Registration Dossier

Administrative data

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:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
(1) Since there was an operation error when using the computer system for animal grouping, a different computer protocol number from the study protocol was used in the data acquisition for females after animal grouping. The data of the females before the animal grouping were transferred to the protocol. It was concluded that this operation did not affect the reliability of this study.
(2) Since there was a failure in the operation of the hydrocarbon monitor on day 59 in males (day 58 for females) of the AC-6000 1000 ppm group, the relative concentration could not be measured for approximately 30 minutes in that group. As the actual concentration did not vary on that day, it was confirmed that the concentration of the origin gas (AC-6000 4000 ppm) did not change. Therefore, it was concluded that the exposure concentration of the AC-6000 1000 ppm group was stable temporally on that day.
Deviations:
yes
Remarks:
see version/remarks
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorooctane
EC Number:
700-684-7
Cas Number:
80793-17-5
Molecular formula:
C8H5F13
IUPAC Name:
1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorooctane
Details on test material:
- Name of test material (as cited in study report): 1,1,1,2,2,3,3,4,4,5,5,6,6-Tridecafluoroctane
- Physical state: liquid
- Analytical purity: 99.87%
- Impurities (identity and concentrations): tridecafluorohexane (0.13%)
Specific details on test material used for the study:
Supplier: Asahi Glass Co., Ltd.
Lot number: 51107202
Purity: 99.936% (non GLP)

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
This strain is widely used in animal toxicity testing using rodents, and there are abundant historical data.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Supplier: Charles River Laboratories Japan, Inc., Kanagawa, Japan
Age at receipt: 6 weeks (quarantined for 9 days from the day of animal receipt)
Acclimation: from animal receipt to the day before dosing
Age at start of dosing: 8 weeks
Body weight at start of dosing: males: 275.7 to 322.2 g; females: 200.1 to 236.4 g

Environmental conditions
Temperature: 21.6° to 23.6°C
Relative humidity: 39.1% to 66.2%
Ventilation: 6 to 20 times/hour, all fresh air
Lighting: 12 hours/day, 7:00 to 19:00 (lighting was turned off during the ophthalmoscopy)
Feeding: ad libitum except the following duration and replaced with new one at the same time as the feeders: during the exposure, during the fresh urine collection, evening on the day before necropsy (17:30)
Water: ad libitum except during the exposure duration and fresh urine collection, the water was replaced with new one at the same time as the water bottles.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
Nose-only inhalation exposure was conducted using a flow-past nose-only inhalation exposure chamber (hereinafter abbreviated as “chamber”, Muenster Ltd.) to animals restrained in restraining tubes (Muenster Ltd.) for nose-only inhalation exposure. The chamber is constructed from stackable tiers, and each tier has 16 exposure ports. Twotier chamber was used in this study. The flow rate of air-supply to the chamber was set at 16 L/min/tier, total of 32 L/min. The exhaust flow rate was set at 29 L/min in the chamber, and it was approximately 10% lower than that of the air-supply to ensure that the test atmosphere was exposed to the animals by positive pressure in the chamber. The exhaust flow was maintained by keeping indication of a flow-meter, which was installed in exhaust line. The set point of the flow meter was obtained by measuring and maintaining exhaust flow rate at 29 L/min with a standard flow meter (Dry test gas meter, DS-6A-T: Shinagawa Co., Ltd., Tokyo, Japan).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Relative concentration of the test substance was monitored continuously in the chamber using a hydrocarbon monitor (HCM-1B: Shimadzu Corp.) connected to the exposure port.

The test atmosphere was collected to a sampling bag (inner volume: 1.55 L, Tedlar bag: GL Science Inc., Tokyo, Japan). The sampling bag was put into a container for vacuum sampling, and connected to the exposure-port. The test atmosphere was sampled by reducing inner pressure of the container with a vacuum pump. The collected air was used as an analytical sample. However, 200 mL of the test atmosphere of the AC-6000 4000 mg/m3 (ppm) group was put into a sampling bag with a 50 mL syringe (SS-05LZ: Terumo Corp., Tokyo, Japan) and diluted with pressurized air (below 0.01 MPa) to prepare an analytical sample (7.75 fold dilution). The test atmosphere was collected from a single exposure port at approximately 0.5, 3, and 5.5 hr after the start of exposure. The test atmosphere was analyzed and quantified with gas chromatography (hereinafter abbreviated as “GC”) and the exposure concentration was calculated. Daily mean concentration was evaluated. The following sections show the analytical method. It was confirmed that relative error of measured concentration was within ±10% of the daily mean concentration at each sampling point. Moreover, it was confirmed that coefficient variation of the daily concentrations was within ±10%.
Duration of treatment / exposure:
6 hours a day for 13 consecutive weeks
Frequency of treatment:
5 days a week
Doses / concentrationsopen allclose all
Dose / conc.:
7 118.61 mg/m³ air
Remarks:
500 ppm
Dose / conc.:
14 237.22 mg/m³ air
Remarks:
1000 ppm
Dose / conc.:
28 474.44 mg/m³ air
Remarks:
2000 ppm
Dose / conc.:
56 948.88 mg/m³ air
Remarks:
4000 ppm
No. of animals per sex per dose:
10/ sex/dose
Control animals:
yes, concurrent no treatment

Examinations

Observations and examinations performed and frequency:
The parameters in the following sections were observed and measured. The first day of dosing was designated as day 1, and the initial week (day 1 to day 7) was designated as week 1.

Clinical signs:
All animals were observed daily from the first day of dosing to the day of necropsy. The observation frequency is shown below.
Dosing day: twice a day (before and after exposure)
Other day: once a day (forenoon)

Body weight
All animals were weighed with an electrical balance (PB3002-S: Mettler-Toledo K.K., Tokyo, Japan) on the following schedule. The measurement was conducted before dosing on the day of exposure. The body weight gain was calculated between measurements on the day of each measurement.
Measurement day: day of start of dosing (day 1), and the following days; 4, 8, 11, 15, 18, 22, 25, 29, 32, 36, 39, 43, 46, 50, 53, 57, 60, 64, 67, 71, 74, 78,
81, 85, 88, and 91
The body weights were measured on day 92 for calculation of relative organ weights (body weight-relative ratio). It was the day of scheduled necropsy.

Food consumption
All feeders of each cage (including diet) were weighed using an electric balance (PB3002-S: Mettler-Toledo K.K., Tokyo, Japan) at the following schedules.
Measurement period: days 1 to 8, 8 to 15, 15 to 22, 22 to 29, 29 to 36, 36 to 43, 43 to 50, 50 to 57, 57 to 64, 64 to 71, 71 to 78, 78 to 85, 88 to 91 (male), or
87 to 91 (female)
Average daily food consumption of each animal was calculated from weight differences between each measurement day.

Ophthalmoscopy
(1) Schedule Once before the start of dosing (male: day -2, female: day -3), and once on week 13
(male: day 90, female: day 89).
(2) Item and method
Items and methods are as follows. Anterior part, optic media, and ocular fundus were examined after instillation of a mydriatic (Midrin-P ophthalmic solution: Santen Pharmaceutical Co., Ltd., Osaka, Japan) after the examination of light reflex.
Light reflex: direct ophthalmoscope (BETA200: Heine Optotechnik Ltd., Herrsching, Germany)
Anterior part and optic media: slit lamp (SL-15: Kowa Company Ltd., Aichi, Japan)
Ocular fundus: binocular indirect ophthalmoscope (OMEGA 200: Heine Optotechnik Ltd.)

Urinalysis
Urinalysis was conducted for 6 animals from each group (ascending number of animals).
(1) Schedule
On week 13 (male: days 86 to 87, female: days 85 to 86)
(2) Sample collection
Urine samples were collected with the autoclaved metabolic cage (Tokiwa Kagaku Kikai
Co., Ltd.) according to the following procedure:
Fresh urine sample: In the morning of inspection day (before dosing), fresh urine samples were collected for 3 hours without food and
water supply.
Accumulated urine sample: On the day of fresh urine sample collection, accumulated urine samples were collected for approximately 15 hours while food and water were being supplied.
(3) Item and method
The paper test and urinary sediment examination were examined with fresh urine samples, and other items were examined with accumulated urine samples. When potassium and chlorine concentration exceeded the upper limit of quantification, the sample was diluted and re-analyzed according to the SOP of the test facility.
(4) Handling of residual samples
The residual urine samples were stored in a freezer at about -80°C (permissible range: -60°C or below) after examination of creatinine and electrolyte, and they were discarded by the completion of the study. The other residual urine samples were discarded after the examination.

Hematology
(1) Schedule
The day of scheduled necropsy (day 92)
(2) Sample collection
Blood samples were obtained from all animals by the following blood collection and processing:
Fasting before blood collection:
one night (from 17:30 on the day before necropsy to the necropsy)
Anesthetization: pentobarbital sodium (Somnopentyl®: Kyoritsu Seiyaku Corp.
Tokyo, Japan, intraperitoneal injection)
Region: inferior vena cava
Sample volume: approximately 4 mL
EDTA treatment: Approximately 0.8 mL of blood was anti-coagulated with EDTA-2K.
Plasma sampling: Approximately 0.6 mL of blood was centrifuged (12,000×g, 3 min, 4°C) after it was anti-coagulated by adding 3.2w/v% citric acid tri-sodium solution, and plasma was collected.

Blood chemistry
(1) Schedule
The day of scheduled necropsy (day 92)
(2) Sample collection
Subjected blood: A portion of blood sample obtained under the condition as described in the section of hematology
Serum sampling: Serum was obtained by centrifuging (about 1,500×g, 10 min,
4°C) after approximately 2 mL of blood was left at room temperature for approximately 30 to 60 min.
(3) Items and methods
The following table shows the items and methods. The serum sample was used.
(4) Handling of residual samples
The residual serum samples were stored in a freezer at about -80°C (permissible range: -60°C or below) and discarded by completion of the study.
Sacrifice and pathology:
See any other information for list of organs and tissues examined.
The hearts of males administered with AC-6000 at 7118.61mg/m3 (500ppm), 14237.22 mg/m3 (1000 ppm) and 28474.44 mg/m3 (2000 ppm) groups were lso examined.

Necropsy
All animals were subjected to necropsy on day 92. The animals were subjected to necropsy after euthanization by exsanguination from the abdominal aorta under anesthetization by intraperitoneal injection of pentobarbital sodium (Somnopentyl®: Kyoritsu Seiyaku Corp.).

Organ weight
The organs and tissues were weighed using an electrical balance (AG204: Mettler-Toledo K.K. and AW120: Shimadzu Corp.).
The bilateral organs were weighed simultaneously for both sides. The pituitary and thyroid/parathyroid were weighed after fixation. Moreover, relative organ weight (body weight-relative) was calculated from body weight measured on the day of necropsy.

Histopathological examination
(1) Collection and preservation of organs and tissues
The organs and tissues were collected from all animals. They were fixed and preserved in 10vol% phosphate-buffered formalin. The lung was instilled with 10vol% phosphate-buffered formalin at 25 cmH2O for 15 min while it was being floated into 10vol% phosphate-buffered formalin. After ligation of the trachea, the lung was immersed and preserved into 10vol% phosphate-buffered formalin. The testes were fixed with Bouin’s solution, and the eyes, optic nerve, and Harderian glands were fixed in Davidson’s solution. Then, they were preserved in 10vol% phosphatebuffered formalin.
(2) Specimen preparation and microscopic examination
The organs and tissues were processed to prepare hematoxylin and eosin stained specimens for all animals in the control and AC-6000 56948.88 mg/m3 (4000 ppm) groups and the grossly abnormalities organs in the all groups by the standard method, and they were examined microscopically. Four levels of the nasal cavity, three levels of larynx, and two levels of trachea specimens were prepared and examined in accordance with OECD guideline (No.413).
Since there were histopathological findings attributable to the test substance in the liver of the males and females of the AC-6000 56948.88 mg/m3 (4000 ppm) group, the livers of all animals were subjected to the histopathological examination.
GROSS PATHOLOGY: Yes (see table) / No / No data
HISTOPATHOLOGY: Yes (see table) / No / No data
Statistics:
The following statistical analyses were conducted. A computer system for toxicology study (Provantis: Instem Plc., Staffordshire, UK) was used in the statistical analyses.
(1) Method
Numerical data:
Initially, variance was assessed using Bartlett's test (significant level: 5%). When the variance was equal, one-way analysis of variance was used; otherwise, the Kruskal-Wallis test (significant level: 5%) was used. When significant differences were seen between the groups, they were evaluated using Dunnett's method (homogeneous variance) or a Dunnett's type multiple comparison test (heterogeneous, Steel test). The multiple comparison tests were conducted by two-sided test with significant level of 5%.
(2) Examined items
The following items were analyzed.
Numerical data:
ˑBody weight
ˑBody weight gain
ˑFood consumption
ˑUrinalysis (volume, specific gravity, and electrolyte)
ˑHematology
ˑBlood chemistry
ˑOrgan weight
ˑRelative organ weight

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no abnormalities attributable to the test substance in any animals.
There were loss of fur in one male exposed to 28474.44 mg/m3 (2000 ppm) AC-6000 and opacity of eyeball in one female exposed to 7118.61 mg/m3 (500 ppm) AC-6000. These changes were considered to be spontaneous changes in rats.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no statistically significant differences in body weight of males or females in the groups exposed to the test substance in comparison with that of the control group. There were statistically significant lower body weight gains in males on day 29 and in females exposed to 28474.44 mg/m3 (2000 ppm) AC-6000 on day 71, and higher body weight gains in males exposed to 7118.61 mg/m3 (500ppm) and 28474.44 mg/m3 (2000 ppm) AC-6000on day 50 in comparison with the control group. Since there were no dose-related patterns in these differences, it was concluded that the differences in body weight gains did not have any toxicological significance.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were statistically significant lower food consumption in males exposed to 7118.61 mg/m3 (500 ppm) AC-6000 on day 22 and higher food consumption in females exposed to 14237.22 mg/m3 (1000 ppm) AC-6000 on day 91 in comparison with the control group. Since there were no dose related patterns in these changes, it was concluded that the differences in food consumption did not have any toxicological significance.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no abnormalities attributable to the test substance. Several findings were observed and described in tables 1 and 2 in any other information on results inc. tables. However, these findings did not change between the pre-dosing period and the dosing period, and were considered to be spontaneous changes.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in any test substance exposure groups in comparison with the control group.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were statistically significant lower value of blood potassium in males exposed 56948.88 mg/m3 (4000 ppm) AC-6000 and lower value of A/G ratio in females exposed to 28474.44 mg/m3 (2000 ppm) AC-6000. Since there was no dose-related pattern in the lower value of A/G ratio, it was concluded that it did not have any toxicological significance.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were statistically significant higher values in urine volume in females exposed to 14237.22 mg/m3 (1000 ppm) and 28474.44 mg/m3 (2000 ppm) AC-6000 in comparison with the control group. Since there were no changes in other parameters, it was concluded that the differences in urine volume did not have any toxicological significance
Behaviour (functional findings):
not examined
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were statistically significant higher values of absolute and relative weights of the liver and lower values of absolute and relative weights of the adrenals weight in males exposed to 56948.88 mg/m3 (4000 ppm) AC-6000 in comparison with the control group. There was statistically significant higher value of relative weight of ovaries weight in females exposed to 28474.44 mg/m3 (2000 ppm) AC-6000 in comparison with the control group. Since there was no dose-related pattern in this change, it was concluded that it did not have any toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were several findings and are described in tables 3 and 4 in any other information on results inc. tables. Since they were observed sporadically without any dose related pattern, it was concluded that the findings were not attributable to the test substance.

Focal myocardial degeneration was observed in 3, 1, and 3 males exposed to 7118.61 mg/m3 (500 ppm), 14237 mg/m3 (1000 ppm) and 2847.44 mg/m3 (2000 ppm) AC-6000. There was no difference in incidence of this finding between the groups; therefore this finding was considered to be a spontaneous lesion.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Hypertrophy of centrilobular hepatocyte was observed in all males and 5 females exposed to 56948.88 mg/m3 (4000 ppm) AC-6000, and 4 males exposed to 28474.44 mg/m3 (2000 ppm) AC-6000. The findings are described in more detail in tables 5 and 6 in any other information on results inc. tables. The other findings were observed in the control or exposed to 56948.88 mg/m3 (4000 ppm) AC-6000. As they were non-specific in rats, or there were no differences in incidence of the findings between the groups, it was concluded that the findings were not attributable to the test substance.
Histopathological findings: neoplastic:
not specified

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
14 237.22 mg/m³ air
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
28 474.44 mg/m³ air
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
not specified
Lowest effective dose / conc.:
14 237.22 mg/m³ air
System:
hepatobiliary
Organ:
liver

Any other information on results incl. tables

Table 1: Ophthalmic Examination in Males- Summary

 Group  Sex  Observation Number of Animals Affected on Day -2*  Number of Animasl Affected on Day 90*
 1  m Particulate opacity in lens  1 2
 1  m Retinal fold  1 -
 2  m Particulate opacity in lens
2  m Persistent hyaloid artery 1
 2 Retinal fold 
 m Particulate opacity in lens  - 1
 4 Particulate opacity in lens  3  3
 4  m Focal opacity in lens  1  1
 5  m Particulate opacity in lens  4  5
 5  m Focal opacity in lens   1  1

*Day number relative to Start Date; total number of animals examined in each group was 10

Group 1- 0 mg/m3 (0 ppm), Group 2 - 7118.61 mg/m3 (500 ppm), Group 3 - 14237.22mg/m3 (1000 ppm), Group 4 - 28474.44 mg/m3 (2000 ppm), Group 5 - 56948.88 mg/m3 (4000 ppm).

Table 2: Ophthalmic Examination in Females- Summary

 Group  Sex  Observation Number of Animals Affected on Day -2*  Number of Animasl Affected on Day 90*
 1  f Particulate opacity in lens  - 1
 1  f Focal opacity in lens 2 2
 2  f Particulate opacity in lens 1
2  f Loss of light reflex -
 2 f Focal opacity in lens 
2 f

Cataract

-

1

 2

f

Not determined

-

1

 3

 f

Particulate opacity in lens

 1

 4

4

 f

Particulate opacity in lens

 2

2

 4

 f

Focal opacity in lens 

 1

 1

 4  f Hyperreflectivity in fundus   -
 5  f Particulate opacity in lens  - 1
 5  f Retinal fold  1  1

*Day number relative to Start Date; total number of animals examine in each group was 10

Group 1- 0 mg/m3 (0 ppm), Group 2 - 7118.61 mg/m3 (500 ppm), Group 3 - 14237.22mg/m3 (1000 ppm), Group 4 - 28474.44 mg/m3 (2000 ppm), Group 5 - 56948.88 mg/m3 (4000 ppm).

Table 3: Necropsy Findings in Males - Summary

Concentration (ppm)  500 1000  2000   4000
Number of animals examined 10 10 10  10  10 
 Necropsy Findings
 Testis: small; unilateral
 Epididymis: aplasia; unilateral
 Skin: loss; hair
 Subcutis: mass

Table 4: Necropsy Findings in Females - Summary

Concentration (mg/m3 (ppm)  500 1000  2000   4000
Number of animals examined 10 10 10  10  10 
 Necropsy Findings
Stomach: nodule, ; serosa, one
 Eyeball:opacity; bilateral -

Table 5: Histopathology Findings in Male Liver - Summary

 Concentration (ppm) 500 1000 2000  4000 
 Number of animals examined 10  9 10  10  10 
 No visible lesion 10 10 

 Hypertrophy, hepatocyte; centrilobular

... minimal

0

0

0

0

0

0

4

10

10 

Necrosis, hepatocyte; focal

... minimal 

0

0

0

3

2

Cell infiltration, inflammatory; focal

... minimal

0

3

3

0

0

0

0

3

3

Table 6: Histopathology Findings in Female Liver - Summary

Concentration (ppm) 500 1000 2000  4000 
 Number of animals examined 10  10 10  10  10 
 No visible lesion 9 10 

 Hypertrophy, hepatocyte; centrilobular

... minimal

0

0

0

0

0

0

0

0

5

Necrosis, hepatocyte; focal

... minimal 

1

0

0

1

1

0

0

0

0

Cell infiltration, inflammatory; focal

... minimal

0

0

1

1

0

0

0

0

0

0

Applicant's summary and conclusion

Conclusions:
The NOAEL of AC-6000 was 14237.22 mg/m3 (1000 ppm) for males and 28474.44 mg/m3 (2000 ppm) for females.
Executive summary:

The subchronic toxicity of AC-6000 was examined when it was repeatedly exposed to Crl:CD(SD) rats for 6 hours a day on 5 days a week for 13 consecutive weeks. The mean actual concentrations were 7628.30 mg/m3 (535.8 ppm), 14993.22 mg/m3 (1053.1 ppm), 29811.31 mg/m3 (2093.9 ppm) and 61370.96 mg/m3 (4310.6 ppm) in the AC-6000 7118.61 mg/m3 (500 ppm), 14237.22 mg/m3 (1000 ppm), 28474.44 mg/m3 (2000 ppm), and 56948.88 mg/m3 (4000 ppm) groups, respectively.


The test substance related effects were evident in the liver. The higher values of absolute and body weight-relative weight in the liver were noted in males exposed to AC-6000 56948.88 mg/m3 (4000 ppm) in comparison with those in the control group. Hypertrophy of centrilobular hepatocyte was observed in males and females exposed to AC-6000 56948.88 mg/m3 (4000 ppm) and in males exposed to AC-6000 28474.44 mg/m3 (2000 ppm). The ground glass cytoplasm was observed in the hypertrophic hepatocyte. It was considered that the hypertrophy of hepatocyte was originated as a result of adaptive changes by induction of drug metabolizing enzymes. There was a low value of potassium in the blood chemistry in males exposed to AC-6000 56948.88 mg/m3 (4000 ppm). Since there was no difference in the potassium range between this study and the background data of the test facility (mean ± 2S.D: 3.8-5.0 mmol/L, Annex 1), it was concluded that the changes were within the physiological fluctuation in the serum potassium. There were low values of absolute and body weight-relative weight in the adrenals in males exposed to AC-6000 56948.88 mg/m3 (4000 ppm). Since there were no abnormalities in the pathological examination, it was concluded that these changes did not have any toxicological significance. There were no changes attributable to the test substance in clinical sign, body weight, food consumption, ophthalmoscopy, urinalysis, hematology, or necropsy. Based on the results, it was concluded that the NOAEL of AC-6000 was 14237.22 mg/m3 (1000 ppm) for males and 28474.44 mg/m3 (2000 ppm) for females under the conditions of this study.