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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed according to EPA and OECD guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EPA OPP 82-4 (90-Day Inhalation Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
no recovery groups included, histopathology only for respiratory tract
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Naphthalene
- Substance type: Aromatic hydrocarbon
- Physical state: Grey-white crystalline solid
- Analytical purity: Assumed to be pure
- Impurities (identity and concentrations): None provided
- Lot/batch No.: LI-1 (LX No: LX158-01)
- Expiration date of the lot/batch: (a) 27 April 1993, (b) 25 June 1993
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
SOURCE:
- Age at study initiation: 8 ½ weeks
- Weight at study initiation: Male 221 – 272g; female 155 – 190g
- Fasting period before study: None
- Housing: Group housed 5/sex/cage
- Diet: ad libitum (while in cages)
- Water: ad libitum
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 – 22°C
- Humidity (%): 30 – 67
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): 12 hr dark / 12 hr light

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: Not applicable; vapour
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION:
-Exposure apparatus: Nose only inhalation chamber (ADG Instruments) with 20 exposure ports and a top section incorporating a tangential air inlet.Each chamber was assembled in 3 sections and formed a 28 cm diameter cylinder with a volume of approximately 50 litres. A separate exposure chamber was used for each group.
- Method of holding animals in test chamber: Molded polycarbonate tubes tapered at one end to allow the nose-only of the rat to protrude from the tapered end of the chamber. The other end was closed by insertion of an expanded plastic bung.
- Source and rate of air: Air was withdrawn from the base of the chamber at a rate of 30 litres/minute. The air was withdrawn by a vacuum pump through filtration media, to remove particulate, and a silica gel to remove excess moisture. The air supply to the chamber was comprised of a carrier (vapour) and a diluent air supply, to a total of 25 litres/minute to allow the exposure chamber to be maintained at a slight negative pressure.
- Method of conditioning air: See above
- System of generating particulates/aerosols: A separate vapour generation system was used for each group. It consisted of a 3-necked round bottom flask containing an aliquot of the test substance. Air was passed through the flask and vapour-laden air passed out through a second neck. The third neck contained a thermometer. At lower concentrations the test atmosphere was generated under ambient temperatures using the carrier airflow to maintain the concentration. At higher concentrations the test substance flask and carrier air was heated in a water bath to assist vaporisation. The vapour-laden air was passed through a clear plastic tube with glass wool (particulate trap) and mixed with diluent air (where applicable) before entering the chamber.
- Temperature, humidity, pressure in air chamber: Temperature 19.7 – 20.1°C; Humidity 45 – 52%; Oxygen concentration 21%
- Air flow rate: 30 litres/minute
- Air change rate: (30 l/min)/50 l; 36/hour
- Method of particle size determination: Not applicable


TEST ATMOSPHERE:
- Brief description of analytical method used: GC analysis of samples reacted with carbon disulfide. Column: 2mm x 1m i.d. glass packed with 10% OV-101 (80 -100 mesh). Column conditions: temperature- injector 200°C, column 120°C, detector 250°C. Gases: helium 30ml/min, hydrogen 33 ml/min, air 300ml/min: Retention time for Naphthalene 1.3 minutes.
- Samples taken from breathing zone: Yes

VEHICLE (if applicable):
- Justification for use and choice of vehicle: Air; Not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples taken at 1, 3 and 5 hours for all treatment groups during each exposure.
Brief description of analytical method used: GC analysis of samples reacted with carbon disulfide. Column: 2mm x 1m i.d. glass packed with 10% OV-101 (80 -100 mesh). Column conditions: temperature- injector 200°C, column 120°C, detector 250°C. Gases: helium 30ml/min, hydrogen 33 ml/min, air 300ml/min: Retention time for Naphthalene 1.3 minutes.
Samples taken from breathing zone: Yes
Duration of treatment / exposure:
13 consecutive weeks
Frequency of treatment:
Six hours exposure for 5 days a week
Doses / concentrations
Remarks:
Doses / Concentrations:
Target 2, 10, 60 ppm: analysed 2, 10, 58 ppm (11, 51, and 304 mg/m3)
Basis:
analytical conc.
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: 4 week inhalation study
Rationale for animal assignment (if not random): Computer distribution based on body weight
Rationale for selecting satellite groups: None
Positive control:
none

Examinations

Observations and examinations performed and frequency:
Observations and examinations performed and frequency

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice a day
- Cage side observations checked in table [No. 9.1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, Weekly

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: Not calculated but data present

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to allocation and during week 13
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13
- Anaesthetic used for blood collection: Yes, light ether anaesthesia
- Animals fasted: Yes
- How many animals: All
- Parameters checked in table [No. 9.7] were examined. PCV, Hb, RBC, MCHC, MCV, WBC, Diff, Plts, TT, Retic, and P, H, A, and R.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13
- Animals fasted: Yes
- How many animals: All
- Parameters checked in table [No. 9.8] were examined. CPK, Glucose, GPT, GOT, g-GT, AP, total protein, Alb, Glob, Urea Nitr, Total bilirubin, Creatine, NA, K, Ca, P, Cl, and Chol.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Bartlett’s for homogeneity, if heterogeneous then Kruskal-Wallis. Analysis of variance (Student’s t test) for dose response.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
3 deaths but not related to treatment. Increase in brown staining of fur but not considered toxicologically significant.
Mortality:
mortality observed, treatment-related
Description (incidence):
3 deaths but not related to treatment. Increase in brown staining of fur but not considered toxicologically significant.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weight gain at 58 ppm in males and females. Reduced body weight gain at 10 ppm in males.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduced food consumption at 10 and 58 ppm in males. Reduced food consumption at 58 ppm in females (not statistically significant).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
Lymphocyte and total WBC were reduced in male rats, not considered treatment-related, as the control values were found at the high end of the normal range.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
All differences were inconsistent between sexes, not dose-related and therefore not considered to be of toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
A decrease in absolute liver weight but increase in relative liver weight to body weight only in high-dose males. In the absence of microscopic changes, it was considered unlikely to be of toxicological significance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Stained body fur at 10 and 58 ppm. Could be the result of method of restraint used.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See "details on results".
Histopathological findings: neoplastic:
no effects observed
Details on results:
Histopathology non-neoplastic:
Degenerative changes in the olfactory epithelium including slight disorganisation, occasional degenerated cells, atrophy and erosion. The changes were more severe at 10 and 58 ppm and generally less severe at mild grades of individual lesions at 2 ppm. Also subepithelial effects were observed in Bowman’s glands at all exposure levels.
Proliferative lesions of the olfactory epithelium at the 58 ppm dose included hyperplasia of basal cells, rosette formation, hyperplasia (with loss of olfactory features), and in a single case early squamous metaplasia. The dose response of the proliferative lesions were not so obvious as of the degenerative changes due to the fact that continuous degenerative damage at the 60 ppm level resulted in increased cell death, thus restricting the proliferative (reparatory) changes.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Remarks:
for nasal inflammation
Remarks on result:
not determinable
Remarks:
no NOAEC identified
Dose descriptor:
NOAEL
Remarks:
for systemic effects
Effect level:
ca. 300 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no direct database for this endpoint, however conclusive In synopsis with results from carcinogenicity (see there) and oral repeated-dose studies.
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Dose descriptor:
LOAEC
Effect level:
0.011 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Histopathology: local degenerative effects in the olfactory region - atrophy, hyperplasia of epithelium, loss of Bowman´s glands
Dose descriptor:
LOAEC
Effect level:
2 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see histopathologic effects above

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Histological Effects of Naphthalene on Nasal Passages

Nasal passages

Males

(animals affected/10)

Females

(animals affected/10)

Dose in ppm

0

2

10

58

0

2

10

58

 

 

 

 

 

 

 

 

 

Olfactory epithelium

Slight disorganisation

0

4

0

0

0

4

0

0

Eosinophilic inclusions (total)

0

1

1

2

0

0

2

0

Minimal

0

1

1

1

0

0

2

0

Moderate

0

0

0

1

0

0

0

0

Occasional degenerate cells (total)

0

4

4

8

0

3

3

8

Trace

0

0

1

0

0

0

0

0

Minimal

0

4

2

5

0

3

3

7

Moderate

0

0

1

3

0

0

0

1

Atrophy (total)

0

9

9

10

0

9

10

10

Trace

0

1

0

0

0

0

0

0

Minimal

0

8

1

0

0

0

0

0

Moderate

0

0

8

8

0

0

9

6

Marked

0

0

0

2

0

0

0

4

Erosion (total)

0

1

4

6

0

0

5

5

Trace

0

0

1

0

0

0

0

1

Minimal

0

1

2

4

0

0

4

1

Moderate

0

0

1

2

0

0

1

3

Hyperplasia of basal cells (total)

0

3

8

6

0

6

7

6

Trace

0

0

1

0

0

0

0

0

Minimal

0

2

4

4

0

5

6

5

Moderate

0

1

3

2

0

1

1

1

Rosette formation (total)

0

3

7

4

0

3

7

6

Trace

0

1

0

1

0

1

0

0

Minimal

0

2

6

2

0

2

6

6

Moderate

0

0

1

1

0

0

1

0

Hyperplasia (total)

0

0

0

0

0

2

3

0

Minimal

0

0

0

0

0

2

3

0

Early squamous metaplasia - minimal

0

0

0

0

0

0

0

1

 

 

 

 

 

 

 

 

 

Bowman’s Glands

Atrophic cells

0

1

6

1

0

4

6

5

Loss of glands around dorsal meatus

0

5

9

10

0

6

9

10

 

 

 

 

 

 

 

 

 

Respiratory epithelium

Dilated gland ducts

0

1

3

1

3

1

1

0

Hypertrophy

0

0

4

10

0

0

6

6

Squamous metaplasia

0

1

0

0

0

0

0

0

Applicant's summary and conclusion

Conclusions:
LOAEL 2 ppm based on minor histopathological changes in the nasal epithelium.
Executive summary:

Rats were exposed to test atmospheres containing naphthalene at concentrations of 2, 10 and 58 ppm. Exposures were 6 hours continuous each day, 5 days a week for 13 weeks.

Statistically significant reduced body weight gain was seen in rats of both sexes exposed to 58 ppm and in male rats exposed to 10 ppm. The reductions in weight gain were associated with reduced food consumption. Body-weight gain and food consumption were also minimally lower than control values for other exposed groups/sexes, but did not reach statistical significance.

Laboratory investigations revealed no evidence of treatment-associated changes or systemic toxicity, and organ-weight analysis and macroscopic findings at necropsy also revealed no adverse changes.

Microscopic examination revealed treatment-related effects on the nasal epithelium at all exposure levels. The severity of the effects was dose-related. At the highest level (58 ppm) changes included erosion of the olfactory epithelium, hyperplasia of basal cells in the olfactory epithelium, loss of Bowman’s glands, hypertrophy of the respiratory epithelium and other inflammatory changes.

At the lowest level of exposure (2 ppm) changes in the olfactory epithelium were less marked but included slight disorganisation, eosinophilic inclusions, occasional degenerate cells, minimal atrophy, minimal erosion (in a single rat), minimal hyperplasia of basal cells, minimal rosette formation, atrophic cells in Bowman’s glands and loss of Bowman’s glands and presence of cysts. In addition dilated gland ducts and squamous metaplasia (in a single rat) were also seen at the low level of exposure (not dose-responsive).

A "No-Effect Level" for the nasal lesions was not established in this study.