protein, related to increased transudation of protein through the alveolar capillary
walls; lactic dehydrogenase (LDH), related to increased permeability of membranes, cell damage and lysis; beta-glucuronidase, related to increased phagocytotic activity of macrophages or lysis of macrophages; and gamma-glutamyl-transferase, membrane protein of pulmonary epithelial cells.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (see table 2)
Other examinations:
Some animals from the control and high-dose group were sacrificed after 7 days, 3 months, and 12 months and their respiratory tracts were histologically examined for cell proliferation and the lungs were weighed.
Statistics:
Body weight and food consumption were analyzed using the "Provantis Decision Tree" where the "best" transformation was determined, the use of possible covariates was checked and the homogeneity of means was assessed by analysis of variance (ANOVA) or analysis of covariance (ANCOVA), the transformed data were tested for dose-related trend and the lowest dose level affected, based on the Williams Test for parametric data or Shirley Test for nonparametric data, and if no trend was found, but the data showed non-homogeneity of means, the data were analyzed by a stepwise Dunnett (parametric) or a modified Steel (non-parametric Dunnett) test. Haematology data were analyzed using analysis of variance. Pair wise comparison of the means were performed using Dunnett's modification of the t-test. For comparisons between two treatment groups, the two-sided t-test. Survival data of the animals were analyzed using the Kaplan-Meier test. Peto‘s analysis was used to compare the tumour incidence. Statistical evaluation of histopathological findings were evaluated as pair wise comparison using Fisher‘s exact test.

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):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: There were no treatment-related effects on clinical signs or mortality.


BODY WEIGHT AND WEIGHT GAIN: Body weight was 3% lower than controls in mid-dose males and 8% lower in mid-dose females from day 119 on. In the high-dose group, there was a 7% reduction in body weight in males and an 8% reduction in females compared to the controls beginning on day 21 in males and day 28 in females. Results were statistically significant at various times throughout the study.


FOOD CONSUMPTION: There were no consistent treatment-related effects on food consumption.


HAEMATOLOGY: Although there was a dose-dependent decrease in white blood cells in males that was significantly reduce in the mid- and high-dose groups, the results were within the range expected for this species, sex, and age; therefore, the results were considered to be of minor toxicological significance.


ORGAN WEIGHTS: There was no treatment-related change in lung weight.


GROSS PATHOLOGY: There were no treatment-related gross pathology findings.


HISTOPATHOLOGY: NON-NEOPLASTIC:At the mid-dose level (20.7 mg/m3) the full histopathology at the termination of the study after 2 years of exposure showed some slight effects in the nasal passages (Table 1). In particular hyperplasia of mucous cells (goblet cells) and eosinophilic cytoplasmic inclusions in the olfactory epithelium was observed. In addition, a statistically significant increased incidence of mononuclear cell infiltrates was seen in the epithelium of the nasal and paranasal cavities in animals of the mid- and high-dose groups. These effects were also seen at a lower incidence in the animals of the control and low-dose groups and are probably adaptive in nature.


HISTOPATHOLOGY: NEOPLASTIC: There were no treatment-related effects in neoplastic histopathology.



OTHER FINDINGS: Lactic dehydrogenase activity in BAL fluid, indicating an increased permeability of cell membranes, was slightly elevated in the exposed females (but not males). However, the absolute values were low and below the values of historical controls and were considered of minor relevance by the investigators. Gamma-Glutamyltransferase levels in BAL fluid, indicative of increased phagocytitic activity of macrophages, were slightly increased in both males and females. Overall results of BAL investigations showed that effects, if any, were very slight to slight. The authors conclude that the broncheoalveolar region of the respiratory tract is not significantly impacted by exposure to bitumen fume. Unit Length Labelling Index was comparable in lung parenchyma of treated and control animals. No consistent effects on cell proliferation were seen for level 1 respiratory epithelium, level 1 non-ciliated epithelium and level 3 olfactory epithelium. The only consistent increase in proliferation was seen in the transitional zone of respiratory to olfactory epithelium in the exposed males, but not females.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Remarks:
Local effects
Effect level:
10.4 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: Based on lack of upper respiratory tract histopathology; 17.2 mg/m³ adjusted
Dose descriptor:
LOAEC
Remarks:
Local effects
Effect level:
20.7 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: Based on upper respiratory tract histopathology; 34.4 mg/m³ adjusted
Dose descriptor:
NOAEC
Remarks:
Systemic effects
Effect level:
> 103.9 mg/L air (analytical)
Sex:
male/female
Basis for effect level:
other: No treatment related findings. 172.5 mg/m³ adjusted

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

 

Table 1. Non-neoplastic lesions of the respiratory tract

 

Sham Control

4 mg/m3

20 mg/m

100 mg/m3

Males

Nasal and paranasal cavities

Olfactory epithelial basal cell hyperplasia

0/50

1/50

1/50

20/50***

Respiratory epithelial hyperplasia

0/50

3/50

3/50

13/50***

Mucous (goblet) cell hyperplasia

1/50

11/50**

25/50***

46/50***

Olfactory epithelial eosinophilic cytoplasmic inclusion(s)

1/50

13/50***

16/50***

31/50***

Respiratory epithelial eosinophilic cytoplasmic inclusion(s)

2/50

5/50

7/50

22/50***

Mucosal mononuclear/inflammatory cell infiltration

2/50

8/50

18/50***

27/50***

Lungs

Bronchiolo-alveolar hyperplasia, bronchiolar type

4/50

1/50

22/50***

46/50***

Alveolar histocytosis

32/50

31/50

47/50***

50/50***

Mononuclear/inflammatory cell infiltration

7/50

2/50

9/50

37/50***

Lung associated lymph nodes

Accumulation of foamy macrophages (histiocytosis)

1/50

1/50

0/50

12/50**

Females

Nasal and paranasal cavities

Olfactory epithelial basal cell hyperplasia

0/50

1/50

3/50

27/50***

Respiratory epithelial hyperplasia

0/50

0/50

2/50

20/50***

Mucous (goblet) cell hyperplasia

7/50

10/50

37/50***

47/50***

Olfactory epithelial eosinophilic cytoplasmic inclusion(s)

12/50

11/50

27/50**

38/50***

Respiratory epithelial eosinophilic cytoplasmic inclusion(s)

7/50

3/50

21/50**

24/50***

Mucosal mononuclear/inflammatory cell infiltration

11/50

5/50

22/50***

34/50***

Lungs

Bronchiolo-alveolar hyperplasia, bronchiolar type

6/50

7/50

21/50**

44/50***

Alveolar histocytosis

39/50

34/50

44/50

50/50***

Mononuclear/inflammatory cell infiltration

2/50

3/50

8/50

39/50***

Lung associated lymph nodes

Accumulation of foamy macrophages (histiocytosis)

1/50

2/50

5/50

26/50**

Significantly different from the control * p<0.05; ** p<0.01, *** p<0.001

Applicant's summary and conclusion

Conclusions:
The NOAEC for systemic effects following inhalation was 103.9 mg/m3(172.5 mg/m3adjusted) based on the absence of any histopathological changes or alterations in haematology parameters. The LOEC for local effects was 20.7 mg/m³ (34.4 mg/m³ adjusted), based on mild irritation effects on the nasal passages. The NOAEC was set at half the LOAEC, that is 10.4 mg/m³ total hydrocarbon concentration (17.2 mg/m³ adjusted) based on the minimal effects and the fact that in the 90-day study and the reproductive toxicity screening studies NOAECs of 20.1 mg/m³ (30.4 mg/m³ adjusted) and 30 mg/m3 were found, respectively.
Executive summary:

Read across for the key inhalation studies is justified because fumes of air-rectified bitumen have been shown to be comparable with fumes of straight-run paving bitumen. In a comparative analytical study, fumes from a straight-run and an air-rectified bitumen were closely similar in terms of composition and physical properties.

A two-year bioassay with fumes from a blended paving bitumen (air-rectified and vacuum residue) was conducted in Wistar rats (Crl:WI(WU)BR). The animals, 50 males and 50 females per dose group, were exposed nose-only to fumes regenerated from the bitumen fume condensate at target concentrations of 0 (clean air), 4, 20 and 100 mg/m3 total hydrocarbon concentration for 6 h/day, 5 days/week for 104 weeks. These concentrations were chosen based on a series of range-finding experiments in which the animals at the highest dose showed signs of slight respiratory irritation. The mean actual concentrations in the study, measured as total hydrocarbon (THC, sum of aerosol and vapour), were 0, 4.1±0.3, 20.7±1.8, and 103.9±9.7 mg/m3 using the methodology described by BIA (Berufsgenossenschaftliches Institut fuer). (Note: taking into account the conversion factor of 1.66 between the absolute concentration of fumes from bitumen and the concentration measured with this method, the concentrations were 0, 6.8 mg/m3, 34.4 mg/m3, and 172.5 mg/m3, respectively.) Additional control animals (36) and animals exposed to the high dose (36) were included in the study to conduct bronchio-alveolar lavage (BAL) and to investigate proliferation of respiratory epithelia, at 7 days, 90 days and 12 months following the start of exposure. In the main study, no statistically significant differences in mortality incidence were observed among the various groups: the mortality prior to final sacrifice was 10, 18, 16 and 14% in the males and 28, 12, 16 and 22% in the females for the control, low, medium and high dose groups, respectively. A statistically significant reduction of body weight gain was observed in the medium dose groups from day 119 (males and females) and in the high dose groups as of day 21 (males) or day 28 (females). The difference at sacrifice averaged –3% (males) and –8% (females) of the medium dose group and –7% (males) and –8% (females) in the high dose group.

 

Lactic dehydrogenase activity in BAL fluid, indicating an increased permeability of cell membranes, was slightly elevated in the exposed females (but not males). However, the absolute values were low and below the values of historical controls and were considered of minor relevance by the investigators.g-Glutamyltransferase levels in BAL fluid, indicative of increased phagocytic activity of macrophages, were slightly increased in both males and females. Overall results of BAL investigations showed that effects, if any, were very slight to slight. The authors conclude that the broncheoalveolar region of the respiratory tract is not significantly impacted by exposure to bitumen fume. Unit Length Labelling Index was comparable in lung parenchyma of treated and control animals. No consistent effects on cell proliferation were seen for level 1 respiratory epithelium, level 1 non-ciliated epithelium and level 3 olfactory epithelium. The only consistent increase in proliferation was seen in the transitional zone of respiratory to olfactory epithelium in the exposed males, but not females. At the mid-dose level (20.7 mg/m3) the full histopathology at the termination of the study after 2 years of exposure showed some slight effects in the nasal passages. In particular hyperplasia of mucous cells (goblet cells) and eosinophilic cytoplasmic inclusions in the olfactory epithelium was observed. In addition, a statistically significant increased incidence of mononuclear cell infiltrates was seen in the epithelium of the nasal and paranasal cavities in animals of the mid- and high-dose groups. These effects were also seen at a lower incidence in the animals of the control and low-dose groups and are probably adaptive in nature.  

 

The NOAEL for systemic effects following inhalation was 103.9 mg/m3(172.5 mg/m3adjusted) based on the absence of any histopathological changes or alterations in haematology. The LOEC for local effects was 20.7 mg/m³ (34.4 mg/m³ adjusted), based on the effects on the nasal passages. The NOAEC was set at half the LOAEC, that is 10.4 mg/m³ total hydrocarbon concentration (17.2 mg/m³ adjusted) based on the minimal effects and the fact that in the 90-day study and the reproductive toxicity screening studies NOAECs of 20.1 mg/m³ (30.4 mg/m³ adjusted) and 30 mg/m3were found, respectively.

 

This study received a Klimisch score of one and is classified as reliable without restrictions because it was an acceptable study that was well documented.