1,4-dioxane

Carcinogenicity

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-noeplastic lesions in the respiratory tract as well as in the liver were observed.

 

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).

 

Carcinogenicity is considered to be caused by a non-genotoxic mode of action.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: 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 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

1981

GLP compliance:

yes

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 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

Post exposure period:

not applicable

Dose / conc.:

500 ppm (nominal)

Remarks:

corresponding to 49 +/- 5 (males) and 66 +/- 10 (females) mg/kg bw/d

Dose / conc.:

2 000 ppm (nominal)

Remarks:

corresponding to 191 +/- 21 (males) and 278 +/- 40 (females) mg/kg bw/d

Dose / conc.:

8 000 ppm (nominal)

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

Dermal irritation (if dermal study):

not examined

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

Relevance of carcinogenic effects / potential:

Please refer to the endpoint-summary of IUCLID Section 7.7.

Key result

Dose descriptor:

NOAEL

Effect level:

500 ppm (nominal)

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

Dose descriptor:

LOAEL

Effect level:

2 000 ppm (nominal)

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:

LOAEL

Effect level:

500 ppm (nominal)

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

Critical effects observed:

no

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.

Reference 2

Endpoint:

carcinogenicity: 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 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

1981

GLP compliance:

yes

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 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

Post exposure period:

not applicable

Dose / conc.:

200 ppm (nominal)

Remarks:

corresponding to 11 +/- 1 (males) and 18 +/-3 (females) mg/kg bw/d

Dose / conc.:

1 000 ppm (nominal)

Remarks:

corresponding to 55 +/- 3 (males) and 83 +/- 14 (females) mg/kg bw/d

Dose / conc.:

5 000 ppm (nominal)

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

Dermal irritation (if dermal study):

not examined

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

Relevance of carcinogenic effects / potential:

Please refer to endpoint-summary in IUCLID Section 7.7.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 ppm (nominal)

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 55 +/- 3 (males) and 83 +/- 14 (females) mg/kg bw/d

Key result

Dose descriptor:

LOAEL

Effect level:

5 000 ppm (nominal)

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 274 +/- 18 (males) and 429 +/- 69 (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

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.

 

Reference 3

Endpoint:

carcinogenicity: 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:

no guideline followed

Principles of method if other than guideline:

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

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 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 (if drinking water study): 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 checked in table were 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-test was used for heamatology parameters, and body and organ weights;
Fisher's Exact probability test was used for analysis of tumours;
Chi-Square contingency tables and Fisher's exact propability test was used for survival comparison.

Clinical signs:

effects observed, treatment-related

Dermal irritation (if dermal study):

not examined

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 examined

Food efficiency:

not examined

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

Remarks:

carcinogenicity

Effect level:

94 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

148 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see 'Remark'

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

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

Remarks:

gernal toxicity

Effect level:

16 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

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

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

Reference 4

Endpoint:

carcinogenicity: 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 451 (Carcinogenicity Studies)

GLP compliance:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

No data

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

Dermal irritation (if dermal study):

not examined

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 examined

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:

not specified

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

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.

Reference 5

Endpoint:

carcinogenicity: 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 451 (Carcinogenicity Studies)

GLP compliance:

no

Species:

rat

Strain:

Osborne-Mendel

Sex:

male/female

Route of administration:

oral: drinking water

Duration of treatment / exposure:

110 weeks

Frequency of treatment:

continuously

Dose / conc.:

0 other: % (v/v)

Remarks:

vehicle control

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

Dermal irritation (if dermal study):

not examined

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 examined

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/female

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:

liver

nasal cavity

Carcinogenicity: via inhalation route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: 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

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 (if applicable):

whole body

Vehicle:

clean air

Details on 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.

Dermal irritation (if dermal study):

not examined

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

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.

Reference 2

Endpoint:

carcinogenicity: 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:

Method: A lifetime 2-year study with rats, with a treated and a control group

GLP compliance:

no

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

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on 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

Post exposure period:

none

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, concurrent vehicle

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

Dermal irritation (if dermal study):

not examined

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 effects were observed

Key result

Critical effects observed:

no

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.

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

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.

1,4-dioxane is legally classified as carcinogenic Cat 1B (H350: "May cause cancer") according to Annex VI of Regulation (EC) No 1272/2008 (CLP Regulation).

Additional information

In the literature there are numerous data available on repeated dose toxicity and carcinogenicity 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
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 /m^3)	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/m^3)	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/m^3)	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/m^3)	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/m^3(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

DW – Drinking Water
WB – Whole Body

wt - weight

h – hour
d – day
wk- week
yr- year

m – males

f - females

 

 

Repeated dose toxicity and carcinogenicity: key value for hazard assessment

There are numerous data available on repeated dose toxicity and carcinogenicity 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)

-      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. 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