Creosote oil, acenaphthene fraction

Endpoint summary

Description of key information

No experimental data is available on wash oil itself. Results from key components as well as from a structure-related tar oil are used to draw conclusions by analogy.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Basic data given, comparable to guideline study (deviations: no satellite groups, interim sacrifices and examinations, only 2 doses tested, limited reporting)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

; no satellite groups, interim sacrifices and examinations, only 2 doses tested, limited reporting

Principles of method if other than guideline:

No guideline stated, but the method used is similar to OECD test guideline 543

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Shizuoka Laboratory Center, Shizuoka, Japan
- Age at study initiation: 6 weeks
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: in plastic cages with wood chips for bedding, 10 animals per cage
- Diet (e.g. ad libitum): powdered CRF-1 diet (Charles River Japan Inc., Kanagawa, Japan), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 55 ± 10
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: feed

Vehicle:

corn oil

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): every month
- Mixing appropriate amounts with (Type of food): appropriate amounts of test substance were dissolved in corn oil and
mixed with powdered CRF-1 diet
- Storage temperature of food: in light-shielded boxes at room temperature

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Amount of vehicle: final concentration in diet 5%

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

81 weeks

Frequency of treatment:

daily (feeding study)

Remarks:

Doses / Concentrations:
0.075% and 0.15%
Basis:
nominal in diet

No. of animals per sex per dose:

50

Control animals:

other: yes, concurrent vehicle; but: only one male and one female control group was used for both studies, the 1-methylnaphthalene study and the 2-methylnaphthalene study.

Details on study design:

- Dose selection rationale: dose selection based on the results of a subchronic (13 weeks) preliminary feeding study
(concentrations 0.0163, 0.049, 0.147, 0.44, and 1.33% test substance in diet)
- Rationale for selecting satellite groups: no satellite groups

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: every week for the first 16 weeks and every other week thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption: measured over three days before every weighing; from a total of 40 measurements total food consumption
and test substance intake and daily intake was determined.

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 data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of exposure period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No
- How many animals: all surviving animals
- Parameters examined: white blood cell count (WBC), red blood cell count (RBC), hemoglobin (Hb), hematocrit (Ht),
mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCVC),
differential white blood cell counts

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the exposure period
- Animals fasted: No
- How many animals: all surviving animals
- Parameters examined: aspartic acid aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP),
lactate dehydrogenase (LDH), cholinesterase, gamma-glutamyl transpeptidase (gamma-GTP), total bilirubin, total protein,
albumin, urea nitrogen, uric acid, creatinine, total lipid, ppospholipid, neutral fat, noesterified fatty acids (NEFA), cholesterol,
esterified cholesterol, HDL-cholesterol, beta-lipoprotein, lipid peroxide, Na, K, Cl, Fe

URINALYSIS: Yes

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Organ weights determined: brain, salivary glands, heart, thymus, lung, liver, pancreas, spleen, kidneys, and testes
- Preparation of organs: organs above and in addition adrenals, trachea, stomach, small intestine, large intestine, seminal vesicle, pituitary gland,
uterus, vagina, mammary gland, skeletal muscle, eye, harderian gland, spinal cord, bone (sternal bone, ribs, vertebral bone) skin and other
tissues with abnormal appearance were fixed in 10% neutralized Formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Particular for lung, a horizontal section per each lobe was made to enable the examination of as much of the lung as possible

HISTOPATHOLOGY: Yes
- Histopatological examinations were performed on all sacrificed animals and also on animals found dead or killed upon becoming moribund

Statistics:

Incidence data on tumor and non tumor lesions were anayzed by the chi² test.
Data for body weights, organ weights, and peripheral blood and serum parameters were analyzed by Student's t test.

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:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
Abnormal clinical signes were not reported.
One female animal receiving the 0.15% diet (high dose) died of leukemia at week 68. One male animal of the control group also died of
leukemia at week 60. The deaths of the two animals are not related to the test substance.

BODY WEIGHT AND WEIGHT GAIN
Both female and male mice receiving 0.15% test substance in diet exhibited slight growth retardation compared with the
control group after 10 weeks, but recovery was complete after 80 and 72 weeks, respectively. Slight temporary growth retardation
is not considered to be an relevant adverse effects.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption did not show any variation among the groups throughout the experimental period for both females and males.
Intake of test substance was calculated based on 40 food weighings (see under Examinations). Intake levels (mg/kg day)
are displayed graphically in the publication individually for both males and females and high and low exposure groups.
Summarized results are tabulated under "Any other information on results incl. tables".

FOOD EFFICIENCY
Not examined

WATER CONSUMPTION
Not examined

OPHTHALMOSCOPIC EXAMINATION
Not examined

HAEMATOLOGY
White and red blood cell counts were not significantly different for controls and test groups. But monocytes were significantly
increased in all treated mice as compared to controls. Increases appeared to be dose related.
Slight but statistically significant increases in Hemoglobin, MCH, and MCHC were observed for both groups of female
but not of male mice treated with test substance as compared with controls. Differences were not dose related.
The biological significance of these effects is not clear.

CLINICAL CHEMISTRY
For some parameters, statistically significant differences between test groups and controls were observed (albumin, total lipids,
phospholipids, neutral fat). But there was no dose relationship. Variations are not considered to be caused by the test substance
but to result from variance due to examination of not fasted animals.

URINALYSIS
Not examined

NEUROBEHAVIOUR
Not examined

ORGAN WEIGHTS
Significantly increased brain weights of male animals treated with 1-MN are due to a low brain weight of male controls, which
were clearly reduced compared to female controls. Brain weights of treated females and males were comparable.
The weight of salivary glands of female mice and hearts of both female and male mice given 1-MN were significantly decreased with
no clear dose-effect relationship. The causes of these increases or decreases are unknown and seem not to be related to test substance
administration.
Increased thymus weights observed in control female mice was due to lymhoma development.

GROSS PATHOLOGY
Pulmonary alveolar proteinosis was diagnosed in numerous mice treated with 1-MN. For incidences see belfow.
Proteinosis appeared as white protuberant nodules of 1-5 mm in diameter.

HISTOPATHOLOGY: NON-NEOPLASTIC
see "Any other information on results incl. tables"

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
see "Any other information on results incl. tables"

HISTORICAL CONTROL DATA (if applicable)
Broncheolar/alveolar adenomas did not occur spontaneously in B6C3F1 mice in an earlier experiment
(Yokose et al 1987, Environ Health Perspect. 76, 205-210).
Liver tumors are known to be the most frequent neoplastic changes that occur spontaneously in male B6C3F1 mice.

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Adverse effects were seen already at the lowest test dose

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Dose descriptor:

LOAEL

Remarks:

(= 0.075 % in diet)

Effect level:

ca. 70 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Gross pathology and histology of the lung

Critical effects observed:

not specified

Intake of test substance

 

Total intake over the experimental period was 42.6 and 40.6 g/kg in the low dose group and 81.5 and 79.5 g/kg in the high dose group for females and males, respectively. The mean daily intake is listed in the table below

 

Mean daily intake [mg /(kg bw *day)]

 

Dose group	Females	Males	Mean
0.075% (low dose)	75.1	71.6	73.4
0.15% (high dose)	143.7	140.2	142.0

 

At the beginning of the study, intake was about 2-fold higher than at the end of the exposure period..

 

 

Non neoplastic lesions

 

Primary non neoplastic lesion was the development of pulmonary alveolar proteinosis (PAP) demonstrating that the main target organ is the lung.

 

Incidences of pulmonary alveolar proteinosis

 

	Females			Males
Dose (% in diet)	0 (controls)	0.075	0.15	0 (controls)	0.075	0.15
No. of mice	50	50	49	49	50	50
Incidence (%)	5 (10)	23 (46)*	17 (34.7)*	4 (8.2)	23 (46)*	19 (38)*

 

* Significantly different from the control value by chi² test p < 0.01

 

No dose dependence was evident for the incidences of PAP.

 

Numbers of mice with PAP with tumors were not statistically correlated.

In addition, the sites of development of PAP and lung tumors were also not always clearly linked.

 

Other non neoplastic lesions were only present at very low incidences and no significant differences were

found between controls and treated animals (data not included in table).

 

 

Neoplastic lesions

 

Incidences of tumors

 

	Incidences (%)
	Females			Males
Dose (% in diet)	0 (controls)	0.075	0.15	0 (controls)	0.075	0.15
No. of mice	50	50	49	49	50	50
Lung
Adenoma	4 (8.0)	2 (4.0)	4 (8.2)	2 (4.1)	13 (26.0)*	12 (24.0)*
Adenocarcinoma	1 (2.0)	0	1 (2.0)	0	0	3 (6.0)
Total	5 (10.0)	2 (4.0)	5 (10.2)	2 (4.1)	13 (26.0)*	15 (30.0)*
Liver
Adenoma	0	2 (4.0)	4 (8.2)	9 (18.4)	1 (2.0)	1 (2.0)
Hepatocell. carcinoma	0	0	1 (2.0)	11 (22.4)	6 (12.0)	3 (6.0)
Total	0	2 (4.0)	5 (10.2)	20 (40.8)	7 (14.0)	4 (8.0)
Lymphoma/Leukemia	4 (8.0)	2 (8.0)	3 (6.1)	1 (2.0)	0	1 (2.0)

 

* Significantly different from the control value by chi² test p < 0.05

Other adverse effects

No other exposure-related adverse effects were observed in any other organs or tissues.

Executive summary:

In a combined chronic toxicity/carcinogenicity feeding study, groups of 50 males and females B6C3F1 mice were exposed for 81 weeks to 1-methylnaphthalene given in concentrations of 0.075 and 0.15% in diet. There were no satellite groups and no interim sacrifices and examinations. Mean test substance intake over the experimental period was 75.1 and 71.6 mg/kg bw/day in the low dose group and 143.7 and 140.2 mg/kg bw/day in the high dose group for females and males respectively.

As main non-neoplastic lesion, pulmonary alveolar proteinosis (PAP) was observed (incidence: low dose group - females and males 46%; high dose group - females 34.7% and males 38%, while 10 and 8 % of the female and male control group, respectively, were affected).

A NOAEL could not be derived. As neoplastic lesions, adenoma and adenocarcinoma were observed. Incidence of adenoma was statistically increased over controls only for male animals (26% and 24% low and high dose group - no dose-effect dependence) but not for females. For adenocarcinoma, there were no statistically significant differences in incidences for both male and female animals. The rate of hepatocellular carcinoma spontaneously occurring in male B6C3F1 mice was not increased in the 1-methynaphthalene treated male dose groups but rather decreased compared to controls.

A BMD has not been derived: Given the less marked effects of 1 -MN at somewhat higher doses as compared to 2 -MN, the BMD of 1 -MN is supposed to be distinctly higher than that for 2 -MN.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

BMD05

4.7 mg/kg bw/day

Study duration:

chronic

Species:

mouse

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

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

Qualifier:

according to guideline

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

Guideline:

EPA OPP 82-4 (90-Day Inhalation Toxicity)

Qualifier:

according to guideline

Guideline:

EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD® BR VAF/PLUS®

Sex:

male/female

Details on test animals or test system 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

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

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

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.

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).

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

22 mg/m³ air (nominal)

Based on:

test mat.

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:

systemic

Effect level:

5.4 mg/m³ air (analytical)

Based on:

test mat.

Remarks:

aerosolic fraction

Sex:

male/female

Basis for effect level:

other: see effects above under nominal 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: local chronic inflammation reactions in the nasal cavity in both sexes

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: see effects above under nominal effect levels

Dose descriptor:

LOAEC

Remarks:

systemic

Effect level:

128 mg/m³ air (nominal)

Based on:

test mat.

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:

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 nominal 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: local chronic inflammation reactions in the nasal cavity in both sexes

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: see effects above under nominal effect levels

Critical effects observed:

not specified

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

22 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

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

Qualifier:

according to guideline

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

Guideline:

EPA OPP 82-4 (90-Day Inhalation Toxicity)

Qualifier:

according to guideline

Guideline:

EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD® BR VAF/PLUS®

Sex:

male/female

Details on test animals or test system 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

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

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

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.

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).

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

22 mg/m³ air (nominal)

Based on:

test mat.

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:

systemic

Effect level:

5.4 mg/m³ air (analytical)

Based on:

test mat.

Remarks:

aerosolic fraction

Sex:

male/female

Basis for effect level:

other: see effects above under nominal 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: local chronic inflammation reactions in the nasal cavity in both sexes

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: see effects above under nominal effect levels

Dose descriptor:

LOAEC

Remarks:

systemic

Effect level:

128 mg/m³ air (nominal)

Based on:

test mat.

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:

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 nominal 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: local chronic inflammation reactions in the nasal cavity in both sexes

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: see effects above under nominal effect levels

Critical effects observed:

not specified

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

22 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable well-documented publication which meets basic scientific principles

Qualifier:

no guideline followed

Principles of method if other than guideline:

Repeated-dose study employing one dose only: Primary goal of the study was to investigate by means of light and electron microscopy the ultrastructure of lesions found in the lungs of mice after induction of pulmonary alveolar proteinosis by dermal application of methylnaphthalene (mixture of 1- and 2-methylnaphthalene).

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Lab., Osaka, Japan
- Age at study initiation: no data
- Weight at study initiation: about 17 g
- Fasting period before study: no
- Housing: no data
- Diet (e.g. ad libitum): pelleted diet (MF, Oriental Yeast Ind., Tokyo, Japan); ad libitum
- Water (e.g. ad libitum): tap water; ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- no data

Type of coverage:

open

Vehicle:

acetone

Details on exposure:

TEST SITE
- Area of exposure: back of mice
- % coverage: no data
- Time intervals for shavings or clipplings: no data

REMOVAL OF TEST SUBSTANCE: no

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): ca. 119 mg/kg bw per application
- Concentration (if solution): 1.2 % (in acetone), test solution was administered to the back skin of mice by means of a syringe
- Constant concentration used: yes

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Amount(s) applied (volume or weight with unit): no data
- Concentration (if solution): 1.2 % test substance in acetone

USE OF RESTRAINERS FOR PREVENTING INGESTION: no

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

--

Duration of treatment / exposure:

30 weeks

Frequency of treatment:

twice weekly

Remarks:

Doses / Concentrations:
119 mg/kg bw per application (ca. 34 mg/kg bw/day)
Basis:
nominal per unit body weight

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: only single dose administered

Observations and examinations performed and frequency:

No observations or examinations are reported for the exposure period.
At the end of the study, final body weights were determined.

Sacrifice and pathology:

GROSS PATHOLOGY: at sacrifice, only lungs were removed, inspected and prepared for microscopic (light and electron microscopy) examination

HISTOPATHOLOGY: Yes; only lungs were examined.
At autopsy, the lungs were removed and fixed in 10% formaline for light microscopy.
For electron microscopy, samples of resected lung were fixed in 300 mM glutaraldehyde containing 0.1% tannic acid buffered
with 100 mM sodium cacodylate, post-fixed in 40 mM OsO4, dehydrated in ethanol, substituted with propylenoxid and embedded
in epoxy resin. Sections from paraffin blocks were stained with haematoxylin and eosin. One µm sections from epoxy resin were
stained with 0.5% toluidine blue. Ultrathin sections were doubly contrasted with 1% uranyl acetate and Reynolds' solution (2%).

Clinical signs:

not specified

Dermal irritation:

not specified

Mortality:

not specified

Body weight and weight changes:

effects observed, treatment-related

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:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

BODY WEIGHT AND WEIGHT GAIN
Average final body weight of mice treated with methylnaphthalene in acetone was 27.2 ± 3.2 g.
Average final body weight of control mice was 31.5 ± 1.7 g.
The average final body weight of treated animals was reduced by about 14% compared to controls.

GROSS PATHOLOGY
The lung surfaces demonstrated multiple, grayish white, soft spots or nodules sharply demarcated from the pinkish-white
surrounding normal tissues, without specific localization. No such lesions were observed in the control group.

HISTOPATHOLOGY: NON-NEOPLASTIC
The alveoli were found to be filled with amorphous eosinophilic material, many mononucleated cells with abundant foamy cytoplasm,
and many clefts corresponding to cholesterol crystals separating the intra-alveolar materials and the lining cells.
The alveolar walls were thickened but no prominent fibrosis was observed. Terminal bronchiols were not markedly affected.

Dose descriptor:

LOAEL

Remarks:

(30 wks, 7 d/wk)

Effect level:

34 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Induction of pulmonary alveolar proteinosis (100% induction, only one dose tested)

Critical effects observed:

not specified

Lung was the only organ examined. Pulmonary alveolar proteinosis was found in all animals treated with methylnaphthalene (incidence 100%).

Executive summary:

Following dermal application of methylnaphthalene (1.2 % in acetone, ca. 119 mg/kg bw per application) to the back skin of female B6C3F1 mice (group of 15 animals) twice weekly for 30 weeks, pulmonary alveolar proteinosis was induced in all of the test animals. Lung lesions were characterized by light and electron microscopy. Besides lungs, no other organs/parameters were examined.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

34 mg/kg bw/day

Study duration:

chronic

Species:

mouse

Quality of whole database:

single dose only, only lung and respiratory tract observed / no NOAEL obtained

Additional information

No experimental data is available for creosote oil, acenaphthene fraction (wash oil) itself. Thus, information from other substances is used as substitute. Repeated dose toxicity data are available for 1- and 2-methylynaphthalene (oral and dermal route), for acenaphthene (oral route), and for the closely structure-related tar oil creosote (inhalation route). These substances will be used as supporting substances.

1- and 2-methylnaphthalene are major constituents amounting to about 30 % of total wash oil (typical concentration). The same holds for acenaphthene as well with a typical concentration of ca. 15 %. Creosote is largely related to wash oil with the same major constituent as in wash oil. Small differences exist for the concentration of methylnaphthalenes (somewhat lower than in creosote) and of phenanthrene to fluoranthene (somewhat higher than in creosote) due to a somewhat different distillation range (210 to 300°C for wash oil compared to 200 to 355°C for creosote).

Oral exposure

2-Methylnaphthalene, a substantial constituent of wash oil, produced pulmonary alveolar proteinosis in an 81 wk feeding study in mice. The LOAEL was about 50 mg/kg bw/day, the lowest dose tested (Murata et al. 1997).

1-Methylnaphthalene caused the same effect. The LOAEL was somewhat higher at about 70 mg/kg bw/day (Murata et al. 1993).

The data of the study with 2-methylnaphthalene (Murata et al. 1997) were used by US EPA to derive a BMD05 by assessing the data and evaluating them with a BMD modelling program (US EPA BMDS Version 1.3.2. software). A BMD05 of 4.7 mg/kg bw/day was established from a quantal-linear model taking into account all available toxicological information (US EPA 2003b, US EPA IRIS 1994).

Acenaphthene, another substantial wash oil component, showed no substance-specific intoxication but an adaptive response of the liver without histopathological manifestation in a 90 d feeding study with rats (US EPA 1994).

Dermal exposure

A 30 wk dermal exposure study with female mice receiving one dose of a mixture of 1- and 2-methylnaphthalene [CAS No. 1321-94-4] (119 mg/kg bw per application, 2x/wk (ca. 34 mg/kg bw/day)) resulted in the same pulmonary effect already noted after oral exposure (Murata et al. 1992).

Inhalation exposure

Inhalation of aerosolic creosote, a structure-related tar oil, caused nasal irritation and chronic inflammation in the nasal cavity of rats exposed for 13 weeks. No particular systemic effects were observed but treatment-related hypertrophic changes of the thyroidal gland, the liver and lung, as well as lung depositions of test material (Hilaski 1995). The NOAEC is reported as 22 mg/m³ (nominal) and includes both the particle and gaseous fraction of creosote. Analytically measured NOEC was determined to be 5.4 mg/m³. The nominal concentration is assessed to be more relevant and representative of the actual exposure as the analytical method only registered material deposited on filter pads, thus very likely underrating exposure because gas phase concentrations were not collected and analysed in the measurements.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
related to 1-methylnaphthalene, major constituent of wash oil, most relevant component for repeated dose toxicity with the lowest N/LOAEL amongst the studies identified. BMD05 value was derived by US EPA, based on the data set of the Murata study using a BMD modelling approach.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
related to the structure-related tar oil creosote providing evidence of adaptive response in liver and thyroid

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
related to the structure-related tar oil creosote providing evidence of chronic local adverse effects (inflammatory response in the nasal cavity)

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
related to 1- and 2-methylnaphthalene (major components)

Repeated dose toxicity: via oral route - systemic effects (target organ) respiratory: lung

Repeated dose toxicity: inhalation - systemic effects (target organ) respiratory: lung; respiratory: nose

Repeated dose toxicity: dermal - systemic effects (target organ) respiratory: lung; respiratory: nose

Justification for classification or non-classification

Adverse effects following prolonged oral or dermal dosing of methylnaphthalenes to mice have primarily been noted in the lung of the test animals. The effective doses (LOAELs) ranged from about 30 to 50 mg/kg bw/d, and hence are of the order of the guidance values for specific target-organ toxicity after repeated dosage of Category 2 (STOT RE 2) (see EU Regulation 1272/2008, Table 3.9.2). This holds for the oral and dermal route. Experimental observations following inhalation exposure to an aerosol of a wash-oil related tar-oil failed to reveal analogous signs of toxicity in the lung of rats, but produced chronic nasal inflammation at about 40 – 50 mg/m³ (local LOAEC), which would also be conform with STOT RE 2.

Methylnaphthalenes comprise of ≥10% in wash oil. This triggers classification of wash oil as STOT RE 2 with H373 (May cause damage to organs <lungs> through prolonged or repeated exposure <dermal, oral>).