Registration Dossier

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
- Detailed examination for clinical signs of toxicity was performed weekly instead of daily.
Qualifier:
according to
Guideline:
EPA OPP 82-4 (90-Day Inhalation Toxicity)
Qualifier:
according to
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
US Type P1/13 based on GC/MS (Selection of Key Compounds)

- Substance type: organic
- Physical state: liquid

Distilled Coal Tar, Complex Hydrocarbon Mixture

Component CAS-No. [%][US P1/13]

Naphthalene 91-20-3 9.0
1-Methylnaphthalene 90-12-0 2.3
2-Methylnaphthalene 91-57-6 5.1
Indene 95-13-6 0.9
Fluorene 86-73-7 4.2
Acenaphthylene 208-96-8 0.3
Acenaphthene 83-32-9 6.1
Phenanthrene 85-01-8 12.2
Anthracene 120-12-7 2.2
Fluoranthene 206-44-0 6.8
Pyrene 129-00-0 6.0
Benz[a]anthracene 56-55-3 0.5
Chrysene 218-01-9 1.5
Benzo(a)pyrene 50-32-8 0.5

Total of 5 remaining EPA-PAH: ca. 1.4

(Benzo[b]fluoranthene, 205-99-2 0.8
Benzo[k]fluoranthene, 207-08-9 0.3
Indeno[1,2,3-cd]pyrene, 193-39-5 0.1
Dibenzo[a,h]anthracene, 53-70-3 not identified
Benzo[ghi]perylene, 191-24-2 <0.1

Test animals

Species:
rat
Strain:
other: Crl:CD® BR VAF/PLUS®
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan, USA
- Age at study initiation: Approx. 6 weeks
- Weight at study initiation: Males: 181-211 g; females: 130-149 g

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: MMAD [µm] (± GSD [µm])
Low dose: 3.0 (1.92)
Mid dose: 2.2 (1.99)
High dose: 2.4 (1.91)
Details on inhalation exposure:
--
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Nominal concentration: Calculated from the amount of test compound used during the exposure period (by weighing the reservoir before and after the exposure period) and dividing the total creosote consumed by the total air volume passed through the chamber.
Analytical aerosol concentration: Determined by gravimetric determination of the oil amount adsorbed onto a 25-mm glass-fibre filter pad, divided by the sample volume.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 h/d, 5 days per week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Mean nominal concentration [mg/m³]: 22, 128, and 221
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
Mean aerosol concentration [mg/m³]: 5.4, 49, 106
Basis:
analytical conc.
No. of animals per sex per dose:
20/sex/group
An additional group (5/sex) was sacrificed pre-test to define a baseline for clinical chemistry and haematological values.
Control animals:
yes, sham-exposed
Details on study design:
--
Positive control:
none

Examinations

Observations and examinations performed and frequency:
Mortality: Yes, twice weekly

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: pre-test and weekly

FOOD CONSUMPTION: Yes - weekly

FOOD EFFICIENCY: No data

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes, prior to terminal necropsy and prior to recovery necropsy.

HAEMATOLOGY: Yes
Number of animals: 5/sex at pre-test, 10/sex/group at termination of study and on surviving recovery animals at the end of the recovery period.
Parameters: haematocrit, haemoglobin, erythrocyte count, total and differential leukocyte count, platelet count, reticulocytes, MCV, MCH, MCHC

CLINICAL CHEMISTRY: Yes
Number of animals: 5/sex at pre-test, 10/sex/group at termination of study and on surviving recovery animals at the end of the recovery period.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Organ weights:
Organs: adrenal, brain, ovary, testis with epididymis, heart, kidney, liver, lung, mammary gland, thymus, thyroid/parathyroid, trachea

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals: external examination; contents of abdominal, thoracic and cranial cavities were examined both in situ and after removal and dissection.

HISTOPATHOLOGY: Yes
All animals: heart, thyroid, nasal tissues, trachea, and lungControl and high dose group, animals that died on study: adrenal, aorta, auditory sebaceous gland, bone with bone marrow (femur), bone marrow smear, brain (fore, mid and hind), eye including optic nerve and contiguous Harderian gland, gastrointestinal tract, gonads, heart, kidney, larynx, lachrymal gland, liver, lung, lymph nodes, mammary gland (females only), nasal tissues, pancreas, pituitary, prostate and seminal vesicle, salivary gland, sciatic nerve, skeletal muscle (thigh), skin, spinal cord, spleen, thymus, thyroid/parathyroid, trachea, tracheal bifurcation, urinary bladder, vagina, uterus and cervix
Other examinations:
Ten animals per dose group and sex were subject to a six-week post-exposure period after which they were sacrificed and examined macroscopically for reversibility of eventual effects.
Statistics:
Analysis of body weights, food consumption, clinical pathology laboratory tests and organ weights were performed as follows:Generally, when the number of animals in any one group was ≤10, non-parametric analysis was conducted using the KRUSKAL-WALLIS one-way analysis of variance, followed by the MANN-WHITNEY U test, where appropriate. In those cases where the number of animals in all groups was greater than ten an the measurements were on at least an interval scale (continuous data), parametric analysis was conducted utilizing BARTLETT’s chi-square test for homogeneity of variance, followed by an analysis of variance and then, where appropriate, by DUNNETT’s t-test.

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 specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY:
One male in the mid-dose group died during the exposure phase (week 4) of this study.

BODY WEIGHT AND WEIGHT GAIN
Body weight decreases were observed in the two highest exposure groups. They were significant in both sexes of the high-dose group and in mid-dose females during exposure week 6. Mean weights for the low-level group were similar to the control group.

FOOD CONSUMPTION:
With the exception of week 1 (all groups significantly less than controls) and week 3 (males in the high-dose group significantly less than controls), food consumption during the 13-week exposures and the six-week post-exposure recovery period was comparable to that of controls.

HAEMATOLOGY:
Several haematological parameters showed significant changes in the two highest exposure levels at the terminal sacrifice including: decreased erythrocytes, haemoglobin, and haematocrit; increased numbers of reticulocytes. These changes were not detectable at the end of the recovery period.

CLINICAL CHEMISTRY:
Serum cholesterol was significantly increased in males of the mid-dose group and in females of the two highest exposure groups. This change was not evident at the end of the recovery period (see table below).

ORGAN WEIGHTS:
Terminal sacrifice: There was a test-article-related and statistically significant increase in the lung/trachea/body weight ratio in males and females of the high-dose group when compared to the respective control values. These increases correlated with the macroscopic observation of grey discolouration of the lungs, and the microscopic observation of pigmented macrophages within the lungs of the animals in the affected groups. In mid- and high-dose males, the adrenal/body weight ratio was increased. No macroscopic or microscopic changes were associated with these changes.
In males, increases in liver weight (mid-dose group) and liver/body weight ratio (mid- and high-dose group) were noted. Relative liver weights were significantly increased at the mid and high dose (about +20 and +25 %, respectively).
In females, there were increases in liver weight (high-dose group), liver/body weight ratio (mid- and high-dose group) and liver/brain weight ratio (mid- and high-dose group) when compared to controls. No macroscopic or microscopic changes were associated with these changes, thus, their toxicbiological significance is uncertain.

Recovery sacrifice: There were no test-article-related changes in organ weight or weight ratios for males in any dose group. In mid-dose females, the mean adrenal weight was significantly decreased compared to controls. No macroscopic or microscopic observations were associated with that finding. Thus, it was deemed not test article related.

GROSS PATHOLOGY:
At the terminal sacrifice, a deposition of the test article consisting of a grey discolouration of the lung was seen at the two highest exposure levels. The discolouration persisted through the recovery period. The control and low-dose group

HISTOPATHOLOGY: NON-NEOPLASTIC:
Microscopic changes were observed in the hearts, lungs, nasal tissues, and thyroid glands of male and female rats at the time of terminal sacrifice. Heart lesions were found in one male of the mid-dose group that died on the study, and in one male and one female of the high-dose group (diffuse myocardial degeneration affecting mainly the right side of the heart). Associated to this change was diffuse arterial medial hypertrophy of small arterioles in the lung, brown pigment within the epithelial cells of the convoluted tubules of the kidney, and in the animal that died on study, alveolar macrophages containing brown pigment consistent with haemosiderin (“heart failure cells”) within the lung and diffuse centrilobular fibrosis within the liver. No heart lesions were found in any dose group at the end of the recovery period.
Note: Cardiac pathology (ie: hemorrhage, lymphocytic infiltration and cardiomyopathy) was noted in all animals of all groups (including controls).

Test-article-related changes in the lung were seen in all animals of the exposed groups (small black pigment granules within alveolar macrophages). Alveolar macrophages containing pigment granules could be detected in all lobes of the lungs indicating a uniform dispersion of the test article throughout the alveolar spaces of the lungs. There were no other changes within the lungs which could be associated with the presence of the granules, such as inflammation, increased number of pulmonary macrophages and/or Type-II pneumocyte hyperplasia. These findings were also seen at recovery sacrifice.

Nasal cavity: Small cystic spaces, containing basophilic mucoid material, within the olfactory epithelium at all levels of the nasal tissues examined and in both sexes, and were considered test article related. Mucoid cysts were seen in mid- and high-dose males, and in low-, mid-, and high-dose females. Similar findings were made at recovery sacrifice.
Other histological changes within the nasal tissues: Squamous metaplasia of respiratory and/or olfactory epithelium and naso­lachrymal duct epithelium, inflammatory cell infiltrates, and glandular dilation within the lamina propria/submucosa of the nasal cavity. These additional changes showed no definitive test article relationship.

Hypertrophy of thyroid follicular cells which resulted in a reduction in the amount of colloid present within the thyroid follicles was seen in both male and female rats of all exposure groups. This anomaly was considered test-article related. No test-article-related effects on the thyroid glands were detected at recovery sacrifice. There was no measurable effect on the mass of the thyroid gland (no changes in absolute and relative weights).

Effect levels

open allclose all
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
5.4 mg/m³ air (analytical)
Based on:
test mat.
Remarks:
aerosolic fraction
Sex:
male/female
Basis for effect level:
other: based on the death of one male, decreases in body-weight gain (>10 %) as well as on the increase in liver weight (>=20 %), and on the increase in hypertrophy of follicular cells of the thyroid gland
Dose descriptor:
NOAEC
Remarks:
local, nasal
Effect level:
5.4 mg/m³ air (analytical)
Based on:
test mat.
Remarks:
aerosolic fraction
Sex:
male/female
Basis for effect level:
other: based on local chronic inflammation reactions in the nasal cavity in both sexes
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
22 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see effects above under NOAEC systemic, analytical effect levels
Dose descriptor:
NOAEC
Remarks:
local, nasal
Effect level:
22 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on local chronic inflammation reactions in the nasal cavity in both sexes
Dose descriptor:
LOAEC
Remarks:
systemic
Effect level:
49 mg/m³ air (analytical)
Based on:
test mat.
Remarks:
aerosolic fraction
Sex:
male/female
Basis for effect level:
other: see effects above under NOAEC systemic, analytical effect levels
Dose descriptor:
LOAEC
Remarks:
local, nasal
Effect level:
49 mg/m³ air (analytical)
Based on:
test mat.
Remarks:
aerosolic fraction
Sex:
male/female
Basis for effect level:
other: based on local chronic inflammation reactions in the nasal cavity in both sexes
Dose descriptor:
LOAEC
Remarks:
systemic
Effect level:
128 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see effects above under NOAEC systemic, analytical effect levels
Dose descriptor:
LOAEC
Remarks:
local, nasal
Effect level:
128 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on local chronic inflammation reactions in the nasal cavity in both sexes

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Results of clinical chemistry and haematology

Parameter changed

Unit

Controls
0 mg/m³

Low dose
5.4 mg/m³

Medium dose
49 mg/m³

High dose
106 mg/m³

Time point

Terminal

Recovery

Terminal

Recovery

Terminal

Recovery

Terminal

Recovery

Males

Haemoglobin

g/dl

15.5

15.2

15.4

15.3

14.3**

15.0

14.6

15.1

Haematocrit

%

44.7

40.0

43.5

40.1

39.7*

39.7

40.3

39.8

Reticulocytes

/ 100 RBC

2.8

2.1

2.8

1.8

4.0

1.6

5.2

1.6

Phosphorus

mg/dl

7.6

7.0

8.9

6.9

8.3

6.7

8.5**

6.8

ALT

U/L

34

33

28

35

28

37

25*

31

Cholesterol

mg/dl

52

70

52

72

74*

69

70

78

Females

Haemoglobin

g/dl

15.4

15.0

15.3

15.5

14.6

15.5

13.5**

15.5

Haematocrit

%

41.4

38.8

40.9

39.6

37.8

39.6

35.0**

39.7

Reticulocytes

/ 100 RBC

2.9

2.3

3.0

2.2

3.4

2.0

7.8*

1.5

g-GT

U/L

1

3

1

1

3*

1

4*

2

Cholesterol

mg/dl

77

94

76

103

109**

99

116**

77

*  p≤ 0.05;              **p≤ 0.01

Applicant's summary and conclusion