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Diss Factsheets

Administrative data

Description of key information

Based on the weight of evidence of available sub-chronic and chronic repeated dose toxicity studies via the oral and the inhalation route in mice as well as in rats with 1,4 -dioxane, predominantly induction of tumors and pre-neoplastic lesions in the respiratory tract as well as in the liver.

The lowest oral NOAEL was found to be 9.6 mg/kg bw/d in rats based on a chronic toxicity study (Kociba et al., 1974).

Regarding inhalation route, the lowest LOAEC (local and systemic) was identified to be 180 mg/m3 (Kasai et al., 2009).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
chronic toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
No specific guideline is mentioned; four groups of rats (60 males and 60 females per group) were maintained on drinking water containing:
0, 0.01, 0.1 and 1.0% dioxane for up to 716 days.
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
- Name of test material: dioxane
- Source: Dow Chemical Co.
- Physical state: colourless liquid
- Storage condition of test material: in amber coloured quart-sized bottles and padded with nitrogen until opened for use.
After opening, dioxane was generally used within one week.

Analysis of various stock samples of dioxane revealed the following:
- Hydrogen peroxide : 10- 340 ppm
- Acetaldehyde : non detectable
- Crotonaldehyd : 220 - 1340 ppm
- 2-Methyl-1,3-dioxolane: 6 - 108 ppm
- Water : 10 - 90 ppm
Acidity: 0.0006 - 0.0042 mequiv./mL
Species:
rat
Strain:
Sherman
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6-8 weeks
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
No data
Route of administration:
oral: drinking water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Drinking water was prepared by dilution in water (1%; 60 mL dioxane added to 5940 mL water).
The drinking water was prepared twice weekly during the first year, and weekly during the second year.
Periodically samples were taken from storage vessels and individual water dispensers, and analysed for dioxane content by gas chromatography.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stock samples were analysed for impurities at 6 different times during the 2 years, by gas chromatography.
Duration of treatment / exposure:
716 days
Frequency of treatment:
continuous
Dose / conc.:
0.01 other: %
Remarks:
corresponding to 9.6 mg/kg bw/day (males) and 19 mg/kg bw/day (females)
Dose / conc.:
0.1 other: %
Remarks:
corresponding to 94 mg/kg bw/day (males) and 148 mg/kg bw/day (females)
Dose / conc.:
1 other: %
Remarks:
corresponding to 1015 mg/kg bw/day (males) and 1599 mg/kg bw/day (females)
No. of animals per sex per dose:
60 male and 60 female rats/dose
Control animals:
yes, plain diet
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations:
twice weekly (first month)
weekly (month 2-7)
biweekly (afterwards)

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily water consumption was recorded daily during 3 periods:
days 1-113, days 114-198, and days 446-460

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: no data
- Anaesthetic used for blood collection: no data
- Animals fasted: no data
- How many animals: no data
- Parameters examined: packed cell volume, total erythrocyte count, Hb, and total and different white blood cell counts.

CLINICAL CHEMISTRY: No data

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, a complete gross pathologic examination was performed.
Organ weights of brain, liver, kidneys, testes, spleen, and heart were recorded.

HISTOPATHOLOGY: Yes
brain
bone and bone marrow
ovaries
pituitary gland
uterus
mesentric lymph nodes
heart
liver
pancreas
spleen
stomach
prostrate
colon
trachea
duodenum
kidneys
esophagus
jejunum
testes
lungs
spinal cord
adrenal gland
parathyroid gland
nasal turbinates
urinary bladder
Statistics:
Student's T-tets was used for Heamatology parameters, and body and organ weights;
Fisher's Exact probability test was used for analysis of tumors;
Chi-Square contigency tables and Fisher's exact propability was used for survival comparison.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Description (incidence):
The concentration of 1% 1,4-dioxane led within two to four months to a severe reduction of survival rates in both sexes, nearly half of the group succumbing after four months. The survival rate after four months was essentially the same for all groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Within 2 days after initiating the study the body weights of both sexes at 1.0% 1,4-dioxane were significantly lower than controls. The body weights remained depressed throughout the study.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
No effects on haematology were observed.
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Significantly increased liver weight in rats receiving 1% 1,4-dioxane.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In rats at 0.1 and 1.0% 1,4-dioxane, gross and histopathological examination revealed variable degrees of renal tubular epithelial and hepatocellular degeneration and necrosis, accompanied by regenerative activities in the liver (hepatocellular hyperplastic nodule formation) and renal tubuli. No effects were seen on male and female reproductive organs.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In rats at 0.1 and 1.0% 1,4-dioxane, gross and histopathological examination revealed variable degrees of renal tubular epithelial and hepatocellular degeneration and necrosis, accompanied by regenerative activities in the liver (hepatocellular hyperplastic nodule formation) and renal tubuli. No effects were seen on male and female reproductive organs.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Male and female rats receiving 0.1% (equivalent to 94 and 148 mg/kg/day respectively) and 0.01 % (equivalent to 9.6 and 19 mg/kg/day respectively) dioxane in drinking water showed no evidence of tumour formation. Only in the highest dose group 1.0% (1055 and 1599 mg/kg/day for males and females respectively) were treatment-related tumours found:
in the liver, carcinomas were found in 10/66 animals surviving at 12 months and cholangiomas in 2/66 animals, while squamous cell carcinomas of the nasal cavities were found in 3/66 animals.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
9.6 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
19 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
LOEL
Effect level:
9.6 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: equivocal necrosis / inflammation in liver
Remarks on result:
other: as re-evaluated by Dourson et al., 2017
Key result
Dose descriptor:
LOEL
Effect level:
148 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: necrosis / inflammation in the liver
Remarks on result:
other: as re-evaluated by Dourson et al., 2017
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
94 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
other: degeneration and necrosis
Treatment related:
yes
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
1981
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
DBF1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc (Kanagawa, Japan)
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 6 weeks
- Weight at study initiation: not specified
- Fasting period before study: not specified
- Housing: individually, stainless steel wire-mesh hanging cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: two weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 24 +/- 1 °C
- Humidity: 55 +/-5 %
- Air changes: 15-17 per hr
- Photoperiod: 12/12 hrs dark / hrs light
Route of administration:
oral: drinking water
Details on route of administration:
The 1,4-dioxane-formulaled drinking water was prepared twice a week, and administered to each animal with a sipper bottle made of glass during a 3- or 4-day period. The volume of the sipper bottle was 35 mL.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
1,4-Dioxane was dissolved in deionized water to a target concentration of 640, 1600, 4000, 10000 or 25000 ppm (wt/wt).


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of 1,4-dioxane in drinking water were determined at the time of preparation with the gas chromatograph, and found to be 94.6-102.9% of the target concentration. Stability of 1,4-dioxane in the drinking water was examined 4 days after preparation of the 1,4-dioxane-formulated drinking water, using the gas chromatograph and found to be 92.8-96.4% of the initial concentrations.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Dose / conc.:
0 ppm
Dose / conc.:
640 ppm
Dose / conc.:
1 600 ppm
Dose / conc.:
4 000 ppm
Dose / conc.:
10 000 ppm
Dose / conc.:
25 000 ppm
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: not specified
- Rationale for animal assignment: random (by body weight)
- Fasting period before blood sampling for clinical biochemistry: overnight
Positive control:
not applicable
Observations and examinations performed and frequency:
Observations included clinical signs, body weight, food and water consumption, haematology, biochemistry, urinalysis, necropsy, organ weights and histopathological examination.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Body weight, water consumption, food consumption, organ weight, and hematological and blood biochemical parameters were analyzed by Dunnett's test. Histopathological findings and urinary parameters were analyzed by chi-square test, A two-sided analysis with p values of 0.05 and 0.01 was performed to determine statistical significance. A no-observed-adverse-effect-level (NOAEL) was determined according to the WHO definition.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the surviving animals, piloerection in the male mice given 25000 ppm were observed.
Mortality:
mortality observed, treatment-related
Description (incidence):
A male mouse, which was given 25000 ppm, died during the 2nd week of the 13-week administration period. Histopathological examination could not reveal the cause of death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Terminal body weight was significantly decreased only in male mice given 25000 ppm.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Significantly decreased food consumption was observed only in the male mice given 25000 ppm.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was decreased dose-dependently in both male and female mice given 10000 ppm and 25000 ppm.
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Red blood cell counts, hemoglobin and hematocrit were significantly increased in male mice given 25000 ppm, whereas those three erythrocyte parameters were not increased in any 1,4-dioxane-dosed females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Both aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were significantly increased in male mice and female mice given 25000 ppm. In addition, ALT was significantly increased in female mice given 10000 ppm. Notably, a significant decrease in the plasma level of glucose was observed in mice of both sexes given 25000 ppm, and in female mice given 10.000 ppm.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Urinary pH was decreased in mice of both sexes given 10000 ppm and 25000 ppm.
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Significantly increased kidney weight was noted in the mice of both sexes given 25000 ppm. Relative lung weight was significantly increased in male mice given 25000 ppm and in female mice given 10000 ppm and 25000 ppm.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No particular macroscopic lesion was found in any 1,4-dioxane-dosed mice of cither sex at terminal necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Positive histopathological findings were observed in the upper and lower respiratory tracts and liver (Table 1).
Nuclear enlargement was a characteristic sign in the upper and lower respiratory tracts. The nuclear size was about 2 times as large in diameter as that in normal nuclei of epithelial cells. The nuclear enlargement appearing in the bronchial epithelium of female mice given 1600 ppm and above was the most sensitive, while the significantly increased incidence of the nuclear enlargement was observed in both the olfactory epithelium and trachea of male and female mice given 4000 ppm and above, and in the bronchial epithelium of male mice given 4000 ppm and above, and in the respiratory epithelium of female mice given 25000 ppm. For the bronchial epithelium the enlarged nuclei were widely distributed over the lower respiratory tract from the main bronchus to terminal bronchioles. In addition to the nuclear enlargement, vacuolic change in the olfactory nerve cells and degeneration in the bronchial epithelium were noted in the male and female mice given 25000 ppm, while the latter degeneration also occurred in the females given 10000 ppm. The vacuolic change in the olfactory nerve cells was mainly observed in the nerve bundles in the lamina propria of dorsal wall at Levels 2 and 3. The degeneration of the bronchial epithelium occurred throughout from the main bronchus to the terminal bronchiole.

Both single cell necrosis and swelling of centrilobular hepatocytes were significantly increased in male and female mice given 4000 ppm and above.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
One male in the 25,000 ppm group died. Body weights and food consumption were slightly reduced in the 10,000 and 25,000 ppm male groups and in the 25,000 ppm female group. Water consumption was decreased in all treated males and in females ≥4,000 ppm. In males, effects on haematology, biochemistry or urinalysis parameters were observed at ≥10,000, ≥4,000 and ≥10,000 ppm, respectively. In females, this occurred at ≥10,000 ppm. Absolute and relative lung weights were increased in males at 25,000 ppm and in females ≥10,000 ppm. In females kidney weight was also increased at these dose levels. Upon histopathology, non-neoplastic lesions were observed in the nasal cavity (nuclear enlargement and eosinophilic change of the olfactory and respiratory epithelium, vacuolic change of the olfactory nerve), trachea (nuclear enlargement of the epithelium), lung (accumulation of foamy cells, degeneration and nuclear enlargement of the bronchial epithelium), and liver (necrosis of single cell and swelling of the central area) in males at 4,000 ppm or greater groups and in females at 1,600 ppm or greater groups. No effects were found on the reproductive organs. Based on the histopathology findings in females at 1,600 ppm, the NOAEL in this study can be established at 640 ppm
Key result
Dose descriptor:
NOAEL
Effect level:
640 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: corresponding to 170 mg/kg bw /d
Key result
Dose descriptor:
NOAEL
Effect level:
1 600 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: corresponding to 231 mg/kg bw/d
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
4 000 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 600 ppm
System:
other: respiratory system: upper and lower
Organ:
bronchi
nasal cavity
trachea
Treatment related:
yes

Table 1 Number of mice bearing the lesions in the respiratory tract, liver, kidneys and brain.

 

Male

Female

Group (ppm)

Control

640

1600

4000

10000

25000

Control

640

1600

4000

10000

25000

No. of animals examined

10

10

10

10

10

9a

10

10

10

10

10

10

Nasal cavity

Nuclear enlargement: respiratory epithelium

0

0

0

2

(1.0)

5

(1.0)

0

0

0

0

3

(1.0)

3

(1.0)

7**

(1.1)

Nuclear enlargement: olfactory epithelium

0

0

0

9**

(1.0)

10**

(1.1)

9**

(1.0)

0

0

0

6*

(1.0)

10**

(1.5)

10**

(2.0)

Vacuolic change olfactory nerve

0

0

0

0

0

9**

(1.1)

0

0

0

0

2

(1.0)

8**

(1.3)

Trachea

Nuclear enlargement: epithelium

0

0

0

7**

(1.0)

9**

(1.0)

9**

(1.1)

0

0

2

(1.0)

9**

(1.0)

10**

(1.0)

10**

(1.3)

Bronchus

Nuclear enlargement: bronchial epithelium

0

0

0

9**

(1.0)

9**

(1.0)

9**

(1.4)

0

0

10**

(1.0)

10**

(1.1)

10**

(2.1)

10**

(3.0)

Degeneration: bronchial epithelium

0

0

0

0

0

8**

(1.1)

0

0

0

0

7**

(1.0)

10**

(1.1)

Liver

Necrosis: single cells

0

0

0

5*

(1.0)

10**

(1.0)

9**

(1.0)

0

0

0

7**

(1.0)

10**

(1.0)

9**

(1.0)

Swelling: centrilobular

0

0

0

10**

(1.1)

10**

(1.0)

9**

(2.0)

0

1

(1.0)

1

(1.0)

10**

(1.0)

10**

(1.0)

9**

(2.0)

Note: Values indicate number of animals bearing lesions.

The values in parantheses indicate the average of severity grade index of the lesion. The average of severity grade was calculated with the following equation.

∑(grade * number of animals with grade) / number of affected animals.

Grade: 1 = slight, 2 = moderate, 3 = severe.

Significant difference:

*: p≤0.05

**: p≤0.01 by chi-square test,

a: One dead animal was excluded for the analysis.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
1981
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc (Kanagawa, Japan)
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 6 weeks
- Weight at study initiation: not specified
- Fasting period before study: not specified
- Housing: individually, stainless steel wire-mesh hanging cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: two weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 24 +/- 1 °C
- Humidity: 55 +/-5 %
- Air changes: 15-17 per hr
- Photoperiod: 12/12 hrs dark / hrs light
Route of administration:
oral: drinking water
Details on route of administration:
The 1,4-dioxane-formulaled drinking water was prepared twice a week, and administered to each animal with a sipper bottle made of glass during a 3- or 4-day period. The volume of the sipper bottle was 200 mL.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
1,4-Dioxane was dissolved in deionized water to a target concentration of 640, 1600, 4000, 10000 or 25000 ppm (wt/wt).



Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of 1,4-dioxane in drinking water were determined at the time of preparation with the gas chromatograph, and found to be 94.6-102.9% of the target concentration. Stability of 1,4-dioxane in the drinking water was examined 4 days after preparation of the 1,4-dioxane-formulated drinking water, using the gas chromatograph and found to be 94.2- 101.1% of the initial concentrations.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Dose / conc.:
0 ppm
Dose / conc.:
640 ppm
Dose / conc.:
1 600 ppm
Dose / conc.:
4 000 ppm
Dose / conc.:
10 000 ppm
Dose / conc.:
25 000 ppm
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: not specified
- Rationale for animal assignment: random (by body weight)
- Fasting period before blood sampling for clinical biochemistry: overnight
Positive control:
not applicable
Observations and examinations performed and frequency:
Observations included clinical signs, body weight, food and water consumption, haematology, biochemistry, urinalysis, necropsy, organ weights and histopathological examination.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
Livers of five 25,000 ppm-dosed and five vehicle-dosed rats of both sexes were sectioned for further examination of altered hepatocellular foci by immunohistochemical staining with antibody of glutathione 5-transferase placental form (anti-GSTP). Focal populations having fifty
or more homogeneously stained brown and agglomerated hepatocytes were defined as the GST-P-positive foci in the present study.
Statistics:
Body weight, water consumption, food consumption, organ weight, and hematological and blood biochemical parameters were analyzed by Dunnett's test as described in detail previously. Histopathological findings and urinary parameters were analyzed by chi-square test, A two-sided analysis with p values of 0.05 and 0.01 was performed to determine statistical significance. A no-observed-adverse-effect-level (NOAEL) was determined according to the WHO definition.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the surviving animals, piloerection and colored fur in both male and female rats given 25000 ppm were observed.
Mortality:
mortality observed, treatment-related
Description (incidence):
A female rat which was given 25000 ppm, died during the 2nd week of the 13-week administration period. Histopathological examination revealed that the death was causally related to renal failure because of marked hydropic degeneration of the proximal tubule.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Terminal body weight was significantly decreased in male rats given 10000 ppm and 25000 ppm and in female rats given 4000 ppm and above.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was significantly decreased in the male rats given 25000 ppm and in the female rats given 10000 ppm and 25000 ppm.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was decreased dose-dependently in both male and female rats given 4000 ppm and above.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Red blood cell counts, hemoglobin and hematocrit were significantly increased in male rats given 25000 ppm, whereas those three erythrocyte parameters were not increased in any 1,4-dioxane-dosed females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Both aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were significantly increased in male rats given 25000 ppm. In addition, AST was significantly increased in female rats given 25000 ppm. Notably, a significant decrease in the plasma level of glucose
was observed in rats of both sexes given 25000 ppm.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Urinary pH was decreased in male rats given 4000 ppm and above, in the female rats given 10000 ppm and 25000 ppm.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Relative liver weight was significantly increased in male rats given 10000 ppm and 25000 ppm and in female rats given 1600 ppm and above. Relative kidney weight was significantly increased in male rats given 4000 ppm and above and in female rats given 1600 ppm and above. Relative lung weight was significantly increased in male rats given 25000 ppm and in female rats given 10000 ppm and 25000 ppm.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No particular macroscopic lesion was found in any 1,4-dioxane-dosed rats of cither sex at terminal necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The upper and lower respiratory tracts, liver, kidneys and brain were affected by oral administration of 1,4-dioxane in drinking water to rats (Table 1). The nuclear enlargement occurring in the nasal respiratory epithelium of male and female rats given 1600 ppm and above was
the most sensitive, and followed by the enlarged nuclei of epithelial cells in the olfactory epithelium and in the tracheal and bronchial epithelia. The nuclear enlargement was morphologically featured by the appearance of epithelial cells having round to oval nuclei which were at least 4 times as large in diameter as the normal nuclei of epithelial cells in the upper and lower respiratory tracts. The enlarged nuclei of the respiratory epithelial cells were distributed over the entire respiratory region at Levels 1 through 3 in the nasal cavity. The enlarged nuclei in the olfactory epithelium occurring in the sustentacular cells were distributed over the entire olfactory region at Levels 2 and 3. The rat nuclear enlargement in the bronchial epithelium was localized only in the main bronchus.

Centrilobular swelling of hepatocytcs was the most sensitive sign appearing in the male rats given 1600 ppm and above. Single cell necrosis, which accompanied inflammatory cell infiltration, significantly increased in male rats given 4000 ppm and 25000 ppm and in female rats given 25000 ppm.
A degenerative change such as vacuolic change in centrilobular hepatocytes occurred at high dose levels. Notably, altered hepatocellular foci stained positively with the anti-GST-P antibody were found in all the five 25000 ppm-dosed rats of both sexes, whereas there were no GST-P-positive foci in the livers of the five male or five female controls each.
However, the hematoxylin-eosin stained altered hepatocellular foci could not be clearly detected in any 1,4-dioxane-dosed rat of either sex.

Nuclear enlargement of epithelial cells in the renal proximal tubules characterized by the appearance of epithelial cells having nuclei which were up to about 2 times as large as the normal nucleus in diameter occurred in the male and female rats given 10000 ppm and 25000 ppm, while hydropic change in the proximal tubules was observed only in the 25000 ppm-dosed males and females.

Vacuolic change in the cerebrum was noted in the 25000 ppm-dosed males and females. The cerebral vacuoles varied in size, up to about 100 µm, and were located in the corpus callosum, hippocampus and dentate gyrus.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
640 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: corresponding to 52 (males) and 83 (females) mg/kg bw/d
Key result
Dose descriptor:
LOEL
Effect level:
1 600 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Remarks on result:
other: as re-evaluated by Dourson et al., 2017
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 600 ppm
System:
other: respiratory system: upper and lower
Organ:
bronchi
nasal cavity
trachea
Treatment related:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 600 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes

Table 1 Number of rats bearing the lesions in the respiratory tract, liver, kidneys and brain.

 

Male

Female

Group (ppm)

Control

640

1600

4000

10000

25000

Control

640

1600

4000

10000

25000

No. of animals examined

10

10

10

10

10

10

10

10

10

10

10

9a

Nasal cavity

Nuclear enlargement: respiratory epithelium

0

0

9**

(1.0)

10**

(2.0)

9**

(2.0)

10**

(2.0)

0

0

5*

(1.0)

10**

(1.0)

10**

(1.0)

8**

(1.0)

Nuclear enlargement: olfactory epithelium

0

0

0

10**

(1.0)

9**

(1.0)

10**

(2.0)

0

0

0

9**

(1.0)

10**

(1.0)

8**

(1.0)

Trachea

Nuclear enlargement: epithelium

0

0

0

10**

(1.0)

10**

(1.0)

10**

(2.8)

0

0

0

9**

(1.0)

10**

(1.0)

9**

(2.1)

Bronchus

Nuclear enlargement: bronchial epithelium

0

0

0

0

1

(1.0)

2

(1.0)

0

0

0

1

(1.0)

1

(1.0)

6*

(1.0)

Liver

Necrosis: single cells

0

0

0

5*

(1.0)

2

(1.0)

10**

(1.1)

2

(1.0)

0

1

(1.0)

5

(1.0)

5

(1.2)

8**

(1.5)

Swelling: centrilobular

0

0

9**

(1.0)

10**

(1.1)

10**

(2.0)

10**

(2.9)

0

0

1

(1.0)

0

9**

(1.0)

9**

(1.7)

Vacuolic change: centrilobular

0

0

1

(1.0)

0

10**

(1.5)

10**

(3.0)

0

0

0

0

0

9**

(2.2)

Kidney

Hydropic change: proximal tubule

0

0

0

0

0

7**

(1.0)

0

0

0

0

0

5*

(1.6)

Nuclear enlargement: proximal tubule

0

0

0

1

(1.0)

5*

(1.0)

9**

(1.0)

0

0

0

0

8**

(1.0)

9**

(1.0)

Brain

Vacuolic change

0

0

0

0

0

10**

(1.2)

0

0

0

0

0

9**

(1.0)

Note: Values indicate number of animals bearing lesions.

The values in parantheses indicate the average of severity grade index of the lesion. The average of severity grade was calculated with the following equation.

∑(grade * number of animals with grade) / number of affected animals.

Grade: 1 = slight, 2 = moderate, 3 = severe.

Significant difference:

*: p≤0.05

**: p≤0.01 by chi-square test,

a: One dead animal was excluded for the analysis.

Endpoint:
chronic toxicity: oral
Remarks:
carcinogenicity study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
1981
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
other: Crj:BDF1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan Inc. (Kanagawa, Japan)
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 6 weeks
- Weight at study initiation: not specified
- Fasting period before study: not specified
- Housing: individually in stainless steel wire-mesh hanging cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 24 +/-1 °C
- Humidity: 50 +/- 5 %
- Air changes: 15-17 per hr
- Photoperiod: 12 / 12 hrs dark / hrs light
Route of administration:
oral: drinking water
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
1,4-Dioxane was dissolved in deionized water at a target concentration of 0 (control), 500, 2000 or 8000 ppm (wt./wt.). The 1,4-dioxane-formulated drinking-water was prepared twice a week and administered using a sipper bottle made of brown glass during a 3- or 4-day period. The volume of the sipper bottle was 35 mL.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of 1,4-dioxane in the drinking-water were determined at the time of preparation by GC, and found to be 94–111% of the target concentrations. The stability of the 1,4-dioxane in the drinking-water was examined 4 days after preparation of the 1,4-dioxane-formulated water and found to remain at 87–92% of the initial concentrations.
Duration of treatment / exposure:
2 years
Frequency of treatment:
daily
Dose / conc.:
500 ppm
Remarks:
corresponding to 49 +/- 5 (males) and 66 +/- 10 (females) mg/kg bw/d
Dose / conc.:
2 000 ppm
Remarks:
corresponding to 191 +/- 21 (males) and 278 +/- 40 (females) mg/kg bw/d
Dose / conc.:
8 000 ppm
Remarks:
corresponding to 677 +/- 74 (males) and 964 +/- 88 (females) mg/kg bw/d
No. of animals per sex per dose:
50
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The highest dose levels were chosen so as not to exceed the MTD, based on both growth rate and toxicity observed in previous 13-week drinking-water studies (Kano et al., 2008)
- Rationale for animal assignment: stratified randomisation (body weight matched groups)


Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for the first 4 weeks; every 2 weeks thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule: weekly for the first 4 weeks; every 4 weeks thereafter

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

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily; daily 1,4-dioxane intake was calculated as the concentration of 1,4-dioxane in drinking-water, multiplied by the volume of drinking-water consumed on a daily basis, and divided by the animal’s body weight.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Survival curves were plotted according to the method of Kaplan–Meier (Kaplan and Meier, 1958). The log-rank test (Peto et al., 1977) was used to test for a statistically significant difference in survival rate between any 1,4-dioxane-dosed group of either sex and the respective control group. Body and organ weights, and food and water consumption were analyzed by Dunnett’s Test. Incidences of non-neoplastic lesions were analyzed by the chi-square test. Incidences of neoplastic lesions were analyzed for a dose–response relationship by Peto’s test (Peto et al., 1980) and for a statistically significant difference from the concurrent control group by Fisher’s exact test. A biologically meaningful increase in the incidence of rare tumors was evaluated by whether or not the observed incidence exceeded the maximum tumor incidence in the JBRC historical control data compiled from 2-year studies of rodent carcinogenicity conducted by the JBRC during a 21-year period from 1987 to 2007. Two-tailed testing was used for all statistical analyses except for Peto’s test. In all cases, statistical analysis with p-values of 0.05 and 0.01 was performed and is indicated in the tables; a p-value of 0.05 was used for statistical significance. NOAEL and LOAEL were determined according to the WHO definition (WHO 1994).
Clinical signs:
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
There was no difference in survival rate between any 1,4-dioxane-dosed male group and the male control. The female groups given 2000 and 8000 ppm, on the other hand, did show significantly decreased survival rates. The decreased survival rates of these two groups were attributed to the increased number of deaths due to hepatic tumors.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Growth rates of the males and females given 2000 and 8000 ppm were significantly retarded and the terminal body weights in these groups were significantly decreased. The decreased body weights were attributed to the increased incidences of malignant liver tumors.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Significantly decreased at 8000 ppm in males and females.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Significantly decreased at 8000 ppm in males and females. The estimated daily intake per body weight of 1,4-dioxane in the males and females given 8000 ppm did not increase proportionally as compared with that of the males and females given 500 or 2000 ppm 1,4-dioxane. This decreased 1,4-dioxane intake was attributed to the decreased water consumption in the 8000 ppm-dosed males and females.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Relative liver weight was significantly increased in the 8000 ppm-dosed group of both sexes and in the 2000 ppm-dosed males.
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
1,4-Dioxane-induced tumors occurred in the nasal cavity and liver. One case each of a nasal esthesioneuroepithelioma and a nasal adenocarcinoma occurred in an 8000 ppm-dosed male and female mouse, respectively. Since these nasal tumors have not been observed in the historical control data of the JBRC (no case of esthesioneuroepithelioma in 1846 male mice and no case of adenocarcinoma in 1847 female mice in 37 carcinogenicity studies), they were judged to be compound-related. These nasal tumors were located in the ethmoturbinate at Level 3. The incidence of nuclear enlargement in the nasal cavity was significantly increased in both respiratory and olfactory epithelia of the 8000 ppm-dosed males and females, and in the olfactory epithelium of the 2000 ppm-dosed males and females.
Hepatocellular carcinomas occurred dose-dependently, and their incidences were significantly increased in the 8000 ppm dosed males and in all 1,4-dioxane-dosed female groups. A significant induction of hepatocellular adenomas occurred in the 2000 ppm-dosed males and in the 500 ppm and 2000 ppm-dosed females.
Other effects:
no effects observed
Key result
Dose descriptor:
LOAEL
Effect level:
500 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: neoplastic
Remarks on result:
other: corresponding to ca. 66 mg/kg bw/d
Remarks:
lowest dose tested
Key result
Dose descriptor:
LOAEL
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
histopathology: neoplastic
Remarks on result:
other: corresponding to ca.191 mg/kg bw/d
Key result
Dose descriptor:
NOAEL
Effect level:
500 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
histopathology: neoplastic
Remarks on result:
other: corresponding to ca.49 mg/kg bw/d
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
2 000 ppm
System:
other: nasal cavity: respiratory and olfactory epithelium
Organ:
nasal cavity
Treatment related:
yes

Table 1. Number of mice bearing selected lesions in the nasal cavity, liver, subcutis, mammary gland and peritoneum.

 

Male

Female

Group (ppm)

Control

500

2000

8000

Petro test

Control

500

2000

8000

Petro test

Number of animals examined

50

50

50

50

 

50

50

50

50

 

Nasal cavity

 

 

 

 

 

 

 

 

 

 

Adenocarcinoma

0

0

0

0

 

0

0

0

1

 

Esthesioneuroepithelioma

0

0

0

1

 

0

0

0

0

 

Respiratory epithelium

Nuclear enlargement

0

0

0

31**

 

0

0

0

41**

 

Olfactory epithelium 

Nuclear enlargement

0

0

9**

49**

 

0

0

41**

33**

 

Liver

 

 

 

 

 

 

 

 

 

 

Hepatocellular adenoma

9

17

23##

11

 

5

31##

20##

3

 

Hepatocellular carcinoma

15

20

23

36##

++

0

6#

30##

45##

++

Hepatocellular adenoma or carcinoma

23

31

37#

40##

++

5

35##

41##

46##

++

Note: Lesions in animals which became moribund or died before the end of the 2-year administration period were also scored.

# Significantly different from control at p < 0.05 by Fisher’s exact test.

## Significantly different from control at p < 0.01 by Fisher’s exact test.

** Significantly different at p < 0.01 by chi-square test.

++ Significantly different at p < 0.01 by Peto’s test.

Endpoint:
chronic toxicity: oral
Remarks:
carcinogenicity study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
1981
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan Inc. (Kanagawa, Japan)
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 6 weeks
- Weight at study initiation: not specified
- Fasting period before study: not specified
- Housing: individually in stainless steel wire-mesh hanging cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 24 +/-1 °C
- Humidity: 50 +/- 5 %
- Air changes: 15-17 per hr
- Photoperiod: 12 / 12 hrs dark / hrs light
Route of administration:
oral: drinking water
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
1,4-Dioxane was dissolved in deionized water at a target concentration of 0 (control), 200, 1000 or 5000 ppm (wt./wt.). The 1,4-dioxane-formulated drinking-water was prepared twice a week and administered using a sipper bottle made of brown glass during a 3- or 4-day period. The volume of the sipper bottle was 200 mL.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of 1,4-dioxane in the drinking-water were determined at the time of preparation by GC, and found to be 90–114% of the target concentrations. The stability of the 1,4-dioxane in the drinking-water was examined 4 days after preparation of the 1,4-dioxane-formulated water and found to remain at 96–97% of the initial concentrations.
Duration of treatment / exposure:
2 years
Frequency of treatment:
daily
Dose / conc.:
200 ppm
Remarks:
corresponding to 11 +/- 1 (males) and 18 +/-3 (females) mg/kg bw/d
Dose / conc.:
1 000 ppm
Remarks:
corresponding to 55 +/- 3 (males) and 83 +/- 14 (females) mg/kg bw/d
Dose / conc.:
5 000 ppm
Remarks:
corresponding to 274 +/- 18 (males) and 429 +/- 69 (females) mg/kg bw/d
No. of animals per sex per dose:
50
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The highest dose levels were chosen so as not to exceed the MTD, based on both growth rate and toxicity observed in previous 13-week drinking-water studies (Kano et al., 2008)
- Rationale for animal assignment: stratified randomisation (body weight matched groups)


Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for the first 4 weeks; every 2 weeks thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule: weekly for the first 4 weeks; every 4 weeks thereafter

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

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily; daily 1,4-dioxane intake was calculated as the concentration of 1,4-dioxane in drinking-water, multiplied by the volume of drinking-water consumed on a daily basis, and divided by the animal’s body weight.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Survival curves were plotted according to the method of Kaplan–Meier (Kaplan and Meier, 1958). The log-rank test (Peto et al., 1977) was used to test for a statistically significant difference in survival rate between any 1,4-dioxane-dosed group of either sex and the respective control group. Body and organ weights, and food and water consumption were analyzed by Dunnett’s Test. Incidences of non-neoplastic lesions were analyzed by the chi-square test. Incidences of neoplastic lesions were analyzed for a dose–response relationship by Peto’s test (Peto et al., 1980) and for a statistically significant difference from the concurrent control group by Fisher’s exact test. A biologically meaningful increase in the incidence of rare tumors was evaluated by whether or not the observed incidence exceeded the maximum tumor incidence in the JBRC historical control data compiled from 2-year studies of rodent carcinogenicity conducted by the JBRC during a 21-year period from 1987 to 2007. Two-tailed testing was used for all statistical analyses except for Peto’s test. In all cases, statistical analysis with p-values of 0.05 and 0.01 was performed and is indicated in the tables; a p-value of 0.05 was used for statistical significance. NOAEL and LOAEL were determined according to the WHO definition (WHO 1994).
Clinical signs:
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
Survival rates of the 5000 ppm-dosed males and females were significantly decreased, and the decreased survival rates were attributed to the increased number of deaths due to nasal tumors and peritoneal mesotheliomas in the males and to nasal and hepatic tumors in the females.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Growth rates of the males and females given 5000 ppm were significantly retarded and the terminal body weights in these groups were significantly decreased. The 20% decrease in the terminal body weight of the 5000 ppm dosed females was attributed to the increased incidences of malignant tumors.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
The estimated daily intake per body weight of 1,4-dioxane increased proportionally with dosage.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Relative liver weight was significantly increased in the 1000 and 5000 ppm-dosed males and in the 5000 ppm-dosed females.
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
1,4-Dioxane-induced tumors occurred in the nasal cavity, liver, subcutis, mammary gland and peritoneum. A dose-dependent increase in the incidences of nasal squamous cell carcinomas in males and females was also observed. The increase in nasal squamous cell carcinomas in the 5000 ppm-dosed females was statistically significant. Squamous cell carcinomas were morphologically well-differentiated and keratinized, and frequently exhibited invasive growth into the submucosal tissue and destruction of the nasal bone or septum. Nasal esthesioneuroepitheliomas, rhabdomyosarcomas and sarcomas NOS (not otherwise specified) were also observed. Since these nasal tumors have not been observed in the historical control data of the JBRC (no cases in 2149 male rats in 43 carcinogenicity studies or in 1997 female rats in 40 carcinogenicity studies), they were judged to be compound-related.

The squamous cell carcinomas arose from the dorsal wall and septum at Level 1 through 3. The other types of nasal tumors were located in the dorsal wall, septum and turbinate at Levels 2 and 3. Proliferative and preneoplastic lesions (squamous cell hyperplasias and squamous cell metaplasias) in the nasal cavity were observed in the 5000 ppm-dosed groups. Squamous cell hyperplasia was characterized by a focal increase in the number of the epithelial cell layers in the squamous epithelium to five or more layers together with keratinization. Squamous cell metaplasia was characterized by replacement of respiratory epithelium with squamous epithelium with or without keratinization. Some of the squamous cell metaplasia was accompanied by atypia that was characterized by irregular size and shape of the cells and irregular arrangement of the cell layer, and were regarded as potentially preneoplastic lesions (Brown, 1990; Monticello et al., 1990).
Furthermore, the incidence of nuclear enlargement was significantly increased in both respiratory and olfactory epithelia of the 5000 ppm-dosed males and females and in the olfactory epithelium of the 1000 ppm-dosed females.

Hepatocellular adenomas and carcinomas occurred dosedependently, and statistically significant increases occurred in the 5000 ppm-dosed males and females. Since the incidences of hepatocellular adenomas in the 1000 ppm-dosed males and females (14% and 12%, respectively) exceeded the maximum incidences of the JBRC historical control data for hepatocellular adenomas (41 cases, 1.9%, in 2149 male rats with a maximum incidence of 8%, and 22 cases, 1.1%, in 1977 female rats with a maximum incidence of 6.1%), the hepatocellular adenomas occurring in the 1000 ppm-dosed males and females were judged to be compound-related. A significant increase in the incidence of altered hepatocellular foci was observed in the 1000 and 5000 ppm-dosed males and in the 5000 ppm-dosed females.

Subcutaneous fibromas occurred dose-dependently in the males, although the tumor incidences were not statistically significant. In the mammary gland, adenomas and fibroadenomas occurred dose-dependently in the females and males, respectively, and the incidence of adenomas was significantly increased in the 5000 ppm-dosed females. Peritoneal mesotheliomas occurred dose-dependently in the males, and a statistically significant increase occurred in the 5000 ppm-dosed group.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
histopathology: neoplastic
mortality
organ weights and organ / body weight ratios
Remarks on result:
other: corresponding to ca. 55 (males) and 83 (females) mg/kg bw/d
Key result
Dose descriptor:
LOAEL
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
histopathology: neoplastic
mortality
organ weights and organ / body weight ratios
Remarks on result:
other: corresponding to ca. 274 (males) and 429 (females) mg/kg bw/d
Dose descriptor:
LOEL
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: liver weight increase (males only); DNA synthesis, hyperplasia or foci
Remarks on result:
other: as re-evaluated by Dourson et al., 2017
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
5 000 ppm
System:
other: respiratory system: upper and lower
Organ:
nasal cavity
other: olfactory and respiratory epithelium
Treatment related:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
5 000 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes

Table 1. Number of rats bearing selected lesions in the nasal cavity, liver, subcutis, mammary gland and peritoneum.

 

Male

Female

Group (ppm)

Control

200

1000

5000

Petro test

Control

200

1000

5000

Petro test

Number of animals examined

50

50

50

50

 

50

50

50

50

 

Nasal cavity

 

Squamous cell carcinoma

0

0

0

3

++

0

0

0

7##

++

Esthesioneuroepithelioma

0

0

0

1

 

0

0

0

1

 

Rhabdomyosarcoma

0

0

0

1

 

0

0

0

0

 

Sarcoma NOS

0

0

0

2

 

0

0

0

0

 

Respiratory epithelium

Nuclear enlargement

0

0

0

26**

 

0

0

0

13**

 

Squamous cell metaplasia

0

0

0

31**

 

0

0

0

35**

 

Squamous cell hyperplasia

0

0

0

2

 

0

0

0

5

 

Olfactory epithelium 

Nuclear enlargement

0

0

5

38**

 

0

0

28**

39**

 

Liver

 

Hepatocellular adenoma

3

4

7

32##

++

3

1

6

48##

++

Hepatocellular carcinoma

0

0

0

14##

++

0

0

0

10##

++

Hepatocellular adenoma or carcinoma

3

4

7

39##

++

3

1

6

48##

++

Altered hepatocellular foci

1

Acidophilic cell foci

2

8

7

5

 

1

1

1

1

 

Basophilic cell foci

7

11

8

16*

 

23

27

31

8**

 

Clear cell foci

3

3

9

8

 

1

1

5

4

 

Mixed cell foci

2

8

14**

13**

 

1

1

3

11*

 

Subcutis

Fibroma

5

3

5

12

++

0

2

1

0

 

Mammary gland

 

Adenoma

0

1

2

2

 

6

7

10

16#

++

Fibroadenoma

1

1

0

4

++

3

2

1

3

 

Adenoma or Fibroadenoma

1

2

2

6

++

8

8

11

18#

++

Peritoneum

Mesothelioma

2

2

5

28##

++

1

0

0

0

 

Note: Lesions in animals which became moribund or died before the end of the 2-year administration period were also scored.

# Significantly different from control at p < 0.05 by Fisher’s exact test.

## Significantly different from control at p < 0.01 by Fisher’s exact test.

** Significantly different at p < 0.01 by chi-square test.

++ Significantly different at p < 0.01 by Peto’s test.

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD 451 (Carcinogenicity Study)
GLP compliance:
no
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Route of administration:
oral: drinking water
Duration of treatment / exposure:
90 weeks
Frequency of treatment:
continuously
Dose / conc.:
0.5 other: % (v/v)
Remarks:
corresponding to 720 (m) and 380 (f) mg/kg bw/d
Dose / conc.:
1 other: % (v/v)
Remarks:
corresponding to 830 (m) and 860 (f) mg/kg bw/d
No. of animals per sex per dose:
50
Control animals:
yes, concurrent no treatment
Details on study design:
Post-exposure period: 3 weeks
Observations and examinations performed and frequency:
Observations included clinical signs, body weight, food and water consumption, necropsy and histopathology.
Clinical signs:
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
Survival rates of the dosed mice (46/50 in low and 45/50 in high dose males, 39/50 in low and 28/50 in high dose females) were lower than those of the controls (48/50 in males and 45/50 in females), but a sufficient number of animals were at risk for development of late-appearing tumours.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights were not consistently affected, although the weight of the high dose females was lower than that of the controls during the second year of the study.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related non-neoplastic lesions in males and females included hepatic cytomegaly, pneumonia and rhinitis. No effects were seen on male and female reproductive organs.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In both sexes an increased incidence in hepatocellular carcinomas was seen. The incidences were 2/49, 18/50 and 24/47 for males and 0/50, 12/48 and 29/37 for females at 0, 0.5 and 1%, respectively. Also an increase in the incidence of hepatocellular adenomas plus carcinomas was seen: at 0, 0.5 and 1% 8/49, 19/50 and 28/47 for males and 0/50, 21/48 and 35/37 for females, respectively. One nasal adenocarcinoma was seen in a low dose female and one in a high dose male.
Other effects:
no effects observed
Key result
Dose descriptor:
LOAEL
Effect level:
<= 380 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
histopathology: neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
380 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
380 mg/kg bw/day (actual dose received)
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes

It is concluded that under the conditions of this bioassay, 1,4-dioxane induced hepatocellular adenomas in female Osborne- Mendel rats. 1,4-Dioxane was carcinogenic in both sexes of rats, producing squamous-cell carcinomas of the nasal turbinates, and in both sexes of B6C3F1 mice, producing hepatocellular carcinomas.

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD 451 (Carcinogenicity Study)
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Osborne-Mendel
Sex:
male/female
Route of administration:
oral: drinking water
Duration of treatment / exposure:
110 weeks
Frequency of treatment:
daily
Dose / conc.:
0 other: % (v/v)
Dose / conc.:
0.5 other: % (v/v)
Remarks:
corresponding to 240 mg/kg bw/d for males and 350 mg/kg bw/d for females
Dose / conc.:
1 other: % (v/v)
Remarks:
corresponding to 530 mg/kg bw/d for males and 640 mg/kg bw/d for females
No. of animals per sex per dose:
35/ sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: 7 weeks
Observations and examinations performed and frequency:
Observations included clinical signs, body weight, food and water consumption, necropsy and histopathology.
Clinical signs:
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
The survival rates of the rats of both dose groups were significantly lower than that of controls, but sufficient animals of each sex were alive at 52 weeks (33/35, 26/35 and 33/35 for males and 35/35, 30/35 and 29/35 for females at 0, 0.5 and 1%, respectively) to be at risk for the development of late-appearing tumours.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights were not consistently affected, although the weight of the high dose animals was lower than that of the controls during the second year of the study.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Nonneoplastic lesions associated with dioxane treatment were observed in the kidney (tubular degeneration), liver (cytomegaly) and stomach (ulceration). A higher incidence of pneumonia and rhinitis occurred in males and females of both dose groups.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Rats of both sexes developed squamous cell carcinomas in the nasal cavities (0/33, 12/33 and 16/34 for control, low and high dosed males and 0/34, 10/35 and 8/35 for control, low and high dosed females, respectively). In one high dose male these carcinomas extended to the retrobulbar tissues of the eye and in one low dose male into the brain. In addition, adenocarcinomas arose from the nasal mucosal epithelium in three high dose males, in one high dose female and one low dose female.

The first nasal carcinomas developed after one year. A follow-up examination localised nasal tumours in the front third of the posterior meatus of the nasal cavities. Also an increase in hepatocellular adenomas was seen in females. The incidence was 0/31, 10/33 and 11/32 for control, low and high dosed females, respectively.
Other effects:
no effects observed
Description (incidence and severity):
No effects were seen on male and female reproductive organs.
Key result
Dose descriptor:
LOAEL
Effect level:
<= 240 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
histopathology: neoplastic
Remarks on result:
other: lowest dose tested (recalculated from 0.5 % v/v in drinking water)
Key result
Dose descriptor:
LOAEL
Effect level:
<= 350 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
histopathology: neoplastic
Remarks on result:
other: lowest dose tested (recalculated from 0.5 % v/v in drinking water)
Dose descriptor:
LOEL
Effect level:
350 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: DNA synthesis, hyperplasia or foci; liver adenomas/carcinomas
Remarks on result:
other: as re-evaluated by Dourson et al., 2017
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
240 mg/kg bw/day (actual dose received)
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
350 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes

It is concluded that under the conditions of this bioassay, 1,4-dioxane induced hepatocellular adenomas in female Osborne- Mendel rats. 1,4-Dioxane was carcinogenic in both sexes of rats, producing squamous-cell carcinomas of the nasal turbinates.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
9.6 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Studies comparable to OECD guideline with acceptable restriction sufficient for assessment.
Organ:
kidney
liver

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
A lifetime 2-year study with rats, with a treated and a control group
GLP compliance:
no
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Dow
- Housing: in groups of 4 or 8 during and between exposure
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
No data
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
Seven-hour daily exposures were given 5 days/week under dynamic exposure conditions in five 3.7 m3 stainless-steel vault-type chambers. A constant air flow of 373 liters/min nitrogen was maintained by means of rotary pump connetcted to the exhaust side of each chamber. The airflow was monitored with calibrated flow meters. The vapour was generated by metering liquid dioxane into an evaporation flaks heated to 100 dergees C.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
(on-line) infrared analysis
Duration of treatment / exposure:
2 years
Frequency of treatment:
7 hours/day, 5 days/week
Dose / conc.:
0.4 mg/L air (analytical)
Remarks:
SD = 0.018 mg/L (5 ppm); corresponding to 111 ppm
No. of animals per sex per dose:
treatment group: 288 male and 288 female animals
control group: 192 male and 192 female animals
Control animals:
yes
Details on study design:
Since the threshold limit value for dioxane at the time of this study was started and up until 1971 was set by the American Conference of Governmental Industrial Hygienists (1971) at 0.36 mg/L (100 ppm v/v), this concentration was selected for the 2-year study.
Selection of this concentration was also supported by previous studies in our laboratory, in which groups of 24 male and 24 female rats, 3 male and 3 female rabbits, and 2 female dogs received 130-136 7-hr exposures in 180-195 days to 50 ppm dioxane vapour in air. In addition, 7 male and 8 female guinea pigs received 82 exposures in 1 1.8 days to 50 ppm dioxane vapor in air, and groups of 12 rats and 2 rabbits of each sex received. 133-136 7-hr exposures to 100 ppm. There were no adverse effects when the exposed groups were compared to control groups on the basis of appearance, demeanor, growth, mortality, hematological and clinical chemical studies, organ weights, or gross and microscopic pathological examination.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes: signs of toxicity including alternations in activity, demeanor, eye and nasal irritation, skin condition, respiratory distress, and tumour formation.
- Time schedule: not excatly indicated; throughout the exposure period.

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: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 16 or 23 months of exposure
- Anaesthetic used for blood collection: No data
- Animals fasted: yes
- How many animals: after 16 months: 232 control rats and 340 exposed rats
after 23 months:115 control rats and 185 exposed rats
Parameters checked : PCV, RBC, Hb, WBC, WBC different; Neut, Lymph, Mono, Eosin, Baso

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination of the study (after 2 years on all surviving rats)
- Animals fasted: Yes
- How many animals:103 control, 151 exposed rats
- Parameters checked: BUN, SGPT, AP and total protein

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
on succumbed and moribund rats, whenever possible moribund rats were killed for for gross examination and tissues were saved for microscopic examination, with special attention to abnormal growths. Following gross examination body weights of liver, spleen and kidneys were recorded.

HISTOPATHOLOGY: Yes:
lungs, trachea, thoracic lymph nodes, heart, liver, pancreas, stomach, intestine, spleen, thyroid, mesentric lumph nodes, kidneys, urinary bladder, pituary, adrenals, tetstes, ovaries, oviduct, uterus, mammary gland, lacrimals gland, lymph nodes, brain, vagina, bone marrow, and any
abnormal growths.
Statistics:
Control and experimental groups were compared statistically using:
Student's T-test (heamatology, clinical chemistry),
Fisher exact probability test (morphological classification of tumours),
Yates corrected Chi-Square test (survival)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Details on results:
No effects were seen on clinical signs (including activity, demeanour, eye and nasal irritation, skin condition and respiratory distress), body weights or mortality.

Some slight changes were observed in haematological values, but these were within the normal physiological limits and not considered of toxicological importance.

BUN and AP values in treated male rats were slightly decreased.

Changes in liver, kidney or spleen weights were not observed.

Upon gross and microscopic examination, no treatment-related non-neoplastic effects were found in tissues/organs, including the reproductive organs. Regarding neoplastic effects, no 1,4-dioxane characteristic nasal and liver tumours, as observed after oral administration, were seen. It is however not clear from the text whether or not the nasal cavity was adequately examined. The incidence of tumours observed in other organs/tissues appeared to be unrelated to exposure. The only difference from the control groups was an increase in lymphoreticular cell sarcomas in males (18% (37/206) versus 12% (18/150)) and in mammary gland adenoma in females (13% (29/217) versus 8% (11/139)), which were not statistically significant.
Key result
Dose descriptor:
NOAEC
Effect level:
> 400 mg/m³ air
Sex:
male/female
Basis for effect level:
other: no adverse health effects were observed
Key result
Critical effects observed:
no
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344/DuCrj
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Japan
- Fasting period before study: no
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 (exposure chamber: 20-24)
- Humidity (%): 50-70 (exposure chamber: 30-70)
- Air changes (per hour): 12
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
Airflow containing 1,4-dioxane vapor at the target concentration was prepared by a vaporization technique. The saturated vapor-air mixture was generated by bubbling clean air through the 1,4-dioxane liquid in a temperature-regulated glass flask (30°C), and by cooling it through a thermostatted condenser at 20 °C. The airflow containing the saturated vapor was diluted with clean air, and then warmed to 30 °C in a thermostatted circulator that served to stabilize the vapor concentration by complete gasification of 1,4-dioxane. The flow rate of the vapor-air mixture was regulated with a flow meter, further diluted with humidity- and temperature-controlled clean air in a spiraling line mixer, and then supplied to an inhalation exposure chamber. Eight inhalation exposure chambers of 1060 L in volume were used in the present study. Each exposure chamber accommodated 20 individual cages for 10 males and 10 females. Chamber concentrations of 1,4-dioxane were monitored every 15 min with a gas chromatograph (GC-148, Shimadzu Corp., Kyoto, Japan), equipped with a hydrogen flame ionization detector and a 1.5-m Shimadzu SBS-120 packed column operated at a column temperature of 90 °C and with a gas injection volume of 2 mL.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
analysis by gas chromography with a flame ionization detector
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
100 ppm (nominal)
Remarks:
analytical conc.: 100.2 (SD 2.1)
Dose / conc.:
200 ppm (nominal)
Remarks:
analytical conc.: 200.7 (SD 4.1)
Dose / conc.:
400 ppm (nominal)
Remarks:
analytical conc.: 403.9 (SD 7.5)
Dose / conc.:
800 ppm (nominal)
Remarks:
analytical conc.: 799.8 (SD 12.1)
Dose / conc.:
1 600 ppm (nominal)
Remarks:
analytical conc.: 1596.4 (SD 27.2)
Dose / conc.:
3 200 ppm (nominal)
Remarks:
analytical conc.: 3198.4 (SD 48.9)
Dose / conc.:
6 400 ppm (nominal)
Remarks:
analytical conc.: 6409.5 (SD 76.3)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: once per week

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

FOOD EFFICIENCY: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the 13 week exposure
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: Yes
- How many animals: 10/sex/group
- Parameters checked with an automated blood cell analyzer: (RBC, Hb, Heamatocrit, MCV were reported)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:t the end of the 13 week exposure
- Animals fasted: Yes
- How many animals:10/sex/group
- Parameters checked (with an automated analyzer and a flame photometer): (AST, ALT, Glucose, Triglyceride were reported)

URINALYSIS: Yes
- Time schedule for collection of urine: the last week of the 13 weeks exposure
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked: Multistix, Ames reagent strip

NEUROBEHAVIOURAL EXAMINATION: No data


Sacrifice and pathology:
GROSS PATHOLOGY: Yes, complete necropsy; all organs were removed, weighed and examined for macroscopic lesions.
HISTOPATHOLOGY: Yes, as designated in the OECD guideline, plus the entire respitratory tract including the nasal cavity, pharynx, larynx, trachea and bronchus.
Other examinations:
Determination of 1,4-dioxane in blood
Three rats of each group on day 3 of week 12 of exposure, 1 hour after termination. Blood was drawn from the tail vain.
Statistics:
Body weight, food consumption, organ weight, and hematological and blood biochemical parameters were analyzed by Dunnett's test as described previously (Aiso et al., 2005). Histopathological findings and urinary parameters were analyzed by chi-square test. A two-sided analysis with p values of .05 and .01 was performed to determine statistical significance.
Clinical signs:
no effects observed
Description (incidence and severity):
In the surviving animals, no abnormal clinical sign was found in any 1,4-dioxanedosed rats of either sex throughout the 13-wk exposure period.
Mortality:
mortality observed, treatment-related
Description (incidence):
All the 6400-ppm-exposed rats of both sexes died during wk 1 of the 13-wk exposure period. The histopathological examination revealed that their deaths were primarily caused by renal failure, because all the 6400-ppm-exposed animals suffered from marked necrosis in the renal tubules. Moreover, lung congestion was observed in the 6400-ppm-exposed males and females. All the other groups survived to the end of the 13-wk exposure period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Terminal body weight significantly decreased in the 200-ppm and 3200-ppm-exposed males and in the females exposed to 200 ppm, 800 ppm, and above.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Some erythrocyte parameters were increased in the 3200ppm-exposed males and females with slight but statistical significance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A slight but statistically significant increase was found in both aspartate aminotransferase (AST) in the 200ppm-and 3200-ppm-exposed females and alanine aminotransferase (ALT) in the 3200-ppm-exposed males and females. The exposure to 3200 ppm decreased blood levels of glucose and triglyceride only in the males, but not in the females.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urinary protein slightly decreased in the males exposed to 3200 ppm.
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Relative liver weight significantly increased in the males and females exposed to 800 ppm and above. Relative kidney weight significantly increased in the 3200-ppm-exposed males and in the females exposed to 800 ppm and above. Relative lung weight increased in the males exposed to 200 ppm and 1600 ppm and above and in the females exposed to 200 ppm and above.
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The upper and lower respiratory tract as well as liver in both males and females and kidneys in females were affected (table 1 and 2). The most sensitive lesion occurred in the nasal cavity, where the incidences and severities of nuclear enlargement in the respiratory epithelium were significantly increased dose-dependently in both males and females exposed to 100 ppm and above. The nuclear enlargement was morphologically characterized by the appearance of the epithelial cells having round to oval or elongated nuclei that were at least four times as huge in diameter as the normal nuclei of respiratory epithelial cells. The enlarged nuclei of respiratory epithelial cells were localized at Level 1 in the males exposed to 100 und 200 ppm and in the females exposed to 100 ppm, and at Levels 1 and 2 in the females exposed to 200 ppm, while the respiratory epithelial cells having the enlarged nuclei were extended over the entire respiratory region at Level 1 through 3 in both males and females exposed to 400 ppm and above. The enlarged nuclei of the sustentacular cells in the olfactory epithelium were significantly increased in both males and females exposed to 200 ppm and above, and were distributed over almost the entire area of the olfactory region al Levels 2 and 3. The incidences and severities of nuclear enlargement in the 1,4-dioxane-exposed males and females tended to decrease along the passage of inspiratory airflow through the upper and lower respiratory tracts. The incidence of degenerative change in the olfactory sensory cells observed as the vacuolic change was significantly increased in the males exposed to 400 ppm and above, while the females exposed to 800 ppm and above exhibited olfactory epithelial atrophy characterized by a decreased number of the olfactory sensory cells and vacuolic change. Vacuolic change in the bronchial epithelium also occurred with statistical significance in the males exposed to 1600 ppm and above. The 1,4-dioxane-induced liver lesions were characterized by significant increases in the incidences of both single-cell necrosis and centrilobular swelling of hepatocytes in the 3200-ppm exposed males, and in the incidence of centrilobular swelling of hepatocytes in the 3200-ppm-exposed females. In addition, the GST-P-positive Iiver foci were observed in three 3200-ppm-exposed males, two 3200-ppm-exposed females, and four 1600-ppm-exposed females out of each ten 1,4-dioxane exposed rats of either sex, whereas the GST-P-positive foci could not be found in any of the 800- and l600-ppm-exposed males and 800-ppm-exposed females and control groups of both sexes. The GST-P-positive hepatocytes exhibited morphologically focal and clonal proliferation. The incidence of hydropic change in the renal proximal tubules was significantly increased in the 3200-ppm-exposed females.
Histopathological findings: neoplastic:
no effects observed
Details on results:
All the 6400-ppm-exposed males and females died during the first week, due to lung congestion and renal failure. Terminal body weight decreased, and relative weights of liver, kidney, and lung increased.
AST increased in the 200 ppm-and 3200-ppm-exposed females, and ALT increased in the 3200-ppm-exposed males and females.
Nuclear enlargement of nasal respiratory epithelial cells occurring in the 100-ppm-exposed males and females was the most sensitive, followed by the enlarged nuclei in the olfactory, tracheal, and bronchial epithelia.
1,4-Dioxane-induced liver lesions occurred at higher exposure concentrations than the nasal lesions did, and were characterized by single-cell necrosis and centrilobular swelling of hepatocytes in males and females.
Glutathione S-transferase placental form (GST-P) positive liver foci were observed in the 1600-ppm-exposed females and 3200-ppm-exposed males and females.
Plasma levels of 1,4-dioxane increased linearly with an increase in the concentrations of exposure to 400 ppm and above.
Key result
Dose descriptor:
LOAEC
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: Lowest dose tested. NOAEC not determinable.
Remarks:
corresponding to 360 mg/m3
Key result
Dose descriptor:
LOEC
Effect level:
ca. 360 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: liver weight increase or hypertrophy
Remarks on result:
other: as re-evaluated by Dourson et al., 2017
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 ppm
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes

Table 1 Incidences and severities of selected lesions in the male rats exposed by inhalation to 1,4 -dioxane vapor at 6 different concentrations or clean air for 13 weeks

 

Male

Group (ppm)

Control

100

200

400

800

1600

3200

No. of animals examined

10

10

10

10

10

10

10

Nasal cavity

 

Nuclear enlargement: respiratory epithelium

0

7**

9**

7**

10**

10**

10**

 

1+:7

1+:9

1+:7

1+:10

2+:10

2+:10

Nuclear enlargement: olfactory epithelium

0

0

5*

10**

10**

10**

10**

 

 

1+:5

1+:10

1+:10

2+:10

2+:10

Vacuolic change: olfactory epithelium

0

1

3

6*

10**

10**

9**

 

1+:1

1+:3

1+:6

1+:10

1+:10

1+:9

Trachea
Nuclear enlargement: epithelium

0

0

0

0

1

10**

10**

 

 

 

 

1+:1

1+:10

1+:10

Bronchus

 

Nuclear enlargement: bronchial epithelium

0

0

0

0

0

9**

10**

 

 

 

 

 

1+:9

1+:10

Vacuolic change: bronchial epithelium

0

0

0

0

4

6*

6*

 

 

 

 

1+:4

1+:6

1+:6

Liver

 

Necrosis: single cells

0

0

0

0

0

1

8**

 

 

 

 

 

1+:1

1+:8

Swelling: centrilobular

0

0

0

0

0

1

10**

 

 

 

 

 

 

1+:10

The number of the animals bearing the lesion in each exposed or control group are shown in the upper column, respectively. The column below indicates values indicate the number of the animals bearing the lesion with each of 4 different grades of severity ie. 1+: sligh; 2+:moderate; 3+: marked; 4+:severe;

Significant difference indicated by * p≤0.05; **p≤0.01 by chi square test.

Data of the 6400 ppm-exposed group are not presented here, because all of them died at the first week of the study.

Table 2 Incidences and severities of selected lesions in the female rats exposed by inhalation to 1,4 -dioxane vapor at 6 different concentrations or clean air for 13 weeks

 

Female

Group (ppm)

Control

100

200

400

800

1600

3200

No. of animals examined

10

10

10

10

10

10

10

Nasal cavity

 

Nuclear enlargement: respiratory epithelium

0

5*

9**

10**

10**

10**

10**

 

1+:5

1+:9

1+:10

1+:10

2+:10

2+:10

Nuclear enlargement: olfactory epithelium

0

2

6*

10**

10**

10**

10**

 

1+:2

1+:6

1+:9; 2+:1

1+:10

1+:7; 2+:3

2+:10

Vacuolic change: olfactory epithelium

0

1

2

3

7**

9**

10**

 

1+:1

1+:2

1+:3

1+:7

1+:9

1+:10

Atrophy: olfactory epithelium

0

0

2

3

5*

5*

4

 

 

1+:2

1+:3

1+:5

1+:5

1+:4

Trachea
Nuclear enlargement: epithelium

0

0

0

0

2

7**

10**

 

 

 

 

1+:2

1+:7

1+:110

Bronchus

 

Nuclear enlargement: bronchial epithelium

0

0

0

0

0

0

10**

 

 

 

 

 

 

1+:10

Vacuolic change: bronchial epithelium

0

0

0

1

1

3

4

 

 

 

1+:1

1+:1

1+:3

1+:4

Liver

 

Necrosis: single cells

0

0

0

0

0

0

3

 

 

 

 

 

 

1+:3

Swelling: centrilobular

0

0

0

0

0

1

8**

 

 

 

 

 

1+:1

1+:8

Kidney

Hydropic change: proximale tubule

0

0

0

0

0

0

6*

 

 

 

 

 

 

1+:6

The number of the animals bearing the lesion in each exposed or control group are shown in the upper column, respectively. The column below indicates values indicate the number of the animals bearing the lesion with each of 4 different grades of severity ie. 1+: sligh; 2+:moderate; 3+: marked; 4+:severe;

Significant difference indicated by * p≤0.05; **p≤0.01 by chi square test.

Data of the 6400 ppm-exposed group are not presented here, because all of them died at the first week of the study.

Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
1981
GLP compliance:
not specified
Remarks:
GLP compliance not stated in the publication
Limit test:
no
Species:
rat
Strain:
Fischer 344
Details on species / strain selection:
Selection of male F344 rats was based on our previous result that 2 year oral administration of 1,4-dioxane in drinking water induced mesotheliomas only in male F344 rats but not in females (Yamazaki et al., 1994).
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc. (Kanagawa, Japan)
- Age at study initiation: 6 weeks
- Weight at study initiation: 150 +/- 5 g
- Housing: individually in stainless steel wire hanging cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 21.9 +/- 0.6 °C
- Humidity: 53.6 +/- 4.9 %
- Air changes: not specified
- Photoperiod: 12/12 hrs dark / hrs light
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
clean air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Vaporization technique:
The saturated vapor–air mixture was generated by bubbling clean air through the 1,4-dioxane liquid in a temperature-regulated glass flask (35°C), and by cooling it through a thermostatted condenser at 20°C. The airflow containing the saturated vapor was diluted with clean air, and
then warmed to 35°C in a thermostatted circulator, which served to stabilize the vapor concentration by complete gasification of 1,4-dioxane. The flow rate of the vapor–air mixture was regulated with a flow meter, further diluted with humidity-and temperature-controlled clean air in a spiraling line mixer, and then supplied to an inhalation exposure chamber.

- Method of holding animals in test chamber: individually

- Temperature, humidity, pressure in air chamber: Temperature: 21.9 +/- 0.6 °C; Humidity: 53.6 +/- 4.9 %

- Air change rate: not specified


TEST ATMOSPHERE
- Brief description of analytical method used: Chamber concentrations of 1,4-dioxane were monitored every 15 min with a GC unit (GC-14B; Shimadzu Corp., Kyoto, Japan) equipped with a hydrogen flame ionization detector and a 1.5-m Shimadzu SBS-120 packed column operated at a column temperature of 90°C and with a gas injection volume of 2 mL, and were maintained constant at 50.2 ± 1.4 (mean ± SD), 250.9 ± 3.2, and 1247.5 ± 18.6 ppm throughout the 2-yr exposure period.

- Samples taken from breathing zone: no

Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
2 years (104 weeks)
Frequency of treatment:
6 hours per day; 5 days per week
Dose / conc.:
50 ppm (nominal)
Remarks:
v/v
Dose / conc.:
250 ppm (nominal)
Remarks:
v/v
Dose / conc.:
1 250 ppm (nominal)
Remarks:
v/v
No. of animals per sex per dose:
50 males per dose / control group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The lowest exposure concentration of 50 ppm was selected as an environmentally relevant level marginally exceeding the occupational exposure limit value of 20 ppm for 1,4-dioxane (ACGIH, 2001). The highest concentration of 1250 ppm was selected not to exceed the maximum tolerated dose (MTD) criteria (Sontag et al., 1976; OECD, 1981; Bannasch et al., 1986), based on both subchronic toxicity and body weight decrement from our 13-wk inhalation exposure study (Kasai et al., 2008). A preliminary study showed that 13-wk inhalation exposure of male F344 rats to 1,4-dioxane at a concentration of 1600 ppm did not induce any death, body weight decrement, or toxicity except histopathological changes in the nasal cavity, trachea, and bronchus. Thus, the highest concentration of 1250 ppm was predicted to elicit signs of minimal toxicity without altering the normal lifespan of the male rats except for the result of neoplastic development, following 2-yr inhalation exposure to 1,4-dioxane.

- Rationale for animal assignment: stratified randomisation (body weight matched)
- Fasting period before blood sampling for clinical biochemistry: no
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: once a week (first 14 weeks); every 4 weeks thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: once a week (first 14 weeks); every 4 weeks thereafter

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of the 2 year exposure period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: no specified

CLINICAL CHEMISTRY: Yes, see heamatology

URINALYSIS: Yes
- Time schedule for collection of urine: last 2 week of 2 year epxosure
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked: Ames reagent strips

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all organs listed in the guideline and the entire respiratory tract)
HISTOPATHOLOGY: Yes
Statistics:
Incidences of neoplastic lesions were analyzed for a dose–response relationship indicated by a significant positive trend with Peto’s test (Peto et al., 1980), and for a significant difference from the clean air-exposed group by Fisher’s exact test.

Incidences of nonneoplastic lesions and urinary parameters were analyzed by Chi2 test. Survival curves were plotted according to the Kaplan–Meier method (Kaplan & Meier, 1958), and both the log-rank test (Peto et al., 1977) and Fisher’s exact test were used to investigate a statistically significant difference in survival rate between any 1,4-dioxane-exposed rat group and the clean air-exposed group. Body weight, organ weight, food consumption, and hematological and blood biochemical parameters were analyzed by Dunnett’s test.

A two-tailed test was used for all statistical analysis except for Peto’s test. In all cases, statistical analysis with p-values of 0.05 and 0.01 was performed and indicated in the tables, while a p-value of 0.05 was used as a level of significance.

A no observed adverse effect level (NOAEL) or a lowest observed adverse effect level (LOAEL) was determined, according to the World Health Organization (WHO) definition (International Programme on Chemical Safety, 1994).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the clinical observation, deformity of the nose was observed at the 79th and 74th wk for one each of the 250 and 1250 ppm-exposed rats, respectively, and these two rats died due to malignant nasal tumor before the end of the 2-yr exposure period.
Mortality:
mortality observed, treatment-related
Description (incidence):
No significant difference in survival rate at any time point was found between the 50 ppm-exposed group and the control. The survival rates of the 250 ppm-exposed group tended to decrease near the end of the 2-yr exposure period, although there was no statistical difference between the 250 ppm-exposed group and the control. The 1250 ppm-exposed group exhibited a statistically significant decrease in survival rate at the 91st wk and thereafter, as compared with the respective controls. The terminal survival rates of the control, 50, 250, and 1250 ppm-exposed groups were 37/50, 37/50, 29/50, and 25/50, respectively. The significantly decreased survival rates in the 1250 ppm-exposed group were attributed to the increased number of deaths due primarily to peritoneal mesotheliomas, although nasal tumors contributed to the causes of death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Although no statistical difference in growth rate between any 1,4-dioxane exposed group and the control was found during the first 22 wk, the growth rates of the 1250 ppm-exposed rats tended to decrease during the latter half of the 2-yr exposure period. A decrease in terminal body weight of the 1250 ppm-exposed group was statistically significant but less than 10% as compared with the control.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption did not decrease in any 1,4-dioxane-exposed group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Significant decreases in hemoglobin, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) were noted in the 1250 ppm-exposed group, although neither red blood cell counts nor hematocrit values were decreased.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma-glutamyltranspeptidase (gamma-GTP) were significantly increased in the 1250 ppm-exposed group. Urinary pH was significantly decreased in the 1250 ppmexposed group.
Urinalysis findings:
not specified
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Relative liver weight was significantly increased in the 1250 ppm-exposed group. A statistically significant increase in relative lung weight was found in the 1250 ppm-exposed group, but this change was considered not to be biologically meaningful, because there was no dose–response relationship for relative lung weight. No remarkable organ weight change was observed in any other organs of 1,4-dioxane-exposed groups.
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Notably, incidences of nuclear enlargement in the respiratory and olfactory epithelia were significantly increased in all 1,4-dioxane-exposed groups, while significantly increased incidences of nuclear enlargement were observed in the liver of the 1250 ppm-exposed group and the kidney of the 250 ppm- and 1250 ppm-exposed groups. Inflammation in the respiratory and olfactory epithelia and atrophy in the olfactory epithelium, hydropic change and sclerosis of lamina propria, and proliferation in the nasal gland occurred at a statistically significant level in the 1,4-dioxane-exposed groups with different exposure concentrations. Necrosis of hepatocytes in the centrilobular region, spongiosis hepatis in the liver, and hydropic change in the renal proximal tubule also occurred in the 1250 ppm exposed group. No exposure-related, nonneoplastic or neoplastic lesion in the pharynx, larynx, trachea, bronchus, or lungs was histopathologically observed in any 1,4-dioxane exposed group.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A dose-dependent increase in tumor incidences as indicated by a significantly positive trend with Peto’s test was observed in nasal squamous cell carcinomas, hepatocellular adenomas, renal cell carcinomas, peritoneal mesotheliomas, fibroadenomas in the mammary gland, and adenomas in the Zymbal gland.

The incidences of nasal squamous cell carcinomas, hepatocellular adenomas, and peritoneal mesotheliomas were significantly increased in the 1250 ppm-exposed rats compared with the concurrent, matched controls by Fisher’s exact test. In addition, a statistically significant increase in the incidences of peritoneal mesotheliomas and subcutaneous fibromas was also noted in the 250 ppm-exposed rats. 1,4-Dioxane-induced nasal squamous cell carcinoma occurred at the dorsal area of levels 1–3 in the nasal cavity, and was histopathologically characterized as well-differentiated and keratinized type, with invasive growth into the surrounding tissue.

Two cases of squamous cell carcinoma destroyed the nasal bone and protruded out of the nose through the nasal bone. Peritoneal mesotheliomas arose from the mesothelium of the scrotal sac, were characterized by complex branching structures with a fibrovascular core covered by single or stratified layers of mesothelial cells, and occasionally had spread throughout the entire surface of the peritoneal cavity and invaded visceral organs including the pancreas. As preneoplastic lesions, squamous cell hyperplasia in the nasal cavity and altered cell foci in the liver were observed in the 1250 ppm-exposed group. The squamous cell hyperplasia occurred primarily on the nasoturbinate, septum, and dorsal wall at levels 1 and 2, was characterized by five or more layers of epithelial cells, and had cellular pleomorphism, irregular arrangement of cell layers, and proliferation of basal cells, resembling an early stage of squamous cell carcinoma. Squamous cell metaplasias that were characterized by replacement of the transitional and respiratory epithelia by the squamous epithelium with or without keratinization occurred in the rats exposed to 250 ppm and above. The squamous cell metaplasia appeared primarily in the focal area of the nasoturbinate, maxilloturbinate, septum, and dorsal and lateral wall at level 1, and occasionally showed slight cellular pleomorphism and proliferation of basal cells, which was indicative of atypia and might be regarded as a potentially preneoplastic lesion.
Other effects:
not specified
Key result
Dose descriptor:
LOAEC
Effect level:
50 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: lowest dose tested
Key result
Dose descriptor:
LOEC
Effect level:
1 250 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: liver weight increase or hypertrophy; necrosis / inflammation; DNA synthesis hyperplasia or foci; liver adenomas / carcinomas
Remarks on result:
other: as re-evaluated by Dourson et al., 2017
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 ppm
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes

Table 1 Incidences of selected histopathological lesions in male F344 rats.

 

Control

50 ppm

250 ppm

1250 ppm

Peto test

No. of animals examined

50

50

50

50

 

Neoplastic lesions

Nasal cavity

 

Squamous cell carcinoma

0

0

1

6#

++

Liver

 

Hepatocellular adenoma

1

2

3

21##

++

Hepatocellular carcinoma

0

0

1

2

 

Kidney

 

Renal cell carcinoma

0

0

0

4

++

Peritoneum
Mesothelioma

2

4

14##

41##

++

Mammary gland

 

Fibroadenoma

1

2

3

5

+

Adenoma

0

0

0

1

 

Zymbal gland
Adenoma

0

0

0

4

++

Subcutis
Fibroma

1

4

9##

5

 

Pre- and non-neoplastic lesions

Nasal cavity

 

Respiratory epithelium

 

Nuclear enlargement

0

50**

48**

38**

 

Squamous cell metaplasia

0

0

7*

44**

 

Squamous cell hyperplasia

0

0

1

10**

 

Inflammation

13

9

7

39**

 

Olfactory epithelium

 

Nuclear enlargement

0

48**

48**

45**

 

Atrophy

0

40**

47**

48**

 

Respiratory metaplasia

11

34**

49**

48**

 

Inflammation

0

2

32**

34**

 

Hydropic change: lamina propria

0

2

36**

49**

 

Sclerosis: lamina propria

0

0

22**

40**

 

Proliferation: nasal gland

0

1

0

6*

 

Liver

 

Nuclear enlargement: centrilobular

0

0

1

30**

 

Acidophilic cell foci

5

10

12

25**

 

Basophilic cell foci

17

20

15

44**

 

Clear cell foci

15

17

20

23

 

Mixed cell foci

5

3

4

14

 

Spongiosis hepatis

7

6

13

19**

 

Necrosis: centrilobular

1

3

6

12**

 

Kidney

 

Nuclear enlargement: proximal tubule

0

1

20**

47**

 

Hydropic change: proximal tubule

0

0

5

6*

 

Note: # and ## significantly different from control at p≤0.05 and p≤0.01 by Fisher’s exact test, respectively. * and ** significantly different from control at p≤0.05 and p≤0.01 by chi square test, respectively. + and ++ significantly different from control at p≤0.05 and p≤0.01 by Peto test, respectively.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
180 mg/m³
Study duration:
chronic
Species:
rat
Quality of whole database:
Studies comparable to OECD guideline with acceptable restriction sufficient for assessment.
Organ:
nasal cavity

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
A lifetime 2-year study with rats, with a treated and a control group
GLP compliance:
no
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Dow
- Housing: in groups of 4 or 8 during and between exposure
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
No data
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
Seven-hour daily exposures were given 5 days/week under dynamic exposure conditions in five 3.7 m3 stainless-steel vault-type chambers. A constant air flow of 373 liters/min nitrogen was maintained by means of rotary pump connetcted to the exhaust side of each chamber. The airflow was monitored with calibrated flow meters. The vapour was generated by metering liquid dioxane into an evaporation flaks heated to 100 dergees C.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
(on-line) infrared analysis
Duration of treatment / exposure:
2 years
Frequency of treatment:
7 hours/day, 5 days/week
Dose / conc.:
0.4 mg/L air (analytical)
Remarks:
SD = 0.018 mg/L (5 ppm); corresponding to 111 ppm
No. of animals per sex per dose:
treatment group: 288 male and 288 female animals
control group: 192 male and 192 female animals
Control animals:
yes
Details on study design:
Since the threshold limit value for dioxane at the time of this study was started and up until 1971 was set by the American Conference of Governmental Industrial Hygienists (1971) at 0.36 mg/L (100 ppm v/v), this concentration was selected for the 2-year study.
Selection of this concentration was also supported by previous studies in our laboratory, in which groups of 24 male and 24 female rats, 3 male and 3 female rabbits, and 2 female dogs received 130-136 7-hr exposures in 180-195 days to 50 ppm dioxane vapour in air. In addition, 7 male and 8 female guinea pigs received 82 exposures in 1 1.8 days to 50 ppm dioxane vapor in air, and groups of 12 rats and 2 rabbits of each sex received. 133-136 7-hr exposures to 100 ppm. There were no adverse effects when the exposed groups were compared to control groups on the basis of appearance, demeanor, growth, mortality, hematological and clinical chemical studies, organ weights, or gross and microscopic pathological examination.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes: signs of toxicity including alternations in activity, demeanor, eye and nasal irritation, skin condition, respiratory distress, and tumour formation.
- Time schedule: not excatly indicated; throughout the exposure period.

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: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 16 or 23 months of exposure
- Anaesthetic used for blood collection: No data
- Animals fasted: yes
- How many animals: after 16 months: 232 control rats and 340 exposed rats
after 23 months:115 control rats and 185 exposed rats
Parameters checked : PCV, RBC, Hb, WBC, WBC different; Neut, Lymph, Mono, Eosin, Baso

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination of the study (after 2 years on all surviving rats)
- Animals fasted: Yes
- How many animals:103 control, 151 exposed rats
- Parameters checked: BUN, SGPT, AP and total protein

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
on succumbed and moribund rats, whenever possible moribund rats were killed for for gross examination and tissues were saved for microscopic examination, with special attention to abnormal growths. Following gross examination body weights of liver, spleen and kidneys were recorded.

HISTOPATHOLOGY: Yes:
lungs, trachea, thoracic lymph nodes, heart, liver, pancreas, stomach, intestine, spleen, thyroid, mesentric lumph nodes, kidneys, urinary bladder, pituary, adrenals, tetstes, ovaries, oviduct, uterus, mammary gland, lacrimals gland, lymph nodes, brain, vagina, bone marrow, and any
abnormal growths.
Statistics:
Control and experimental groups were compared statistically using:
Student's T-test (heamatology, clinical chemistry),
Fisher exact probability test (morphological classification of tumours),
Yates corrected Chi-Square test (survival)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Details on results:
No effects were seen on clinical signs (including activity, demeanour, eye and nasal irritation, skin condition and respiratory distress), body weights or mortality.

Some slight changes were observed in haematological values, but these were within the normal physiological limits and not considered of toxicological importance.

BUN and AP values in treated male rats were slightly decreased.

Changes in liver, kidney or spleen weights were not observed.

Upon gross and microscopic examination, no treatment-related non-neoplastic effects were found in tissues/organs, including the reproductive organs. Regarding neoplastic effects, no 1,4-dioxane characteristic nasal and liver tumours, as observed after oral administration, were seen. It is however not clear from the text whether or not the nasal cavity was adequately examined. The incidence of tumours observed in other organs/tissues appeared to be unrelated to exposure. The only difference from the control groups was an increase in lymphoreticular cell sarcomas in males (18% (37/206) versus 12% (18/150)) and in mammary gland adenoma in females (13% (29/217) versus 8% (11/139)), which were not statistically significant.
Key result
Dose descriptor:
NOAEC
Effect level:
> 400 mg/m³ air
Sex:
male/female
Basis for effect level:
other: no adverse health effects were observed
Key result
Critical effects observed:
no
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344/DuCrj
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Japan
- Fasting period before study: no
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 (exposure chamber: 20-24)
- Humidity (%): 50-70 (exposure chamber: 30-70)
- Air changes (per hour): 12
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
Airflow containing 1,4-dioxane vapor at the target concentration was prepared by a vaporization technique. The saturated vapor-air mixture was generated by bubbling clean air through the 1,4-dioxane liquid in a temperature-regulated glass flask (30°C), and by cooling it through a thermostatted condenser at 20 °C. The airflow containing the saturated vapor was diluted with clean air, and then warmed to 30 °C in a thermostatted circulator that served to stabilize the vapor concentration by complete gasification of 1,4-dioxane. The flow rate of the vapor-air mixture was regulated with a flow meter, further diluted with humidity- and temperature-controlled clean air in a spiraling line mixer, and then supplied to an inhalation exposure chamber. Eight inhalation exposure chambers of 1060 L in volume were used in the present study. Each exposure chamber accommodated 20 individual cages for 10 males and 10 females. Chamber concentrations of 1,4-dioxane were monitored every 15 min with a gas chromatograph (GC-148, Shimadzu Corp., Kyoto, Japan), equipped with a hydrogen flame ionization detector and a 1.5-m Shimadzu SBS-120 packed column operated at a column temperature of 90 °C and with a gas injection volume of 2 mL.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
analysis by gas chromography with a flame ionization detector
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
100 ppm (nominal)
Remarks:
analytical conc.: 100.2 (SD 2.1)
Dose / conc.:
200 ppm (nominal)
Remarks:
analytical conc.: 200.7 (SD 4.1)
Dose / conc.:
400 ppm (nominal)
Remarks:
analytical conc.: 403.9 (SD 7.5)
Dose / conc.:
800 ppm (nominal)
Remarks:
analytical conc.: 799.8 (SD 12.1)
Dose / conc.:
1 600 ppm (nominal)
Remarks:
analytical conc.: 1596.4 (SD 27.2)
Dose / conc.:
3 200 ppm (nominal)
Remarks:
analytical conc.: 3198.4 (SD 48.9)
Dose / conc.:
6 400 ppm (nominal)
Remarks:
analytical conc.: 6409.5 (SD 76.3)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: once per week

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

FOOD EFFICIENCY: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the 13 week exposure
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: Yes
- How many animals: 10/sex/group
- Parameters checked with an automated blood cell analyzer: (RBC, Hb, Heamatocrit, MCV were reported)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:t the end of the 13 week exposure
- Animals fasted: Yes
- How many animals:10/sex/group
- Parameters checked (with an automated analyzer and a flame photometer): (AST, ALT, Glucose, Triglyceride were reported)

URINALYSIS: Yes
- Time schedule for collection of urine: the last week of the 13 weeks exposure
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked: Multistix, Ames reagent strip

NEUROBEHAVIOURAL EXAMINATION: No data


Sacrifice and pathology:
GROSS PATHOLOGY: Yes, complete necropsy; all organs were removed, weighed and examined for macroscopic lesions.
HISTOPATHOLOGY: Yes, as designated in the OECD guideline, plus the entire respitratory tract including the nasal cavity, pharynx, larynx, trachea and bronchus.
Other examinations:
Determination of 1,4-dioxane in blood
Three rats of each group on day 3 of week 12 of exposure, 1 hour after termination. Blood was drawn from the tail vain.
Statistics:
Body weight, food consumption, organ weight, and hematological and blood biochemical parameters were analyzed by Dunnett's test as described previously (Aiso et al., 2005). Histopathological findings and urinary parameters were analyzed by chi-square test. A two-sided analysis with p values of .05 and .01 was performed to determine statistical significance.
Clinical signs:
no effects observed
Description (incidence and severity):
In the surviving animals, no abnormal clinical sign was found in any 1,4-dioxanedosed rats of either sex throughout the 13-wk exposure period.
Mortality:
mortality observed, treatment-related
Description (incidence):
All the 6400-ppm-exposed rats of both sexes died during wk 1 of the 13-wk exposure period. The histopathological examination revealed that their deaths were primarily caused by renal failure, because all the 6400-ppm-exposed animals suffered from marked necrosis in the renal tubules. Moreover, lung congestion was observed in the 6400-ppm-exposed males and females. All the other groups survived to the end of the 13-wk exposure period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Terminal body weight significantly decreased in the 200-ppm and 3200-ppm-exposed males and in the females exposed to 200 ppm, 800 ppm, and above.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Some erythrocyte parameters were increased in the 3200ppm-exposed males and females with slight but statistical significance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A slight but statistically significant increase was found in both aspartate aminotransferase (AST) in the 200ppm-and 3200-ppm-exposed females and alanine aminotransferase (ALT) in the 3200-ppm-exposed males and females. The exposure to 3200 ppm decreased blood levels of glucose and triglyceride only in the males, but not in the females.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urinary protein slightly decreased in the males exposed to 3200 ppm.
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Relative liver weight significantly increased in the males and females exposed to 800 ppm and above. Relative kidney weight significantly increased in the 3200-ppm-exposed males and in the females exposed to 800 ppm and above. Relative lung weight increased in the males exposed to 200 ppm and 1600 ppm and above and in the females exposed to 200 ppm and above.
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The upper and lower respiratory tract as well as liver in both males and females and kidneys in females were affected (table 1 and 2). The most sensitive lesion occurred in the nasal cavity, where the incidences and severities of nuclear enlargement in the respiratory epithelium were significantly increased dose-dependently in both males and females exposed to 100 ppm and above. The nuclear enlargement was morphologically characterized by the appearance of the epithelial cells having round to oval or elongated nuclei that were at least four times as huge in diameter as the normal nuclei of respiratory epithelial cells. The enlarged nuclei of respiratory epithelial cells were localized at Level 1 in the males exposed to 100 und 200 ppm and in the females exposed to 100 ppm, and at Levels 1 and 2 in the females exposed to 200 ppm, while the respiratory epithelial cells having the enlarged nuclei were extended over the entire respiratory region at Level 1 through 3 in both males and females exposed to 400 ppm and above. The enlarged nuclei of the sustentacular cells in the olfactory epithelium were significantly increased in both males and females exposed to 200 ppm and above, and were distributed over almost the entire area of the olfactory region al Levels 2 and 3. The incidences and severities of nuclear enlargement in the 1,4-dioxane-exposed males and females tended to decrease along the passage of inspiratory airflow through the upper and lower respiratory tracts. The incidence of degenerative change in the olfactory sensory cells observed as the vacuolic change was significantly increased in the males exposed to 400 ppm and above, while the females exposed to 800 ppm and above exhibited olfactory epithelial atrophy characterized by a decreased number of the olfactory sensory cells and vacuolic change. Vacuolic change in the bronchial epithelium also occurred with statistical significance in the males exposed to 1600 ppm and above. The 1,4-dioxane-induced liver lesions were characterized by significant increases in the incidences of both single-cell necrosis and centrilobular swelling of hepatocytes in the 3200-ppm exposed males, and in the incidence of centrilobular swelling of hepatocytes in the 3200-ppm-exposed females. In addition, the GST-P-positive Iiver foci were observed in three 3200-ppm-exposed males, two 3200-ppm-exposed females, and four 1600-ppm-exposed females out of each ten 1,4-dioxane exposed rats of either sex, whereas the GST-P-positive foci could not be found in any of the 800- and l600-ppm-exposed males and 800-ppm-exposed females and control groups of both sexes. The GST-P-positive hepatocytes exhibited morphologically focal and clonal proliferation. The incidence of hydropic change in the renal proximal tubules was significantly increased in the 3200-ppm-exposed females.
Histopathological findings: neoplastic:
no effects observed
Details on results:
All the 6400-ppm-exposed males and females died during the first week, due to lung congestion and renal failure. Terminal body weight decreased, and relative weights of liver, kidney, and lung increased.
AST increased in the 200 ppm-and 3200-ppm-exposed females, and ALT increased in the 3200-ppm-exposed males and females.
Nuclear enlargement of nasal respiratory epithelial cells occurring in the 100-ppm-exposed males and females was the most sensitive, followed by the enlarged nuclei in the olfactory, tracheal, and bronchial epithelia.
1,4-Dioxane-induced liver lesions occurred at higher exposure concentrations than the nasal lesions did, and were characterized by single-cell necrosis and centrilobular swelling of hepatocytes in males and females.
Glutathione S-transferase placental form (GST-P) positive liver foci were observed in the 1600-ppm-exposed females and 3200-ppm-exposed males and females.
Plasma levels of 1,4-dioxane increased linearly with an increase in the concentrations of exposure to 400 ppm and above.
Key result
Dose descriptor:
LOAEC
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: Lowest dose tested. NOAEC not determinable.
Remarks:
corresponding to 360 mg/m3
Key result
Dose descriptor:
LOEC
Effect level:
ca. 360 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: liver weight increase or hypertrophy
Remarks on result:
other: as re-evaluated by Dourson et al., 2017
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 ppm
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes

Table 1 Incidences and severities of selected lesions in the male rats exposed by inhalation to 1,4 -dioxane vapor at 6 different concentrations or clean air for 13 weeks

 

Male

Group (ppm)

Control

100

200

400

800

1600

3200

No. of animals examined

10

10

10

10

10

10

10

Nasal cavity

 

Nuclear enlargement: respiratory epithelium

0

7**

9**

7**

10**

10**

10**

 

1+:7

1+:9

1+:7

1+:10

2+:10

2+:10

Nuclear enlargement: olfactory epithelium

0

0

5*

10**

10**

10**

10**

 

 

1+:5

1+:10

1+:10

2+:10

2+:10

Vacuolic change: olfactory epithelium

0

1

3

6*

10**

10**

9**

 

1+:1

1+:3

1+:6

1+:10

1+:10

1+:9

Trachea
Nuclear enlargement: epithelium

0

0

0

0

1

10**

10**

 

 

 

 

1+:1

1+:10

1+:10

Bronchus

 

Nuclear enlargement: bronchial epithelium

0

0

0

0

0

9**

10**

 

 

 

 

 

1+:9

1+:10

Vacuolic change: bronchial epithelium

0

0

0

0

4

6*

6*

 

 

 

 

1+:4

1+:6

1+:6

Liver

 

Necrosis: single cells

0

0

0

0

0

1

8**

 

 

 

 

 

1+:1

1+:8

Swelling: centrilobular

0

0

0

0

0

1

10**

 

 

 

 

 

 

1+:10

The number of the animals bearing the lesion in each exposed or control group are shown in the upper column, respectively. The column below indicates values indicate the number of the animals bearing the lesion with each of 4 different grades of severity ie. 1+: sligh; 2+:moderate; 3+: marked; 4+:severe;

Significant difference indicated by * p≤0.05; **p≤0.01 by chi square test.

Data of the 6400 ppm-exposed group are not presented here, because all of them died at the first week of the study.

Table 2 Incidences and severities of selected lesions in the female rats exposed by inhalation to 1,4 -dioxane vapor at 6 different concentrations or clean air for 13 weeks

 

Female

Group (ppm)

Control

100

200

400

800

1600

3200

No. of animals examined

10

10

10

10

10

10

10

Nasal cavity

 

Nuclear enlargement: respiratory epithelium

0

5*

9**

10**

10**

10**

10**

 

1+:5

1+:9

1+:10

1+:10

2+:10

2+:10

Nuclear enlargement: olfactory epithelium

0

2

6*

10**

10**

10**

10**

 

1+:2

1+:6

1+:9; 2+:1

1+:10

1+:7; 2+:3

2+:10

Vacuolic change: olfactory epithelium

0

1

2

3

7**

9**

10**

 

1+:1

1+:2

1+:3

1+:7

1+:9

1+:10

Atrophy: olfactory epithelium

0

0

2

3

5*

5*

4

 

 

1+:2

1+:3

1+:5

1+:5

1+:4

Trachea
Nuclear enlargement: epithelium

0

0

0

0

2

7**

10**

 

 

 

 

1+:2

1+:7

1+:110

Bronchus

 

Nuclear enlargement: bronchial epithelium

0

0

0

0

0

0

10**

 

 

 

 

 

 

1+:10

Vacuolic change: bronchial epithelium

0

0

0

1

1

3

4

 

 

 

1+:1

1+:1

1+:3

1+:4

Liver

 

Necrosis: single cells

0

0

0

0

0

0

3

 

 

 

 

 

 

1+:3

Swelling: centrilobular

0

0

0

0

0

1

8**

 

 

 

 

 

1+:1

1+:8

Kidney

Hydropic change: proximale tubule

0

0

0

0

0

0

6*

 

 

 

 

 

 

1+:6

The number of the animals bearing the lesion in each exposed or control group are shown in the upper column, respectively. The column below indicates values indicate the number of the animals bearing the lesion with each of 4 different grades of severity ie. 1+: sligh; 2+:moderate; 3+: marked; 4+:severe;

Significant difference indicated by * p≤0.05; **p≤0.01 by chi square test.

Data of the 6400 ppm-exposed group are not presented here, because all of them died at the first week of the study.

Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
1981
GLP compliance:
not specified
Remarks:
GLP compliance not stated in the publication
Limit test:
no
Species:
rat
Strain:
Fischer 344
Details on species / strain selection:
Selection of male F344 rats was based on our previous result that 2 year oral administration of 1,4-dioxane in drinking water induced mesotheliomas only in male F344 rats but not in females (Yamazaki et al., 1994).
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc. (Kanagawa, Japan)
- Age at study initiation: 6 weeks
- Weight at study initiation: 150 +/- 5 g
- Housing: individually in stainless steel wire hanging cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 21.9 +/- 0.6 °C
- Humidity: 53.6 +/- 4.9 %
- Air changes: not specified
- Photoperiod: 12/12 hrs dark / hrs light
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
clean air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Vaporization technique:
The saturated vapor–air mixture was generated by bubbling clean air through the 1,4-dioxane liquid in a temperature-regulated glass flask (35°C), and by cooling it through a thermostatted condenser at 20°C. The airflow containing the saturated vapor was diluted with clean air, and
then warmed to 35°C in a thermostatted circulator, which served to stabilize the vapor concentration by complete gasification of 1,4-dioxane. The flow rate of the vapor–air mixture was regulated with a flow meter, further diluted with humidity-and temperature-controlled clean air in a spiraling line mixer, and then supplied to an inhalation exposure chamber.

- Method of holding animals in test chamber: individually

- Temperature, humidity, pressure in air chamber: Temperature: 21.9 +/- 0.6 °C; Humidity: 53.6 +/- 4.9 %

- Air change rate: not specified


TEST ATMOSPHERE
- Brief description of analytical method used: Chamber concentrations of 1,4-dioxane were monitored every 15 min with a GC unit (GC-14B; Shimadzu Corp., Kyoto, Japan) equipped with a hydrogen flame ionization detector and a 1.5-m Shimadzu SBS-120 packed column operated at a column temperature of 90°C and with a gas injection volume of 2 mL, and were maintained constant at 50.2 ± 1.4 (mean ± SD), 250.9 ± 3.2, and 1247.5 ± 18.6 ppm throughout the 2-yr exposure period.

- Samples taken from breathing zone: no

Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
2 years (104 weeks)
Frequency of treatment:
6 hours per day; 5 days per week
Dose / conc.:
50 ppm (nominal)
Remarks:
v/v
Dose / conc.:
250 ppm (nominal)
Remarks:
v/v
Dose / conc.:
1 250 ppm (nominal)
Remarks:
v/v
No. of animals per sex per dose:
50 males per dose / control group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The lowest exposure concentration of 50 ppm was selected as an environmentally relevant level marginally exceeding the occupational exposure limit value of 20 ppm for 1,4-dioxane (ACGIH, 2001). The highest concentration of 1250 ppm was selected not to exceed the maximum tolerated dose (MTD) criteria (Sontag et al., 1976; OECD, 1981; Bannasch et al., 1986), based on both subchronic toxicity and body weight decrement from our 13-wk inhalation exposure study (Kasai et al., 2008). A preliminary study showed that 13-wk inhalation exposure of male F344 rats to 1,4-dioxane at a concentration of 1600 ppm did not induce any death, body weight decrement, or toxicity except histopathological changes in the nasal cavity, trachea, and bronchus. Thus, the highest concentration of 1250 ppm was predicted to elicit signs of minimal toxicity without altering the normal lifespan of the male rats except for the result of neoplastic development, following 2-yr inhalation exposure to 1,4-dioxane.

- Rationale for animal assignment: stratified randomisation (body weight matched)
- Fasting period before blood sampling for clinical biochemistry: no
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: once a week (first 14 weeks); every 4 weeks thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: once a week (first 14 weeks); every 4 weeks thereafter

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of the 2 year exposure period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: no specified

CLINICAL CHEMISTRY: Yes, see heamatology

URINALYSIS: Yes
- Time schedule for collection of urine: last 2 week of 2 year epxosure
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked: Ames reagent strips

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all organs listed in the guideline and the entire respiratory tract)
HISTOPATHOLOGY: Yes
Statistics:
Incidences of neoplastic lesions were analyzed for a dose–response relationship indicated by a significant positive trend with Peto’s test (Peto et al., 1980), and for a significant difference from the clean air-exposed group by Fisher’s exact test.

Incidences of nonneoplastic lesions and urinary parameters were analyzed by Chi2 test. Survival curves were plotted according to the Kaplan–Meier method (Kaplan & Meier, 1958), and both the log-rank test (Peto et al., 1977) and Fisher’s exact test were used to investigate a statistically significant difference in survival rate between any 1,4-dioxane-exposed rat group and the clean air-exposed group. Body weight, organ weight, food consumption, and hematological and blood biochemical parameters were analyzed by Dunnett’s test.

A two-tailed test was used for all statistical analysis except for Peto’s test. In all cases, statistical analysis with p-values of 0.05 and 0.01 was performed and indicated in the tables, while a p-value of 0.05 was used as a level of significance.

A no observed adverse effect level (NOAEL) or a lowest observed adverse effect level (LOAEL) was determined, according to the World Health Organization (WHO) definition (International Programme on Chemical Safety, 1994).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the clinical observation, deformity of the nose was observed at the 79th and 74th wk for one each of the 250 and 1250 ppm-exposed rats, respectively, and these two rats died due to malignant nasal tumor before the end of the 2-yr exposure period.
Mortality:
mortality observed, treatment-related
Description (incidence):
No significant difference in survival rate at any time point was found between the 50 ppm-exposed group and the control. The survival rates of the 250 ppm-exposed group tended to decrease near the end of the 2-yr exposure period, although there was no statistical difference between the 250 ppm-exposed group and the control. The 1250 ppm-exposed group exhibited a statistically significant decrease in survival rate at the 91st wk and thereafter, as compared with the respective controls. The terminal survival rates of the control, 50, 250, and 1250 ppm-exposed groups were 37/50, 37/50, 29/50, and 25/50, respectively. The significantly decreased survival rates in the 1250 ppm-exposed group were attributed to the increased number of deaths due primarily to peritoneal mesotheliomas, although nasal tumors contributed to the causes of death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Although no statistical difference in growth rate between any 1,4-dioxane exposed group and the control was found during the first 22 wk, the growth rates of the 1250 ppm-exposed rats tended to decrease during the latter half of the 2-yr exposure period. A decrease in terminal body weight of the 1250 ppm-exposed group was statistically significant but less than 10% as compared with the control.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption did not decrease in any 1,4-dioxane-exposed group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Significant decreases in hemoglobin, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) were noted in the 1250 ppm-exposed group, although neither red blood cell counts nor hematocrit values were decreased.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma-glutamyltranspeptidase (gamma-GTP) were significantly increased in the 1250 ppm-exposed group. Urinary pH was significantly decreased in the 1250 ppmexposed group.
Urinalysis findings:
not specified
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Relative liver weight was significantly increased in the 1250 ppm-exposed group. A statistically significant increase in relative lung weight was found in the 1250 ppm-exposed group, but this change was considered not to be biologically meaningful, because there was no dose–response relationship for relative lung weight. No remarkable organ weight change was observed in any other organs of 1,4-dioxane-exposed groups.
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Notably, incidences of nuclear enlargement in the respiratory and olfactory epithelia were significantly increased in all 1,4-dioxane-exposed groups, while significantly increased incidences of nuclear enlargement were observed in the liver of the 1250 ppm-exposed group and the kidney of the 250 ppm- and 1250 ppm-exposed groups. Inflammation in the respiratory and olfactory epithelia and atrophy in the olfactory epithelium, hydropic change and sclerosis of lamina propria, and proliferation in the nasal gland occurred at a statistically significant level in the 1,4-dioxane-exposed groups with different exposure concentrations. Necrosis of hepatocytes in the centrilobular region, spongiosis hepatis in the liver, and hydropic change in the renal proximal tubule also occurred in the 1250 ppm exposed group. No exposure-related, nonneoplastic or neoplastic lesion in the pharynx, larynx, trachea, bronchus, or lungs was histopathologically observed in any 1,4-dioxane exposed group.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A dose-dependent increase in tumor incidences as indicated by a significantly positive trend with Peto’s test was observed in nasal squamous cell carcinomas, hepatocellular adenomas, renal cell carcinomas, peritoneal mesotheliomas, fibroadenomas in the mammary gland, and adenomas in the Zymbal gland.

The incidences of nasal squamous cell carcinomas, hepatocellular adenomas, and peritoneal mesotheliomas were significantly increased in the 1250 ppm-exposed rats compared with the concurrent, matched controls by Fisher’s exact test. In addition, a statistically significant increase in the incidences of peritoneal mesotheliomas and subcutaneous fibromas was also noted in the 250 ppm-exposed rats. 1,4-Dioxane-induced nasal squamous cell carcinoma occurred at the dorsal area of levels 1–3 in the nasal cavity, and was histopathologically characterized as well-differentiated and keratinized type, with invasive growth into the surrounding tissue.

Two cases of squamous cell carcinoma destroyed the nasal bone and protruded out of the nose through the nasal bone. Peritoneal mesotheliomas arose from the mesothelium of the scrotal sac, were characterized by complex branching structures with a fibrovascular core covered by single or stratified layers of mesothelial cells, and occasionally had spread throughout the entire surface of the peritoneal cavity and invaded visceral organs including the pancreas. As preneoplastic lesions, squamous cell hyperplasia in the nasal cavity and altered cell foci in the liver were observed in the 1250 ppm-exposed group. The squamous cell hyperplasia occurred primarily on the nasoturbinate, septum, and dorsal wall at levels 1 and 2, was characterized by five or more layers of epithelial cells, and had cellular pleomorphism, irregular arrangement of cell layers, and proliferation of basal cells, resembling an early stage of squamous cell carcinoma. Squamous cell metaplasias that were characterized by replacement of the transitional and respiratory epithelia by the squamous epithelium with or without keratinization occurred in the rats exposed to 250 ppm and above. The squamous cell metaplasia appeared primarily in the focal area of the nasoturbinate, maxilloturbinate, septum, and dorsal and lateral wall at level 1, and occasionally showed slight cellular pleomorphism and proliferation of basal cells, which was indicative of atypia and might be regarded as a potentially preneoplastic lesion.
Other effects:
not specified
Key result
Dose descriptor:
LOAEC
Effect level:
50 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: lowest dose tested
Key result
Dose descriptor:
LOEC
Effect level:
1 250 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: liver weight increase or hypertrophy; necrosis / inflammation; DNA synthesis hyperplasia or foci; liver adenomas / carcinomas
Remarks on result:
other: as re-evaluated by Dourson et al., 2017
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 ppm
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes

Table 1 Incidences of selected histopathological lesions in male F344 rats.

 

Control

50 ppm

250 ppm

1250 ppm

Peto test

No. of animals examined

50

50

50

50

 

Neoplastic lesions

Nasal cavity

 

Squamous cell carcinoma

0

0

1

6#

++

Liver

 

Hepatocellular adenoma

1

2

3

21##

++

Hepatocellular carcinoma

0

0

1

2

 

Kidney

 

Renal cell carcinoma

0

0

0

4

++

Peritoneum
Mesothelioma

2

4

14##

41##

++

Mammary gland

 

Fibroadenoma

1

2

3

5

+

Adenoma

0

0

0

1

 

Zymbal gland
Adenoma

0

0

0

4

++

Subcutis
Fibroma

1

4

9##

5

 

Pre- and non-neoplastic lesions

Nasal cavity

 

Respiratory epithelium

 

Nuclear enlargement

0

50**

48**

38**

 

Squamous cell metaplasia

0

0

7*

44**

 

Squamous cell hyperplasia

0

0

1

10**

 

Inflammation

13

9

7

39**

 

Olfactory epithelium

 

Nuclear enlargement

0

48**

48**

45**

 

Atrophy

0

40**

47**

48**

 

Respiratory metaplasia

11

34**

49**

48**

 

Inflammation

0

2

32**

34**

 

Hydropic change: lamina propria

0

2

36**

49**

 

Sclerosis: lamina propria

0

0

22**

40**

 

Proliferation: nasal gland

0

1

0

6*

 

Liver

 

Nuclear enlargement: centrilobular

0

0

1

30**

 

Acidophilic cell foci

5

10

12

25**

 

Basophilic cell foci

17

20

15

44**

 

Clear cell foci

15

17

20

23

 

Mixed cell foci

5

3

4

14

 

Spongiosis hepatis

7

6

13

19**

 

Necrosis: centrilobular

1

3

6

12**

 

Kidney

 

Nuclear enlargement: proximal tubule

0

1

20**

47**

 

Hydropic change: proximal tubule

0

0

5

6*

 

Note: # and ## significantly different from control at p≤0.05 and p≤0.01 by Fisher’s exact test, respectively. * and ** significantly different from control at p≤0.05 and p≤0.01 by chi square test, respectively. + and ++ significantly different from control at p≤0.05 and p≤0.01 by Peto test, respectively.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
180 mg/m³
Study duration:
chronic
Species:
rat
Quality of whole database:
Studies comparable to OECD guideline with acceptable restriction sufficient for assessment.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information





In the literature there are numerous data available on repeated dose toxicity of 1,4-dioxane via oral and inhalation route of exposure in rats and mice. Only reliable and relevant data (i. e. Klimisch score 1 or 2) are taken into account in this dossier and are summarized in the following table.


 


Table 1: Relevant repeated dose toxicity and carcinogenicity data with 1,4-dioxane
























































































































































Species



Exp. Dur.



Route



Dose levels



Other findings



NOAEL or LOAEL



Source



IUCLID Section



Comments



Rat



2 yrs



Oral (DW)



0.01, 0.1, 1 %


 


 


m:


9.6, 94, 1015


mg/kg bw/d


 


 


f:


19, 148, 1599


mg/kg bw/d



Liver carcinomas; cholangiomas; squamous cell carcinomas of the nasal cavities;


 


 


Renal tubular epithelial and hepatocellular degeneration and necrosis, accompanied by regenerative activities in the liver (hepatocellular hyperplastic nodule formation) and renal tubuli



NOAEL =


 


m:


9.6 mg/kg bw/d  


 


f:


19 mg/kg bw/d



Kociba et al., 1974



7.5.1 & 7.7



NOAEL not based on carcinomas but liver and kidney effects



Rat



2 yrs



Oral (DW)



200; 1000; 5000 ppm


 


m:


11; 55; 274 mg/kg bw/d


 


 


f:


18; 83; 429 mg/kg bw/d


 



5000 ppm:


m & f:


Liver: hepatocellular adenomas and / or carcinomas


 


Nasal cavity: Squamous carcinoma


 


Respiratory and olfactory epithelium: nuclear enlargement;


 


f:


Mammary gland: adenoma and/or fibroadenoma;


 


m:


Peritoneum: Mesenthelioma



NOAEL =


 


m:


55 mg/kg bw/d


 


f:


83 mg/kg bw/d



Kano et al., 2009



7.5.1 & 7.7



re-evaluation of full study report by Dourson et al., (2017):


non-cancer effects found indicating a non- genotoxic MoA based on cytotoxicity and regenerative hyperplasia



Rat



13 wks



Oral (DW)



0, 0.064, 0.16, 0.4, 1, 2,5 %


 


m:


0, 52, 126, 274, 657, 1554 mg/kg


bw/d


 


f:


0, 83, 185, 427, 756, 1614 mg/kg bw/d


 



126/185 mg/kg bw/d:


m & f:


nose: nuclear enlargement in respiratory epithelium ↑,


 


m:


liver: centrilobular enlargement of hepatocytes ↑,


 


f:


rel. Liver wt ↑, rel. Kidney wt ↑;


 


274/427 mg/kg bw/d:


m & f:


water consumption ↓, nose: nuclear enlargement in the olfactory epithelium ↑, trachea: nuclear enlargement in the epithelium ↑,


 


m:


rel. Kidney wt ↑, urine pH ↓, liver: single cell necrosis ↑,


 


f:


bw ↓, liver: single cell necrosis ↑;


 


657/756 mg/kg bw/d


m & f:


kidney: nuclear enlargement in proximal tubuli-epithelial cells,


 


m:


bw ↓, rel. Liver wt. ↑, liver: centrilobular vacuoles ↑,


 


f:


food consumption ↓, rel. Lung wt ↑, urine pH ↓, liver: centrilobular enlargement of hepatocytes ↑;


 


1554/1614 mg/kg bw/d


m & f:


ruffled fur, glucose in blood ↓, kidney:


Hydropic changes in proximal tubuli epithelial cells, brain: vacuolic changes cerebrum,


 


m:


food consumption ↓, rel. Liver wt ↑, erythrocytes, hemoglobin hematocrit ↑, plasma AST & ALT ↑,


 


f:


mortality 1/10 (kidney failure), plasma AST ↑, liver: single cell necrosis and centrilobular vacuoles ↑, lung: nuclear enlargement in the bronchial epithelium ↑



NOAEL =


 


m:


52 mg/kg bw/d


 


f:


83 mg/kg bw/d



Kano et al., 2008



7.5.1 & 7.7



re-evaluation of full study report by Dourson et al., (2017):


non-cancer effects found indicating a non- genotoxic MoA based on cytotoxicity and regenerative hyperplasia



Rat



110 wks



Oral (DW)



0.5; 1.0 v/v %


 


m:


240; 530 mg/kg bw/d


 


 


 


f:


350; 640 mg/kg bw/d



f & m:


squamous cell carcinomas in the nasal cavities;


partially extended to the retrobulbar tissues of the eye and into the brain;


adenocarcinomas in nasal mucosal epithelium


 


f:


hepatocellular adenomas


 



LOAEL = 0.5 % (v/v)


 


m: 240 mg/kg bw/d


 


f: 350 mg/kg bw/d



NCI, 1978



7.5.1 & 7.7



Dourson et al. (2014)


 


re-read of slides revealed identification on non-cancer effects such as necrosis inflammation



Mouse



90 wks



Oral (DW)



0.5; 1.0 v/v %


 


m:


720; 830 mg/kg bw/d


 


 


 


f:


380; 860 mg/kg bw/d



f & m:


squamous cell carcinomas in the nasal cavities;


hepatocellular carcinomas


 



LOAEL = 0.5 % (v/v)


 


m: 720 mg/kg bw/d


 


f: 380 mg/kg bw/d



NCI, 1978



7.5.1 & 7.7



Dourson et al. (2014)


 


re-read of slides revealed identification on non-cancer effects such as necrosis inflammation



Mouse



2 yrs



Oral (DW)



500, 2000, 8000 ppm


 


m:


49; 191; 677 mg/kg bw/d


 


 


f:


66; 278; 964 mg/kg bw/d


 



Liver:


Hepatocellular adenoma and / or carcinoma


 


Nasal cavity:


Nuclear enlargement in respiratory and olfactory epithelium



NOAEL =


 


m:


49 mg/kg bw/d


 


LOAEL=


 


f:


66 mg/kg bw/d (lowest dose tested)



Kano et al., 2009



7.5.1 & 7.7



re-evaluation of full study report by Dourson et al. (2017):


pre-cancer effects found indicating a non-genotoxic MoA based on cytotoxicity and regenerative hyperplasia



Mouse



13 wks



Oral (DW)



0, 0.064, 0.16, 0.4, 1,


2.5 %


 


m:


0, 86,


231, 585, 882, 1570 mg/kg


bw/d


 


f:


0, 170, 387, 898, 1620, 2669 mg/kg bw/d


 


 



387 mg/kg bw/d


f:


lung: nuclear enlargement in bronchial epithelium ↑;


 


585/898 mg/kg bw/d


m & f:


nose: nuclear enlargement in the olfactory epithelium ↑, trachea: nuclear enlargement in the epithelium ↑, liver: single cell necrosis, enlargement of centrilobular hepatocytes ↑,


 


m:


lung: nuclear enlargement in the bronchial epithelium ↑;


 


882/1620 mg/kg bw/d


m & f:


water consumption ↓, urine pH ↓,


 


f:


rel. Lung wt. ↑, ALT ↑, glucose ↓, lung: degeneration in bronchial epithelium ↑;


 


1570/2669 mg/kg bw/d:


m & f:


nose: nuclear enlargement in respiratory epithelium ↑, vacuolic changes in the olfactory epithelium ↑, rel. kidney wt. ↑, plasma AST & ALT ↑,


 


m:


mortality 1/10, piloerection, BW ↓, food consumption ↓, rel. lung wt. ↑, erythrocytes, hemoglobin and hematocrit ↑, glucose ↓, lung: degeneration in bronchial epithelium ↑



NOAEL = 


 


m:


231 mg/kg bw/d


 


f:


170 mg/kg bw/d



Kano et al., 2008



7.5.1



 



Mouse



13 weeks



oral (DW)



0,40, 200, 600, 2000, 6000 ppm



6000 ppm:



  • increase in hepatocellular proliferation

  • increase in relative liver weights

  • increased blood levels of 1,4-DX

  • Moderate centrilobular hypertrophy and increased apoptosis in liver



NOAEL= 2000 ppm



Lafranconi et al., 2020



7.5.1



NOAEL not based on carcinomas but liver weight changes and histopathological findings in liver at 6000 ppm



 



Rat



2 yrs



7 h/d


 


5 d/wk,



Inhal (WB)



111 ppm


 


(0.4 mg/L)



No adverse effects observed in


-      Growth,


-      Mortality,


-      Hematology


-      Clinical chemistry



NOAEC = 111 ppm (400 mg /m3)


 



Torkelson et al.,1974



7.5.2 & 7.7



Nasal cavity not microscopically examined



Rat



2 yrs

6 h/d

5 d/wk



Inhal (WB)



50, 250, 1250 ppm


 


(180, 900, 1800 mg/m3)



nasal squamous cell carcinomas; hepatocellular adenomas; renal cell carcinomas; peritoneal mesotheliomas; fibroadenomas in the mammary gland; adenomas in the Zymbal gland


 


 


50 ppm:


Nose: Incidence of nuclear enlargement and inflammation↑, atrophy, respiratory metaplasia olfactory epithelium, hydropic changes and sclerosis lamina propria, glandular proliferations


 


250 ppm:


mortality ↑, nose: epithelial cell –Metaplasia, kidneys: Incidence nuclear enlargement ↑,


 


1250 ppm:


BW ↓, nose: epithelial-Hyperplasia, kidney: Hydropic changes renal proximal tubuli, liver: rel. weight ↑, Incidence of nuclear enlargement ↑, Necrosis hepatocytes, changes in cell foci, plasma: ALT-, AST- and γ-GTP-Activities ↑


 


 


 àNuclear enlargement of epithelial cells over the entire region of respiratory and olfactory epithelia (no anterior-posterior gradient)



LOAEC = 50 ppm (180 mg/m3)



Kasai et al., 2009



7.5.2 & 7.7



Males only



Rat



13 wks


 


6 h/d


 


5 d/wk



Inhal (WB)



0, 100, 200, 400, 800, 1600, 3200, 6400 ppm


 


(0,360, 721, 1441, 2883, 5765, 11530, 23060 mg/m3)


 



100 ppm:


m & f:


nose: Incidence of nuclear enlargement respiratory epithelium ↑


 


200 ppm:


m & f: BW ↓,


 


m: rel. Lung weight ↑, AST ↑;


 


above 200 ppm:


m & f:


Nose: Incidence of nuclear enlargement olfactory epithelium ↑,


 


f: rel. Lung weight ↑;


 


400 ppm:


m: Nose: degeneration olfactory epithelium;


 


800 ppm:


m & f:


rel. Liver weight ↑,


 


f:


BW ↓, rel. Liver weight ↑, Nose: degeneration olfactory epithelium;


 


1600 ppm:


m & f:


lung: degeneration bronchial epithelium


 


m: rel. Lung weight ↑,


f: Liver: GST-P positive Foci;


 


3200 ppm:


m & f:


liver: single cell necrosis centrilobular


hepatocyte swelling, ALT ↑,


 


m:


BW ↓, rel. Liver weight ↑, Liver: GST-P positive Foci, Triglycerides & Glucose in blood ↓, protein in urine ↓,


 


f: AST ↑;


 


6400 ppm:


m & f:


Mortality 10/10 within the 1st week (kidney failure and congestions in the lungs)



LOAEC =


 


100 ppm or 100 mL/m3(lowest concentration tested)



Kasai et al., 2008



7.5.2



incidence and severity of enlarged nuclei of epithelial cells decreased along the passage of inspiratory flow through the upper and lower respiratory tracts, and the respiratory epithelial area having enlarged nuclei was expanded from the anterior portion (level 1) to the entire region (levels 1, 2, and 3) with an increase in concentration of inhalation exposure



 Mouse



 



 



 



 



 



 



 



 



DW – Drinking Water
WB – Whole Body


wt - weight


h – hour
d – day
wk- week
yr- year


m – males


f - females


 


 


Repeated dose toxicity: key value for hazard assessment


There are numerous data available on repeated dose toxicity with 1,4-dioxane via the oral as well as via the inhalation route in either rat or mouse. As most relevant endpoint in regards to repeated dose toxicity, tumor formation and pre-neoplastic lesions in the nasal cavity as well as in the liver caused by exposure to 1,4-dioxane were identified.


These findings and respective mode of action were extensively discussed in the literature as well as by evaluating authorities (US EPA (2013 and 2019 (Draft only)), RAC (2019), SCOEL (2004) and German MAK Committee (2019)).


 


The following considerations are taken into account being scientifically sound and regulatory agreed arguments:


Based on toxicokinetic data (as provided in detail in IUCLID section 7.1) 1,4-dioxane was demonstrated to follow a non-linear toxicokinetic. More specifically, this means that metabolism and/or excretion processes of 1,4-dioxane become saturated at higher doses and subsequently accumulation occurs. The dose levels, at which saturation of toxicokinetics is reached, was found to be in the range of 9.6 – 42 mg/kg bw/d for rats and 57 – 66 mg/kg bw/d in mice (Dourson et al., 2017; IUCLID Section 7.12).
Liver tumor formation and / or pre-neoplastic findings, such as nuclear enlargement, necrosis or hyperplasia were observed only at dose levels clearly exceeding the linear range of toxicokinetics. These effects should thus be treated with caution when deriving a no-adverse-effect- level.
Carcinomas and related precursor findings in the nasal cavity of rats are considered to be caused by local irritation events. 1,4-dioxane is legally classified for respiratory irritation (STOT RE 3) according to CLP. It is noted that due to their natural behavior, for rats exposed to 1,4-dioxane via drinking water local exposure of the respiratory tract cannot be excluded.



Against this background effects on nasal cavity are thus of limited relevance for derivation of a systemic no-effect- level as well as the human exposure by oral or dermal route.


In brief, repeated dose toxicity hazard assessment for systemic effects with 1,4-dioxane takes into account the following considerations:


-      1,4-dioxane follows non-linear toxicokinetics (see IUCLID 7.1)


-      Its metabolism and/or excretion becomes saturated at high dose levels (see IUCLID 7.1)


-      Tumors and pre-neoplastic findings were observed at doses clearly exceeding the linear toxicokinetic only (IUCLID Section 7.5 and 7.7)


-      As mode of action cytotoxicity and subsequent regenerating hyperplasia are considered to be the main processes leading to neoplasia and tumor formation (IUCLID Section 7.12). This model is supported  by the observations from Lafranconi et al. 2020 (7.5.1). The results from this study indicate an early onset of a direct mitogenic stimulus occurring prior to the development of cytotoxicity, necrosis and the regenerative processes in the liver after saturation of metabolic clearance pathways of 1,4-dioxane after 90-days of exposures of mice between 2000 ppm and 6000 ppm 1,4-dioxane.


-      A genotoxic mode of action is unlikely for carcinogenesis induced by 1,4 -dioxane (see IUCLID Section 7.6)


 


In conclusion, a safe threshold, below which no adverse effects are expected, can be derived for 1,4-dioxane (see also IUCLID Section 7).


 


The above considerations are in line with the German Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area (MAK Commission).


The MAK Commission conducted a re-evaluation of the national OEL (MAK) for 1,4-Dioxane in 2019 (MAK Commission, 2019) and concluded on an OEL value of 37 mg/m3.


The following main arguments were considered by the MAK Commission (2019):


“The critical effect is nasal toxicity and irritation as well as carcinogenic effects in the nose, liver, and kidneys. New carcinogenicity studies with 1,4-dioxane in drinking water confirm the previous tumour findings in rats and mice. Squamous cell carcinomas in the rat nose, also occurring in a long-term rat inhalation study at 1250 ml/m3, are a result of direct tissue contact with 1,4-dioxane in the drinking water. At 50 ml/m3 (LOAEC, lowest observed adverse effect level), no increase in tumour incidences, but nuclear enlargement, atrophy, and respiratory metaplasia in the nasal cavity were noted. The mechanisms involved in the tumour development in the nose are most likely cytotoxicity, inflammation, regenerative cell proliferation and hyperplasia. As the primary mode of action is non-genotoxic and genotoxic effects play no or at most a minor part at cytotoxic doses, 1,4-dioxane remains in Carcinogen Category 4 [Annotation: according to MAK Categories]. A NAEC of 16.67 ml/m3 (LOAEC / 3) for effects in the nose was calculated from the long-term rat inhalation study, which is in the same range as the NOAEC of 20 ml/m3 from studies with 2- to 8-hour inhalation exposure of volunteers. To provide additional protection from tumour induction in the nose, the MAK value is lowered to 10 ml/m3. As the critical effect of 1,4-dioxane is local and no irritation was observed in the study with 2-hour exposure of volunteers to 20 ml/m3, Peak Limitation Category I and the excursion factor of 2 are retained.”


 


References not provided as RSS in this dossier


-      U.S. Environmental Protection Agency (US EPA), TOXICOLOGICAL REVIEW OF 1,4-DIOXANE (CAS No. 123-91-1) In Support of Summary Information on the Integrated Risk Information System (IRIS), September 2013


-      U.S. Environmental Protection Agency (US EPA), Draft Risk Evaluation for 1,4-Dioxane CASRN: 123-91-1, June 2019


-      The MAK Collection for Occupational Health and Safety 2019, Vol4, No2


-      SCOEL/SUM/112 June 2004


-      Committee for Risk Assessment, RAC, Opinion proposing harmonised classification and labelling at EU level of 1,4-dioxane, CLH-O-0000001412-86-264/F, 15 March 2019





Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The NOAEC and NOAEL in the repeated dose toxicity tests are above the cut-off values. As a result classification of the substance as STOT RE is not warranted under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.