Registration Dossier

Administrative data

Description of key information

NOAEC(inhal.) of 5 mg/m3 could be identified for local respiratory effects from a 90d study.
The NOAEC(90 d) for systemic respiratory effects was 300 mg/m3 (highest concentration tested).
By the dermal route, a NOAEL for systemic toxicity of 1000 mg/kg/day (the highest dose tested) has been identified in a 90-day rat study. Slight changes at the treated skin site were noted.
By the oral route, NOAELs in rat and mice for systemic effects were 200 mg/kg bw/d, based on the absence of histopathologic changes; however, based on unspecific clinical effects, the NOAELs came down to 100 mg/kg/day.(considered a NOEL)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study following accepted scientific standards; although no guideline provided, acceptable for assessment.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
no documentation of organ weights
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Portage, Michigan
- Age at study initiation: 6 weeks
- Weight at study initiation: Males: 170 - 215 g; females: 135-155 g
- Fasting period before study: no data
- Housing: groups of 5 animals per polycarbonate cages
- Diet (e.g. ad libitum): yes (added as needed)
- Water (e.g. ad libitum): yes (via automatic watering devices)
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 23 °C
- Humidity (%): 40 - 60 % relative humidity
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Stock solution was prepared by placing a weighed portion of naphthalene into a mixing graduated cylinder and adding corn oil to make up the proper volume. Selected doses of 400, 200, 100, 50 and 25 mg/kg were achieved by preparing a stock solution of the highest concentration and sequential dilution in order to reach the appropriate dose level. The respective mixture quantities were prepared on a weekly basis.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no justification provided
- Concentration in vehicle: 400, 200, 100, 50, 25 mg/5 mL solution
- Amount of vehicle (if gavage): 0.5 mL solution per 100 g body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
One analysis for the accuracy of the dose was performed: results were within +/- 10% of the concentrations for all dose levels, except 200 mg/kg which had a deviation of 1.76 % from this range.

Duration of treatment / exposure:
13 weeks

Frequency of treatment:
1x/d, five consecutive days per week.

Remarks:
Doses / Concentrations:
25, 50, 100, 200, 400 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Ten animals of each sex per dose level.

Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Dose selection was performed in order to determine maximum tolerated doses for a subsequent 104-week chronic toxicity study. Dose selections for this sub-chronic study were based on clinical and pathological findings which were observed during a prior two-week repeated dose study.

- Other: Animals were administered 5 ml/kg bw with the respective mixture.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice each day (with at least six hours between observation)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice each day (with at least six hours between observation)

BODY WEIGHT: Yes
- Time schedule for examinations: weekly
Sacrifice and pathology:
Detailed necropsy examinations were performed on all rats in this study. The following tissues were examined: mandibular lymph node, salivary gland, femur, thyroid, parathyroid, small intestine, colon, liver, prostate, testes, ovaries, lungs and mainstem brochi, mammary gland, eyes, heart, oesophagus, stomach, uterus, brain (3 sections), thymus, trachea, pancreas, spleen, kidneys, adrenals, urinary bladder, pituitary gland.

For histopathology, the tissues obtained from the highest dosage groups and from the control group were examined in a routine manner: they were sectioned at 5 µm, stained with haematoxylin and eosin and examined microscopically.
Since lesions had been observed in the highest dosage groups, which may have been substance-specific, kidneys from males and thymus glands from females were examined from the 200 mg/kg dosage groups.
Other examinations:
Haematology
Statistics:
Yes (in regard of body weight and haematology)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Signs of discomfort and disorder observed in the highest dosage groups of both sexes. 2/10 males died during the last week of the study.
Mortality:
mortality observed, treatment-related
Description (incidence):
Signs of discomfort and disorder observed in the highest dosage groups of both sexes. 2/10 males died during the last week of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Dose-related trend especially at the two highest doses.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Marginal decrease in haemoglobin and haematocrit at 400 mg/kg bw in the male and female group.
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Lesions in kidneys (male rats) and thymus glands (female rats) were observed at highest dosage groups.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY:
Diarrhoea, lethargy, hunched posture and roughened haircoats were observed in animals of both sexes of the highest dosage groups. Mortality was noted for two male rats in the highest dose groups in the last week of the experiment. In one of the dead animals, renal lesions were found.

BODY WEIGHT AND FOOD CONSUMPTION:
Effects on body weights were observed at the 400 mg/kg and 200 mg/kg dose groups: in male rats significant decreases in body weight of -60% and -24% were seen in the 400 mg/kg and 200 mg/kg dose groups, respectively. In female rats, both doses caused a decrease in body weight of -69% and -15%, respectively. No substance-specific trends in diet consumption were evident.

HAEMATOLOGY
Marginal decrease in haemoglobin and haematocrit in the male and female group of 400 mg/kg bw. Considerable effects on lymphocyte and neutrophil values were observed in males of the highest dosage groups (400 mg/kg bw).

HISTOPATHOLOGY:
Lesions in kidneys from male animals (score 3 = moderate) and thymus glands from female animals (score 3 = moderate) have been observed in the highest dosage groups (400 mg/kg bw).


Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on decrease in body-weight gain
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on clinical signs, mortality (males), haematological effect, and some evidence of histological changes (kidney, thymus).
Dose descriptor:
LOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
not specified

Table 1: Summary of necropsy data by group and sex 

Note: Only organs that exhibited anomalies in any group have been documented.

Organ and Observation

Dose Group

0 mg/kg

25 mg/kg

50 mg/kg

100 mg/kg

200 mg/kg

400 mg/kg

Sex

M

F

M

F

M

F

M

F

M

F

M

F

Number in Group

10

10

10

10

10

10

10

10

10

10

10

10

Anus, inflamed

1

Eye, left, yellow material in anterior chamber or on lens

1

Heart, enlarged

1

Liver, middle lobe, small nodule at diaphragmatic hiatal area

1

1

1

Liver, middle lobe, nodular protrusion into hiatus of diaphragm

1

Lung, all lobes haemorrhagic

1

Ovarian sac, left, red, small amount of fluid

2

Ovary, left, small amount of fluid in parovarian sac

1

Ovary, right, small parovarian cyst

1

Periovarian fat, 5 mm red nodule on fibrous stalk

1

Seminal vesicles, small

3

Testes, two-thirds normal size

1

Uterine horns filled with clear fluid (estrus)

3

3

3

2

Uterine horns, slightly reddened and slightly enlarged (estrus)

1

Table 2: Summary of histopathology data by group and sex 

Note: Only organs that exhibited anomalies in any group have been documented.

Organ and Observation

Dose Group

0 mg/kg

200 mg/kg #

400 mg/kg

Sex

M

F

M

F

M

F

Number in Group

10

10

10

10

10

10

Adrenal glands, congestion

1

Bone narrow, reticulum cell hyperplasia, focal

2

Colon, parasitosis

1

2

Eye, retina, retinal rosette

1

Eye, retina, focus of vacuolated cells

1

Heart, papillary muscle, histiocytic infiltrate, focal

1

Heart, left ventricle, myocardium, myocarditis, fibrosis, focal

1

Heart, myocarditis, subacute, multifocal

1

Heart, myocardium, myocarditis, multifocal

1

Kidney, cortex, lymphocytic infiltrate, focal

1

Kidney, cortex, tubular degeneration, diffuse

1

Kidney, tubular, regeneration, focal

1

Liver, centrilobular areas, vacuolization, diffuse

3

Liver, centrilobular areas, hepatocellular vacuolization, multifocal or diffuse

1

1

Liver, subcapsular area, fibrosis, focal

1

Lung, pneumonia, granulomatous, focal, gavage induced

1

Lung, pneumonia, histiocytic, focal or multifocal, gavage induced

2

Lung, pneumonia, subacute, focal

1

Parathymic adipose tissue, congestion and haemorrhage

1

Pleural cavity, mast cell and neutrophil accumulation

1

Pleural cavity, inflammation, fibrinous

1

Thymus, lymphoid depletion

2

Thyroid gland, lymphocytic infiltrate, focal

1

Trachea, tracheitis, acute

1

# 200 mg/kg bw: Unscheduled histopathology only of kidney (males) and thymus (females) examined due to increased effect at 400 mg/kg bw.

Conclusions:
After 13 weeks of oral exposure by gavage, unspecific adverse effects, predominantly loss in body-weight gain, considered to be substance-related were noted from 200 mg/kg bw/d onwards.
Executive summary:

Doses of 0, 25, 50, 100, 200, and 400 mg naphthalene/kg bw/d were administered orally in corn oil to Fischer 344 rats (60 rats per sex) for five days a week for 13 weeks. Up to 200 mg/kg bw/d, the only signs - obviously substance-related - were decrease in body weight of >10 % in both sexes. At 400 mg/kg bw/d, in addition, clear clinical signs of discomfort and disorder (lethargy, roughened haircoats, hunched posture, and diarrhoea) emerged, but also mortality was slightly increased (2/10 males during the last week of the study, one with renal lesion), shifts in the lymphocyte count and apparently an increasing trend in organ damage (kidney in males and there noted, and the thymus) were observed.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
acceptable, NOAEL based on histopathological observations, also corresponds to the NOAEL in mice.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
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.
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
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

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
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
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.
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.

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
Critical effects observed:
not specified

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

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
300 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
on its own not evaluable for systemic effects, but see justification of selection.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to US EPA guideline.
Qualifier:
according to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Deviations:
yes
Remarks:
Histopathology only for respiratory tract
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Kingston, NY
- Age at study initiation: approx. 6-7 weeks of age on receipt
- Weight at study initiation: no data
- Fasting period before study: none
- Housing: singly housed in stainless steel wire mesh cages (Hazelton inhalation racks, Lab Products, Seaford, DE) in a climate-controlled room
- Diet: pelleted food (NIH-07, Zeigler Bros., Gardners, PA), ad libitum
Food was removed during inhalation exposure
- Water: reverse-osmosis purified; ad libitum
- Acclimation period: Acclimated in the animal facility for approximately two weeks prior to beginning the inhalation exposure


Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: nitrogen gas as carrier of naphthalene vapour into the exposure chamber
Remarks on MMAD:
MMAD / GSD: not applicable (No aerosol or particle formation was observed)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1 m3 whole body exposure chambers (H1000, Lab Products, Seaford, DE) using Hazelton R-24 cage units.
- Method of holding animals in test chamber: One chamber for each concentration including a control chamber
- Source and rate of air: 95% HEPA-filtered
- System of generating particulates/aerosols: Naphthalene vapors were generated by heating solid naphthalene and using nitrogen gas to carry the vapor into the 1 m3 chamber inlet. Concentrations of test substance in the chambers were controlled by adjusting a combination of the heating
temperature and nitrogen flow rate through the generators(see publication p 71 for details).
- Temperature: 73-75 °F; humidity: 45-56 %
- Air flow rate: 300 l/min)
- Air change rate: Approx. 18 air changes per hour
- Method of particle size determination: Prior to and during animal exposures, a check for aerosol formation was conducted.
A scanning mobility particle sizer (SMPS, Model 3934, TSI, Inc., Shoreview, MN) was used to measure particle concentration and size distribution in
the size range from 4 nm to 1000 nm.
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used: During exposures, naphthalene concentration was measured using a gas chromatograph mass
spectrometer (GC/MS, Model GCMS-QP2010 Plus, Shimadzu, Kyoto, Japan) equipped with a Rxi-5ms 15 m x 0.32 mm id x 0.25 µm film thickness
capillary column and mass spectrometer detection system.
- Samples taken from breathing zone: yes, at 9 locations in the exposure chamber
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Purity check by gas chromatography with flame ionization detector (GC/FID) of the test substance was conducted pre- and post-exposure to check for stability.

Duration of treatment / exposure:
90 d
Frequency of treatment:
Animals were exposed to the target concentration for 6h/day, 5d/wk for a total of 65 or 66 exposure days (for approximately a 90-day period).
Remarks:
Doses / Concentrations:
0 (control), 0.1, 1.0, 10, 30 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.00 +-0.00(control); 0.10+-0.00; 0.97+-0.06; 9.97+-1.2; 29.8+-1.7 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
10 (additional 5/ sex/ group evaluated for recovery 4wk post-exposure)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on previous investigations
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: 4 wk
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice per day: Observation for mortality and overt clinical signs prior to and immediately following daily exposure.

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Once per week: Animals were weighed prior to the start of exposures.

ORGAN WEIGHTS: yes (liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, thymus, spleen, brain, and heart)

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
(Food and water consumption was measured in a subset of rats assigned as recovery animals (5/sex/group) by transferring animals to
polycarbonate caging following the end of the weekly exposure period)

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: Yes
- Time schedule for examinations: see above

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (see table 1 and 2, attached document: only focussed on the head with nasal region: Cross-sectional blocks of the nasal tissues were prepared at 6 levels after preparation.)
Statistics:
Continuous variables (e.g. body weight, food and water consumption, and organ weights) were analysed using the default statistical method of the Provantis software system (NT2000, versions 8.2.0.1 or 8.2.0.6, Instem/PA). A result of p<0.05 was considered significant.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Concentration-related decrease at the end of the 90 d study in the 10 and 30 ppm groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
for absolute but not relative organ weights (see Tab. 3 and 4, below)
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see tables 1 and 2 (attached documents: only shown for males, similar for females)
Histopathological findings: neoplastic:
no effects observed
Details on results:
FOOD and WATER CONSUMPTION: Decreased in animals exposed at 30 ppm. The decreased consumption appears to be correlated with the
observed decreased body weights in animals at the higher exposure concentrations of naphthalene.

ORGAN WEIGHTS:
- Males:The absolute mean weights of the spleen, left testis, and thymic region of male rats exposed to 30 ppm were statistically significantly lower
(p< 0.05 or p< 0.01) than the control mean values (Table 3).
Mean right testis weight was slightly lower than control, but not statistically significant.
The magnitude of the organ weight decreases compared to control means was approximately 5% (spleen and left testis) and 17% (thymus).
When expressed relative to body weight, these organ weights were similar to control values (Table 3).
Similar observations were observed in the organ weights of the 10 ppm-exposed males.
- Females: In female rats, the absolute mean weights of the heart, liver, and thymic region were statistically significantly lower (p<0.05 or p< 0.01)
than the control mean weights in all exposed groups (except the heart weight of the 10ppm group, the liver weight of the 1ppm group, and the
thymus weight of the 0.l ppm group) (Table 4). magnitude of these decreases (5-7% for heart and liver; 16% for thymus) was not exposure
concentration-dependent
Relative to body weight, they were similar to control values (Table 4).
A decrease in mean weight of adrenal glands in the l0ppm exposed females only was considered spurious.
- 4-weeks recovery: All statistically significant differences in organ weight values (absolute, relative to body weight or to brain) observed immediately following the 90-day exposure regimen were diminished in magnitude and no longer statistically significantly different from control values
following 4 weeks recovery

HISTOPATHOLOGY: NON-NEOPLASTIC
- Respiratory and olfactory epithelium: Microscopic evaluation showed naphthalene-induced lesions.
Squamous epithelium or transitional/respiratory epithelium: Minimal to slightly mild inflammation of the was observed in the nasal tip and level 1
areas of all exposure groups, including controls (Table 1 for male data). Low grade inflammation was also observed in all exposure groups,
including controls, after a 4 week recovery period (Table 2 for male data). This observation was not considered to be related to exposure.
- Respiratory epithelium: No lesions were observed in animals exposed to 0.1ppm naphthalene. Lesions related to exposure were observed
beginning at level 2 in the nose.
- Transitional/respiratory epithelium: At a concentration of 1ppm, hyperplasia was observed in males and females with an average severity of 1.0
(minimal). The severity of the lesion progressed to mild hyperplasia at l0ppm (average severity 1.4 in males, average severity of 1.6 in females)
and 30 ppm (average severity of 1.4 in males and average severity of 1.5 in females) (Figures 2 and 3; Table 1 for male data).
Minimal squamous metaplasia of the transitional/respiratory epithelium was also observed in level 2 sections of male and female nasal cavities
with average severity of 0.9 and 0.8 for l0ppm and 30ppm, respectively (Table 1 for male data).
- Olfactory epithelium: Olfactory epithelial degeneration and necrosis was observed at the 10- and 30-ppm exposure concentrations in males and
females (see severity scores: table 1, attached document). Associated with the degenerative olfactory epithelium, there was a prominent basal cell
hyperplasia characterized by an increase in the number of basal cells resulting in thickening of this layer of cells immediately under the
degenerative epithelium (see publication, fig. 6)

The incidence and severity of both the olfactory epithelium degeneration and basal cell hyperplasia lesions decreased in exposed males and females after the 4-week recovery period (Table 2, attached document; publication, fig. 7).
There were no olfactory epithelial effects seen in male or female rats exposed at 0.1 or 1.0ppm (Table 1 for male data).


Dose descriptor:
NOEC
Effect level:
ca. 0.1 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Histopathology: Absence of lesions in the olfactory and respiratory epithelia
Dose descriptor:
NOAEC
Effect level:
1 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Histopathology: Absence of lesion in the olfactory epithelium, minimal hyperplasia in the repiratory epithelium in all animals, but reversible during recovery period
Dose descriptor:
LOAEC
Effect level:
10 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Histopathology: Clear increase in the frequency of olfactory effects (hyperplasia, degeneration and necrosis) in 6/10 to 10/10 animals (males) graded from minimal to mild, regressing in severity and number within 4-wk recovery but not completely.
Critical effects observed:
not specified

The absolute weights of the spleen, left testis (ca. -5%), and thymic region (ca. -17%) of male rats exposed to 10 or 30 ppm were significantly lower than the control values (see Tab. 3 + 4 below), but no difference was noted for the relative organ-weights.

Mean right testis weight was slightly lower than control without statistical significance.

In females, sporadic decreases in absolute liver, heart, and thymus weight were observed, but when related to body-weight differences disappeared.

Table 3:  Select absolute and relative organ weights for male F344 rats immediately following the 90-day study

(from: Dodd et al., 2012)

Organ

Target exposure concentration (ppm)

0

0.1

1.0

10

30

Spleen (g)

0.710 ± 0.039 (a)

0.709 ± 0.021

0.746 ± 0.075

0.676 ± 0.034

0.670* ± 0.037

Spleen (%) (b)

0.21 ± 0.01

0.21 ± 0.01

0.22 ± 0.03

0.22 ± 0.01

0.22 ± 0.01

Testis, left (g)

1.567 ± 0.046

1.565 ± 0.041

1.535 ± 0.078

1.491** ± 0.069

1.507* ± 0.046

Testis, left (%)

0.47± 0.02

0.47 ± 0.02

0.45 ± 0.02

0.48 ± 0.04

0.49 ± 0.03

Thymic region (g)

0.350 ± 0.074

0.319 ± 0.038

0.340 ± 0.064

0.291* ± 0.035

0.289* ± 0.039

Thymic region (%)

0.ll ± 0.02

0.10 ± 0.01

0.10 ± 0.02

0.09 ± 0.01

0.09 ± 0.01

(a) Mean ± standard deviation for n =10,

(b) Percentage of body weight,

* p<0.05 compared to control,

** p<0.01 compared to control

Table 4:   Select absolute and relative organ weights for female F344 rats immediately following the 90-day study

(from: Dodd et al., 2012)

Organ

Target exposure concentration (ppm)

0

0.1

1.0

10

30

Heart (g)

0.595 ±0.018 (a)

0.562* ± 0.036

0.559* ± 0.041

0.570 ± 0.040

0.564* ± 0.017

Heart (%) (b)

0.31 ± 0.02

0.31 ± 0.02

0.30 ± 0.02

0.31 ± 0.02

0.31 ± 0.02

Liver (g)

6.060 ± 0.301

5.628* ± 0.319

5.790 ± 0.516

5.383** ± 0.343

5.646* ± 0.432

Liver (%)

3.17 ± 0.17

3.09 ± 0.16

3.10 ± 0.19

2.94 ± 0.19

3.11 ± 0.19

Thymic region (g)

0.267 ± 0.029

0.238 ± 0.044

0.226* ± 0.042

0.225* ± 0.031

0.224* ± 0.045

Thymic region (%)

0.14 ± 0.02

0.13 ± 0.02

0.12 ± 0.03

0.12 ± 0.02

0.12 ± 0.02

(a) Mean ± standard deviation for n =10,

(b) Percentage of body weight,

* p<0.05 compared to control,

** p<0.01 compared to control
Conclusions:
No histopathological adverse effects were noted at 0.1 ppm naphthalene: Therefore, this exposure concentration is adopted as NOEC.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
5 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
high

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Dec 16, 1985 – May 7, 1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP conform study according to OECD guideline.
Qualifier:
according to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
SOURCE:
- Age at study initiation: 7 weeks
- Weight at study initiation: Males 219.8 – 277.4g; females 152.7 – 197.2g
- Fasting period before study: None
- Housing: Singly in stainless steel cages with solid sides and wire mesh floors
- Diet: ad linitum, ground Purina Certified Rodent Chow #5002
- Water: ad libitum
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 3 °C
- Humidity (%): 30 – 70%
- Air changes (per hr): 20 – 30/hr
- Photoperiod (hrs dark / hrs light): 12 light/12 dark
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: 2x2 inches (1 inch = 2.54 cm)
- % coverage: approximately 10% of body surface
- Type of wrap if used: Coban Action Wrap secured with Scotchrap Veterninary Elastic Tape
- Time intervals for shavings or clipplings: Not specified

REMOVAL OF TEST SUBSTANCE
- Washing (if done): No, wiped clean
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): applied neat ranging from 100 – 1000 mg/kg body weight
- Concentration (if solution): not applicable
- Constant volume or concentration used: not applicable
- For solids, paste formed: no

VEHICLE
- Justification for use and choice of vehicle (if other than water): not applicable

USE OF RESTRAINERS FOR PREVENTING INGESTION: No
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Verification of starting purity. Was applied neat.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
5 days/week
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg
Basis:
no data
No. of animals per sex per dose:
10/sex/dose with a satellite group of an additional 10/sex for the control and high dose
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Tested to limit dose
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: random
- Post-exposure recovery period in satellite groups: 4 weeks
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and general clinical signs
- Cage side observations checked in table were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

DERMAL IRRITATION (if dermal study): No, other than gross observations
- Time schedule for examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-dose and post-dosing
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: pre-dose, 4 weeks and 13 weeks
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 10/sex/group pre-dose and 5/sex/group during dosing periods
- Parameters checked in table were examined: Leukocyte and erythrocyte count, hemoglobin, hematocrit, MCV, MCH, MCHC, platelet count, differential leukocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: pre-dose, 4 weeks and 13 weeks
- Animals fasted: Yes
- How many animals: 10/sex/group pre-dose and 5/sex/group during dosing periods
- Parameters checked in table were examined: Fasted glucose, urea nitrogen, creatinine, AST, ALT, total protein, albumin, total bilirubin, direct bilirubin, calcium, phosphorus, sodium, potassium, chloride

URINALYSIS: Yes
- Time schedule for collection of urine: Pre-dose, week 4 and week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table were examined: pH, protein, glucose, ketone, color, volume, bilirubin, blood, urobilinogen, specific gravity, turbidity

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Yes. Food consumption, body and organ weight were intercompared for the dose groups and control group by Levene’s test for homogeneity of variances, analysis of variance and individual t-tests (if significance in the analysis of variance).
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Excortiated skin and papules in treatment area of high dose male and female animals. This effect was also observed in the control animals and would appear to have been exacerbated by treatment but not caused by treatment.
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 specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Excoriated skin and papules at 1000 mg/kg.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
not specified
Conclusions:
Naphthalene has no effect on toxicity in rats when applied dermally for 13 weeks.
Executive summary:

Male and female Sprague-Dawley CD® rats were exposed to naphthalene via the dermal route at 0, 100, 300, and 1000 mg/kg body weight/day. The test material was applied under occlusion for 6 hours per day, 5 days per week for 13 weeks. Following the exposure period, animals from each of the control and high dose groups were followed for 4 weeks during a recovery phase. No treatment-related effects on the incidence of clinical observations, ophthalmological changes, gross anatomic alterations, or histopathologic changes were observed. Cutaneous exposure to naphthalene did not affect food consumption, body weight, or clinical measurements conducted for clinical chemistry, haematology, and urinalysis parameters. Testes weights in the high dose group were slightly reduced at the 13-week sacrifice. However, this change was not noted at the 17-week recovery sacrifice. No other organ weight changes were observed. The decreased testes weight was considered to be of negligible biological significance based on the small magnitude of the change and the absence of any corroborating evidence of tissue alteration upon histologic examination. Based on the results of this study, cutaneous exposure of rats to crystalline naphthalene at or below 1000 mg/kg body weight/day is not considered to result in any biologically significant responses. The no observable effect level (NOEL) is considered to be at least 300 mg/kg body weight/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
high

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Dec 16, 1985 – May 7, 1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP conform study according to OECD guideline.
Qualifier:
according to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
SOURCE:
- Age at study initiation: 7 weeks
- Weight at study initiation: Males 219.8 – 277.4g; females 152.7 – 197.2g
- Fasting period before study: None
- Housing: Singly in stainless steel cages with solid sides and wire mesh floors
- Diet: ad linitum, ground Purina Certified Rodent Chow #5002
- Water: ad libitum
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 3 °C
- Humidity (%): 30 – 70%
- Air changes (per hr): 20 – 30/hr
- Photoperiod (hrs dark / hrs light): 12 light/12 dark
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: 2x2 inches (1 inch = 2.54 cm)
- % coverage: approximately 10% of body surface
- Type of wrap if used: Coban Action Wrap secured with Scotchrap Veterninary Elastic Tape
- Time intervals for shavings or clipplings: Not specified

REMOVAL OF TEST SUBSTANCE
- Washing (if done): No, wiped clean
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): applied neat ranging from 100 – 1000 mg/kg body weight
- Concentration (if solution): not applicable
- Constant volume or concentration used: not applicable
- For solids, paste formed: no

VEHICLE
- Justification for use and choice of vehicle (if other than water): not applicable

USE OF RESTRAINERS FOR PREVENTING INGESTION: No
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Verification of starting purity. Was applied neat.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
5 days/week
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg
Basis:
no data
No. of animals per sex per dose:
10/sex/dose with a satellite group of an additional 10/sex for the control and high dose
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Tested to limit dose
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: random
- Post-exposure recovery period in satellite groups: 4 weeks
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and general clinical signs
- Cage side observations checked in table were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

DERMAL IRRITATION (if dermal study): No, other than gross observations
- Time schedule for examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-dose and post-dosing
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: pre-dose, 4 weeks and 13 weeks
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 10/sex/group pre-dose and 5/sex/group during dosing periods
- Parameters checked in table were examined: Leukocyte and erythrocyte count, hemoglobin, hematocrit, MCV, MCH, MCHC, platelet count, differential leukocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: pre-dose, 4 weeks and 13 weeks
- Animals fasted: Yes
- How many animals: 10/sex/group pre-dose and 5/sex/group during dosing periods
- Parameters checked in table were examined: Fasted glucose, urea nitrogen, creatinine, AST, ALT, total protein, albumin, total bilirubin, direct bilirubin, calcium, phosphorus, sodium, potassium, chloride

URINALYSIS: Yes
- Time schedule for collection of urine: Pre-dose, week 4 and week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table were examined: pH, protein, glucose, ketone, color, volume, bilirubin, blood, urobilinogen, specific gravity, turbidity

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Yes. Food consumption, body and organ weight were intercompared for the dose groups and control group by Levene’s test for homogeneity of variances, analysis of variance and individual t-tests (if significance in the analysis of variance).
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Excortiated skin and papules in treatment area of high dose male and female animals. This effect was also observed in the control animals and would appear to have been exacerbated by treatment but not caused by treatment.
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 specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Excoriated skin and papules at 1000 mg/kg.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
not specified
Conclusions:
Naphthalene has no effect on toxicity in rats when applied dermally for 13 weeks.
Executive summary:

Male and female Sprague-Dawley CD® rats were exposed to naphthalene via the dermal route at 0, 100, 300, and 1000 mg/kg body weight/day. The test material was applied under occlusion for 6 hours per day, 5 days per week for 13 weeks. Following the exposure period, animals from each of the control and high dose groups were followed for 4 weeks during a recovery phase. No treatment-related effects on the incidence of clinical observations, ophthalmological changes, gross anatomic alterations, or histopathologic changes were observed. Cutaneous exposure to naphthalene did not affect food consumption, body weight, or clinical measurements conducted for clinical chemistry, haematology, and urinalysis parameters. Testes weights in the high dose group were slightly reduced at the 13-week sacrifice. However, this change was not noted at the 17-week recovery sacrifice. No other organ weight changes were observed. The decreased testes weight was considered to be of negligible biological significance based on the small magnitude of the change and the absence of any corroborating evidence of tissue alteration upon histologic examination. Based on the results of this study, cutaneous exposure of rats to crystalline naphthalene at or below 1000 mg/kg body weight/day is not considered to result in any biologically significant responses. The no observable effect level (NOEL) is considered to be at least 300 mg/kg body weight/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
4 mg/cm²
Study duration:
subchronic
Species:
rat
Quality of whole database:
high: area-specific dose not available, calculated from the highest dose of 300 mg/kg bw/d on a site of approx. 75 cm2/kg bw

Additional information

There are no verifiable epidemiological studies on the human health effects of naphthalene, and the only human information available derives from a limited number of early case-reports which provide no quantitative data on the levels or duration of exposure. A principal human health effect is haemolytic anaemia which in some cases reached marked severity in humans exposed by inhalation to naphthalene vapour and by ingestion of solid naphthalene. Dermal exposure to the solid and vapour was also likely in these cases.

Animal studies reveal species differences in response to naphthalene. Haemolytic anaemia was noted in a dog following oral dosing of 220 mg/kg/day for 7 days, but not in rodents even with high/prolonged exposures. Cataract formation was the principal effect seen in pigmented rats and rabbits following oral exposure to 700 and 1000 mg/kg/day, respectively, in studies ranging from 10-180 days, but this effect was not seen in mice with similar exposures. The lack of reliable reports of cataracts in humans despite the widespread use of naphthalene and accidental exposure to high doses suggests that cataract formation is unlikely to be a significant health effect in humans.

Very minor signs of nasal olfactory inflammation, not yet considered adverse, were reported in a 28 -and a 90 day rat inhalation study at 5 mg/m3 (1 ppm) (Coombs et al. 1993a). More marked changes in the olfactory epithelium were noted in a 90-day rat inhalation study at the lowest exposure level of 10 mg/m3 (2 ppm) (Coombs et al. 1993). These nasal effects became more marked with increasing levels of naphthalene exposure (Dodd et al. 2012). In a 104-week carcinogenicity study in mice, signs of nasal, olfactory and pulmonary inflammation were noted at 50 mg/m3, the lowest exposure concentration used. A NOEC could not be identified for local respiratory effects in these studies, but 1 ppm (5 mg/m3) is considered as a NOAEC.

General signs of toxicity, including death, were reported in rats and rabbits orally dosed with 700 and 1000 mg/kg/day, respectively. It is apparent that mice are more susceptible to naphthalene than rats or rabbits following oral treatment.

No histopathologic changes were reported in two oral 90-d studies in rats and mice at a dose of 200 mg/kg/d each; however, clinical signs of discomfort and decrease in body-weight gain were noted at the lower dose of 100 mg/kg bw/d (BCL 1980a,b).

An LD50 of 354 mg/kg was estimated in female mice treated with naphthalene once daily by gavage for 8 consecutive days (Plasterer et al. 1985, not shown). The dose response curve appeared to be very steep, because no deaths occurred at 250 mg/kg/day, but all animals died with a dose of 500 mg/kg/day. In a different study with a 14-day dosing period, 10% of the males and 5% of the females died at a dose of 267 mg/kg/day, but none were affected by doses of 27 and 53 mg/kg/day (Shopp et al. 1984b). In an increasing dose study, male rats were treated with naphthalene at doses beginning at 100 mg/kg/day and raised the dose weekly to a final level of 750 mg/kg/day over 6 weeks. Doses were then kept constant for an additional 3 weeks. The animals tolerated 750 mg/kg/day with no mortalities (Germansky and Jamall 1988). No increase in mortality was observed in rats administered naphthalene at 41 mg/kg/day in a 2-year feeding study (Schmahl 1955). For systemic effects, a NOAEL of 133 mg/kg/day was identified in a 90-day oral mouse study (Shopp et al. 1984a).

In the 90-day rat inhalation study, there was no evidence of systemic toxicity at exposures up to 300 mg/m3, although a reduction in body weight gain of up to 43% associated with reduced food consumption was observed at this top dose (Coombs et al. 1993). By the dermal route, a NOAEL for systemic toxicity of 1000 mg/kg/day (the highest dose tested) was identified in a 90-day rat study.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Relevant and comprehensive 90d study by the oral route of exposure

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
relevant 90-d study by route of inhalation in synopsis with carcinogenicity (inhalation) and oral repeated-dose study (oral).

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
identifiable NOAEC

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Only valid study available, comprehensive guideline study

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
The only valid study available, comprehensive guideline study

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

Justification for classification or non-classification

Naphthalene is already classified as Carc. Cat. 3; R40 and Xn; R22 (according to 67/548/EEC or 1999/45/EC) /

Carc. 2, H351, Suspected of causing cancer; Acute toxic 4, H302, Harmful if swallowed (according to CLP Regulation (EC) No 1272/2008). No further classification is required.