1,4-dioxane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1,4 -dioxane was clearly negative in all available in vitro mutagenicity studies: Ames test (OECD 471); Mammalian gene mutation assay (OECD 476) and Chromosomal aberration test (OECD 473).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

21 Mar 1979 - 23 Mar 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(neither Escherichia coli strain WP2 uvrA pKM101a nor S. typhimurium strain TA102 tested)

Principles of method if other than guideline:

The study was conducted according to the method described by Ames BN et al. (1975). Mutation Research 31: 347-364.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

His -

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S-9 mix

Test concentrations with justification for top dose:

4, 20, 100, 500, 2500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqua dest.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: With S-9: 2-aminoanthracene (TA100, TA98, TA1538, TA1537, TA1535); without S-9: N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) (TA100, TA98, TA1537, TA1535)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 48 h


NUMBER OF REPLICATIONS: 4 plates


DETERMINATION OF CYTOTOXICITY
- Method: clearing or diminution of the background lawn (= reduced his- background growth)

Evaluation criteria:

Positive results
A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S 9 mix or after adding a  metabolizing system.
A test substance is generally considered nonmutagenic in this test if: The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Statistics:

No statistics were performed.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Evaporation from medium: presumably (up to 40 % test substance over 48 h test period)
- Water solubility: yes
- Precipitation: none

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

20 Mar 1979 - 22 Mar 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(neither Escherichia coli strain WP2 uvrA pKM101a nor S. typhimurium strain TA102 tested)

Principles of method if other than guideline:

The study was conducted according to the method described by Ames BN et al. (1975). Mutation Research 31: 347-364.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

His -

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S-9 mix

Test concentrations with justification for top dose:

4, 20, 100, 500, 2500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqua dest.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: With S-9: 2-aminoanthracene (TA100, TA98, TA1538, TA1537, TA1535); without S-9: N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) (TA100, TA98, TA1537, TA1535)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 48 h


NUMBER OF REPLICATIONS: 4 plates


DETERMINATION OF CYTOTOXICITY
- Method: clearing or diminution of the background lawn (= reduced his- background growth)

Evaluation criteria:

Positive results
A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S 9 mix or after adding a  metabolizing system.
A test substance is generally considered nonmutagenic in this test if: The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Statistics:

No statistics were performed.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Evaporation from medium: presumably (up to 40 % test substance over 48 h test period)
- Water solubility: yes
- Precipitation: none

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14 Mar 1979 - 16 Mar 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(neither Escherichia coli strain WP2 uvrA pKM101a nor S. typhimurium strain TA102 tested)

Principles of method if other than guideline:

The study was conducted according to the method described by Ames BN et al. (1975). Mutation Research 31: 347-364.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

His -

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S-9 mix

Test concentrations with justification for top dose:

4, 20, 100, 500, 2500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqua dest.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: +S9: 2-aminoanthracene (TA100, TA98, TA1538, TA1537, TA1535); -S9: N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) (TA100, TA98, TA1537, TA1535)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 48 h


NUMBER OF REPLICATIONS: 4 plates


DETERMINATION OF CYTOTOXICITY
- Method: clearing or diminution of the background lawn (= reduced his- background growth)

Evaluation criteria:

Positive results
A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S 9 mix or after adding a  metabolizing system.
A test substance is generally considered nonmutagenic in this test if: The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Statistics:

No statistics were performed.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Evaporation from medium: presumably (up to 40 % test substance over 48 h test period)
- Water solubility: yes
- Precipitation: none

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

His -

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain / cell type:

E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 from SD rat liver

Test concentrations with justification for top dose:

0, 156, 313, 625, 1250, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in the presence of S9 for TA1535, TA100, E.coli WP2, and E.coli WP2 urvA pKM101)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

Standard plate and pre-incubation test.
The plates were incubated at 37 degrees Celsius for 2 days and revertant colonies were counted. Triplicate plates were used for treatments and positive controls, five plates were used for vehicle controls.

Evaluation criteria:

Positive results:
A dose-related and reproducible increase in the number of revertant colonies,  either without S 9 mix or after adding a  metabolizing system. In comparison to negative/ and or positive control, using Dunnet's test.

Statistics:

The data were analyzed statistically by Dunnet’s test.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo, there were positive and negative findings observed in both, gene mutation and chromosomal aberration tests.

However, positive results, i. e. genotoxicity, were only observed at dose levels clearly exceeding the linear toxicokinetic range of 1,4-dioxane.

Therefore, and although it cannot be completely excluded that 1,4-dioxane may induce micronuclei via the oral route with a low potency and at high doses only, 1,4-dioxane is considered a non-genotoxic compound based on the total weight of evidence evaluation of all relevant in vivo and in vitro studies.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

July - October 1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test procedure was based on the method described by Roehrborn G and Vogel F (1967). Deutsche Medizinische Wochenschrift 92: 2315-2321

GLP compliance:

no

Type of assay:

rodent dominant lethal assay

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: WIGA, Sulzfeld
- Age at study initiation: approx. 14-16 weeks old
- Weight at study initiation: 28.4 g (mean)
- Assigned to test groups randomly: yes
- Housing: 1 male/cage (except during mating: 1 male and 3 females/cage)
- Diet (ad libitum): Altromin-R (Altromin GmbH, Lage/Lippe)
- Water (ad libitum): tap water


ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 55 ± 5 %
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: aqua bidest.

Details on exposure:

- Applied volume: 10 mL/kg bw

Duration of treatment / exposure:

single administration

Frequency of treatment:

single administration

Post exposure period:

not applicable

Dose / conc.:

2 575 mg/kg bw/day (actual dose received)

Remarks:

corresponding to 2500 µL/kg bw (Recalculation based on relative density = 1.03)

No. of animals per sex per dose:

20

Control animals:

yes, concurrent no treatment

Positive control(s):

none

Evaluation criteria:

Mutagenicity index:
MI = ((dead foetuses + deciduomata + early resorptions) / total implants) x 100

Statistics:

Chi-square-test (* >= 95 %, ** >= 99 %, compared to the untreated control)

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Male mice:

- Clinical signs of toxicity:

The animals showed immediately after i.p. application of the test substance an increase in spontaneous motor function for a period of approx. 15 seconds. Apart from these unspecific symptoms no test substance-related clinical signs were observed. Untreated and vehicle-control animals did not demonstrate any clinical symptoms.

- Body weights:

Treated males showed body weight development comparable to untreated and vehicle-control animals.

- Gross-pathological examination:

At necropsy treated, untreated, and vehicle-control animals did not show any abnormalities.

Female mice:

- Conception rate:

The conception rate varied among test groups. No substance-related alteration could be detected.

- Mutagenicity Index (implants, dead implants):

No substance-related difference between test and control groups could be observed.

Control untreated:

Mating week	Conception rate	Implantations (mean no.)	Living fetuses (%)	Dead fetuses (%)	Deciduomata (%)	Early resorptions (%)	Dead implantations (%)	Corpora lutea	Mutagenicity
1	83.33	12.24	91.67	0.65	7.68	0.00	8.33	0.0	8.33
2	88.33	11.89	87.62	0.48	10.95	0.95	12.38	0.0	12.38
3	93.33	12.91	91.42	0.55	7.88	0.14	8.58	0.0	8.58
4	93.33	13.00	89.70	0.82	9.48	0.00	10.30	0.0	10.30
5	88.33	12.72	88.58	1.04	10.09	0.30	11.42	0.0	11.42
6	88.33	13.47	90.34	0.14	9.10	0.42	9.66	0.0	9.66
7	90.00	13.46	89.27	0.55	10.18	0.00	10.73	0.0	10.73
8	81.67	13.47	88.48	1.06	8.48	1.97	11.52	0.0	11.52

Control water:

Mating week	Conception rate	Implantations (mean no.)	Living fetuses (%)	Dead fetuses (%)	Deciduomata (%)	Early resorptions (%)	Dead implantations (%)	Corpora lutea	Mutagenicity
1	90.00	12.46	90.04	1.49	8.32	0.15	9.96	0.0	9.96
2	91.67	13.49	91.24	0.94	7.82	0.00	8.76	0.0	8.76
3	95.00	13.81	87.93	0.89	11.18	0.00	12.07	0.0	12.07
4	100.00	13.63	89.49	0.61	9.66	0.24	10.51	0.0	10.51
5	95.00	13.25	90.46	0.79	8.74	0.00	9.54	0.0	9.54
6	95.00	14.07	92.14	0.12	7.73	0.00	7.86	0.0	7.86
7	93.33	13.71	89.71	0.52	9.77	0.00	10.29	0.0	10.29
8	91.67	13.18	93.93	0.28	5.79	0.00	6.07	0.0	6.07*

* Significant

Treatment group: 2500 µL/kg b.w. Dioxane

Mating week	Conception rate	Implantations (mean no.)	Living fetuses (%)	Dead fetuses (%)	Deciduomata (%)	Early resorptions (%)	Dead implantations (%)	Corpora lutea	Mutagenicity
1	85.00	11.80	90.37	0.33	9.14	0.17	9.63	0.0	9.63
2	88.33	13.09	89.63	0.72	9.65	0.00	10.37	0.0	10.37
3	90.00	13.44	90.63	0.14	9.23	0.00	9.37	0.0	9.37
4	96.67	13.57	90.98	0.51	8.51	0.00	9.02	0.0	9.02
5	93.33	13.46	93.24	0.13	6.63	0.00	6.76	0.0	6.76*
6	93.33	12.84	91.38	0.70	7.93	0.00	8.62	0.0	8.62
7	95.00	13.12	89.97	0.13	9.89	0.00	10.03	0.0	10.03
8	86.67	13.56	89.79	0.85	9.36	0.00	10.21	0.0	10.21

* Significant

 

 

Reference 2

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Remarks:

gpt delta transgenic F344 rat

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

gpt delta transgenic F344 rats as well as wildetype F344 rats were administered 1,4-dioxane at different doses in the drinking water for 16 weeks.

GLP compliance:

not specified

Type of assay:

transgenic rodent mutagenicity assay

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

gpt delta transgenic and wildtype

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: transgenic animals: Japan SLC Inc.; wildtype animals: Charles River Japan, Inc.
- Age at study initiation: 6 weeks
- Fasting period before study: no
- Housing: polycarbonate cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C
- Humidity: 55 +/-5 %
- Air changes: not specified
- Photoperiod: 12 / 12 hrs dark / hrs light

Route of administration:

oral: drinking water

Vehicle:

- Vehicle/solvent used: distilled water

Details on exposure:

- fresh distilled water containing the test item was changed three times a week

Duration of treatment / exposure:

daily

Frequency of treatment:

16 weeks

Post exposure period:

not applicable

Dose / conc.:

0 ppm

Remarks:

Experiment 1; control group

Dose / conc.:

200 ppm

Remarks:

Experiment 1
corresponding to 18.7 mg/kg bw/d

Dose / conc.:

1 000 ppm

Remarks:

Experiment 1
corresponding to 92.3 mg/kg bw/d

Dose / conc.:

5 000 ppm

Remarks:

Experiment 1
corresponding to 440.2 mg/kg bw/d

Dose / conc.:

0 ppm

Remarks:

Experiment 2; control group

Dose / conc.:

0.2 ppm

Remarks:

Experiment 2
corresponding to 0.02 mg/kg bw/d

Dose / conc.:

2 ppm

Remarks:

Experiment 2
corresponding to 0.2 mg/kg bw/d

Dose / conc.:

20 ppm

Remarks:

Experiment 2
corresponding to 1.9 mg/kg bw/d

Dose / conc.:

0 ppm

Remarks:

Experiment 3; control group

Dose / conc.:

2 ppm

Remarks:

Experiment 3

Dose / conc.:

20 ppm

Remarks:

Experiment 3

Dose / conc.:

200 ppm

Remarks:

Experiment 3

Dose / conc.:

2 000 ppm

Remarks:

Experiment 3

Dose / conc.:

5 000 ppm

Remarks:

Experiment 3

No. of animals per sex per dose:

Experiment 1
Dose group number of animals (gpt delta transgenic)
0 8 males
200 7 males
1000 7 males
5000 8 males

Experiment 2
5 gpt delta transgenic males per dose group

Experiment 3
30 wildtype males per dose group

Control animals:

yes, concurrent vehicle

Positive control(s):

not applicable

Tissues and cell types examined:

liver

Details of tissue and slide preparation:

Experiment 1, 2 and 3:
At necropsy, livers were excised and weighed. A total of 3 sections of liver tissue (one section each from the left lateral lobe, right middle lobe, and caudate lobe) were fixed in phosphate buffered formalin, embedded in paraffin, and processed for hematoxylin/eosin and immunohistochemical staining. The remaining liver tissues were snap frozen with liquid nitrogen and stored at − 80 °C for mutation assays, oxidative DNA damage, and gene expression analyses.

In addition,for Experiment 3: One hour prior to sacrifice, ten rats from each group were injected i.p. with 100 mg BrdU/kg body weight for evaluation of cell proliferative activity in their livers.

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

yes

Remarks:

reduced body weight and relative liver weight increase (at 5000 ppm); histopathological changes in the liver (hypertrophy, swelling, necrosis, apoptosis, or fatty changes)

Vehicle controls validity:

not specified

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: Experiment 1

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not specified

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: Experiment 2

Key result

Sex:

male

Genotoxicity:

not determined

Toxicity:

yes

Remarks:

body weight decrease at 5000 ppm

Vehicle controls validity:

not specified

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: Experiment 3

The overall mutation frequency (MF) and A:T- to -G:C transitions and A:T- to -T:A transversions in the gpt transgene were significantly increased by administration of 5000 ppm 1,4 -dioxane (experiment 1).

A:T- to -T:A transversions were also significantly increased by administration of 1000 ppm 1,4 -dioxane (experiment 1).

Furthermore, the DNA repair enzyme MGMT was significantly induced at 5000 ppm 1,4-dioxane, implying that extensive genetic damage exceeded the repair capacity of the cells in the liver and consequently led to liver carcinogenesis (experiment 1).

No evidence supporting other MOAs, including induction of oxidative stress, cytotoxicity, or nuclear receptor activation, that could contribute to the carcinogenic effects of 1,4-dioxane were found.

Table 1 gpt transgene MFs and induction of GST-P positive foci in the livers of gpt delta transgenic F344 rats administered high doses of 1,4-dioxane for 16 weeks (Experiment 1; n=8 for 0 and 5000 ppm group; n=7 for 200 and 1000 ppm group)

1,4-dioxane (ppm)	Average MF (x10^-5)	GST-P positive foci (No./cm^2)
0	0.43 +/- 0.26	1.3 +/- 1.4
200	0.65 +/- 0.31	1.6 +/- 1.0
1000	0.79 +/- 0.29	2.5 +/- 1.5
5000	1.32 +/- 0.71^a	13 +/- 5.5^b

^aSignificantly different from the control group (0 ppm) at p < 0.05

^bSignificantly different from the control group (0 ppm) at p < 0.001

Reference 3

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

Method:
- according to Butterworth, B.E. et al. (1987). A protocol and guide for the in vivo rat hepatocyte DNA repair assay. Mutation Research 189, 123-133 (hepatocyte DNA-repair assay)
- according to Bermudez and Allen (1984). The assessment of DNA damage and repair in rat nasal epithelial cells. Carcinogenesis 5: 1453-1458 (respiratory epithelial cells assay)

GLP compliance:

not specified

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Lab. Inc. Raleigh NY
- Age at study initiation: 10-12 weeks
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72+/- 2
- Humidity (%): 50 +/- 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

other: oral: drinking water in combination with gavage

Vehicle:

water

Duration of treatment / exposure:

- liver hepatocytes: a single application of 1000 mg/kg bw or treatment with 1% 1,4-dioxane in drinking water for 2 weeks or treatment with 2% 1,4-dioxane for 1 week
- respiratory epithelial cells: 1% 1,4-dioxane in drinking water for 8 days, or 1% in drinking water for 8 days with an additional single gavage dose of 0, 10, 100 and 1000 mg/kg 12 h prior to sacrifice

Frequency of treatment:

continuous and once by gavage

Dose / conc.:

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

Remarks:

1% in drinking water with an additional single gavage

Dose / conc.:

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

Remarks:

1% in drinking water with an additional single gavage

Dose / conc.:

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

Remarks:

1% in drinking water with an additional single gavage

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

1% in drinking water with an additional single gavage

Dose / conc.:

1 other: %

Remarks:

concentration in drinking water

Dose / conc.:

2 other: %

Remarks:

concentration in drinking water

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Positive control(s):

- hepatocyte DNA repair assay: DMN and 2-AAF
- nasal epithelial cell DNA repair assay: MMS

Tissues and cell types examined:

liver hepatocytes and nasal epithelial cells

Evaluation criteria:

Labelled cells were recognized by by the dense pattern of silver grains overlying the nuclei, cells with a nuclei density of >= 10 were scored as undergoing replicative DNA-synthesis. The labeling index(LI) was calculated by dividing the number of labeled nuclei by the total counted.


The autoradiograms were stained and scored for the number of grains over the nucleus for 100 cells.

Statistics:

for the hepatocytes : P <= 0.05 by the Newman-Keuls multicomparison test.
for the nasal epithelium: unpaired t-test at significance level of P<= 0.05

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Neither a single application of 1000 mg/kg bw, nor treatment with 1% 1,4-dioxane in drinking water for 2 weeks or with 2% 1,4-dioxane for 1 week, induced unscheduled DNA synthesis in primary rat hepatocytes. Negative results for unscheduled DNA synthesis were also found in rat nasal respiratory epithelial cells (from the nasoturbinate or the maxilloturbinate) after treatment of rats with 1% 1,4-dioxane in drinking water for 8 days, or after treatment with 1% in the drinking water for 8 days with an additional single gavage dose of up to 1000 mg/kg bw 1,4-dioxane.

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

bone marrow and liver cell micronucleus (cytogenicity) and in vivo mutagenicity in Pig-a gene mutation assay in peripheral blood

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:

no guideline followed

Principles of method if other than guideline:

The ability of 1,4-dioxane to induce micronuclei in the liver and bone marrow of rats was investigated.
The juvenile animal method and two methods using partial hepatectomy (PH), dosing before PH or dosing after PH were applied for the liver micronucleus test.

In vivo mutagenicity of 1,4-dioxane was also studied by Pig-a gene mutation assay using rat peripheral blood.

GLP compliance:

not specified

Type of assay:

other: bone marrow and liver cell micronucleus (cytogenicity) and in vivo mutagenicity in Pig-a gene mutation assay in peripheral blood

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc. (Yokohama, Japan)
- Age at study initiation: 4 - 8 weeks (liver micronucleus); 6 - 7 weeks (bone marrow micronucleus and Pig-a assay)
- Assigned to test groups randomly: not specified
- Fasting period before study: not specified
- Housing: Econ PC cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1week

ENVIRONMENTAL CONDITIONS
- Temperature: 23 +/- 3 °C
- Humidity: 30-70 %
- Air changes: not specified
- Photoperiod: 12 / 12 hrs dark / hrs light

Route of administration:

oral: unspecified

Vehicle:

- Vehicle/solvent used: Water

Duration of treatment / exposure:

Bone marrow MNT: Once on day 1

Liver MNT:
Juvenile rat method: Dosing on day 1 and 2
Dosing before partial hepatectomy (PH) method: Once on day 1 (PH on day 2)
Dosing after PH method: Once on day 1 (PH on day -1)

Pig-a Mutation assay: Once on day 1 only

Frequency of treatment:

once a day

Post exposure period:

Bone marrow MNT: treatment on day 1; bone marrow sampling on day 3

Liver MNT:
Juvenile rat method: treatment on day 1 and 2; liver sampling on day 6
Dosing before partial hepatectomy (PH) method: treatment on day 1 (PH on day 2); liver sampling on day 6 (4 days after PH)
Dosing after PH method: treatment on day 1 (PH on day -1); liver sampling on day 4 (4 days after PH)

Pig-a Mutation assay: treatment on day 1; blood sampling on days 15 and 30

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Dose / conc.:

3 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Bone marrow MNT: 5 males per dose group

Juvenile rat method: 4 males per dose group
Dosing before partial hepatectomy (PH) method: 4 males per dose group
Dosing after PH method: 4 males per dose group

Pig-a Mutation assay: 5 males per dose group

Control animals:

yes

Positive control(s):

Bone marrow MNT: Cyclophosphamide (CP)

Juvenile rat and PH methods: diethylnitrosamine (DEN) & carbendazim

Pig-a Mutation assay: 7,12-dimethylbenz[a]anthracene (DMBA)

Tissues and cell types examined:

liver, bone marrow and peripheral blood

Evaluation criteria:

not specified

Statistics:

Statistical analyses were performed by EXSUS Ver. 7.7 with significance level at 5%.

In the liver micronucleus tests (juvenile rat and PH methods), the incidence of MNH and dose dependency across all groups including the vehicle control group were analyzed by two-tailed Fisher’s exact test and two-tailed Cochran-Armitage trend test, respectively.

The relative liver weight was analyzed by a tree-type algorithm for quantitative data.

In the bone marrow micronucleus test, the incidence of MNIE and dose dependency across all groups including the vehicle control group were analyzed by two-tailed Fisher’s exact test and two-tailed Cochran-Armitage trend test, respectively. For the percentage of IE, two-tailed Wilcoxon’s rank sum test was used. The body weight was analyzed by two-tailed Dunnett’s test.

In the Pig-a assay, the incidence of CD59 negative red blood cells was analyzed by Bartlett’s test to evaluate the homogeneity of variance. The parameter was further analyzed by a parametric Dunnett’s test when the variance was homogeneous or by a Steel’s (nonparametric Dunnett’s) test when it was not.

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

no effects

Vehicle controls validity:

not specified

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Liver micronucleus test by juvenile rat method

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

no effects

Vehicle controls validity:

not specified

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: liver micronucleus test by both PH methods

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

decreased body weight at 3000 mg/kg bw/d

Vehicle controls validity:

not specified

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: bone marrow micronucleus

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not specified

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Pig-a assay

Reference 5

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

GLP compliance:

no

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10-16 weeks
- Weight at study initiation: within +/-2 gm of a common weight

ENVIRONMENTAL CONDITIONS
No data

Route of administration:

intraperitoneal

Vehicle:

saline (phosphate buffered saline)

Duration of treatment / exposure:

- three daily injections
- single injection

Frequency of treatment:

once daily

Post exposure period:

- three daily injections: 24 hrs after the last injection
- single injection: 24 and 48 hrs after the last injection

Dose / conc.:

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

Remarks:

ip injected
(three daily injections)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

ip injected
(three daily injections)

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

ip injected
(three daily injections)

Dose / conc.:

2 000 mg/kg bw (total dose)

Remarks:

ip injected (single injection)

Dose / conc.:

3 000 mg/kg bw (total dose)

Remarks:

ip injected (single injection)

Dose / conc.:

4 000 mg/kg bw (total dose)

Remarks:

ip injected (single injection)

No. of animals per sex per dose:

n=5 and n=6

Control animals:

yes

Positive control(s):

mitomycin C

Tissues and cell types examined:

bone marrow erythrocytes

Details of tissue and slide preparation:

Bone marrow smears were stained in acridine orange and examined under fluorescent microscopy.

Evaluation criteria:

Two observers each scored 1000 PCEs from each mouse on separate, coded slides, and determined the proportion of PCEs among 200 randomly selected erythrocytes. Data for the number of micronucleated PCEs were analyzed by a 1-tailed trend test (Margolin et al ., 1986), while values for the proportion of PCEs in the erythrocyte population were subjected to analysis of variance . Pairwise comparisons between each treatment group and their concurrent controls were also made using the t-test.

Statistics:

Statistical significance was set at an alpha level of 0 .05 but to adjust for multiple statistical analyses, i.e., a trend analysis and multiple pairwise comparisons, a p value of 0.025 is required for significance in the trend test and 0 .008 is required for significance of a pairwise comparison when three dosed groups are included in a trial.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

the ratio PCE/NCE was decreased

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Reference 6

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

GLP compliance:

no

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

other: C57BL6 and Balb/c

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female C57BL6 mice and male BALB/c mice were used and maintained as described earlier (Ashby and Mirkova, 1987).

Route of administration:

oral: gavage

Vehicle:

distilled water

Duration of treatment / exposure:

once

Frequency of treatment:

once

Post exposure period:

24 or 48 hrs after treatment

Dose / conc.:

450 mg/kg bw (total dose)

Dose / conc.:

900 mg/kg bw (total dose)

Dose / conc.:

1 800 mg/kg bw (total dose)

Dose / conc.:

3 600 mg/kg bw (total dose)

Dose / conc.:

5 000 mg/kg bw (total dose)

No. of animals per sex per dose:

0, 3600 mg/kg: males 10 per dose (24 hr sampling time)
0, 5000 mg/kg: females 10 per dose (5 for 24 hr sampling time and 5 for 48 hr sampling time)
0, 450, 900, 1800, 3600 mg/kg, 24 hr sampling time: males 10 per dose (for 3600 mg/kg 48 hr sampling time 5 males)
0, 900, 1800, 3600 mg/kg, 24 hr sampling time: males 4 per dose (for 3600 mg/kg 48 hr sampling time 4 males)

Control animals:

yes

Positive control(s):

cyclophosphamide

Tissues and cell types examined:

bone marrow erythrocytes

Evaluation criteria:

Bone marrow smears were prepared and assessed for the incidence of micronucleated polychromatic erythrocytes (MPE) among polychromatic
erythrocytes (PE) 24 h and 48 h after dosing as described in detail earlier (Ashby and Mirkova, 1987). 2000 PE were assessed for MPE per animal.

Statistics:

one-sided Student 's t- test

Key result

Sex:

male/female

Genotoxicity:

negative

Remarks:

Balb/c mice

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Sex:

male/female

Genotoxicity:

positive

Remarks:

C57BL6 mice

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

In addition, the authors describe a negative result when using 5000 mg/kg for Balb/c mice (a dose at which death occured 1/6).

Reference 7

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

no

Type of assay:

mammalian bone marrow chromosome aberration test

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan.
- Age at study initiation: 9 weeks
- Weight at study initiation: 32.6 - 39.7 gram
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 1 week

ENVIRONMENTAL CONDITIONS
No data

Route of administration:

intraperitoneal

Vehicle:

saline for injection

Duration of treatment / exposure:

96 hours

Frequency of treatment:

animals were dosed twice (24 hr apart)

Dose / conc.:

500 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

2 000 mg/kg bw (total dose)

Dose / conc.:

3 200 mg/kg bw (total dose)

No. of animals per sex per dose:

5 males per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C

Tissues and cell types examined:

peripheral erythocytes

Details of tissue and slide preparation:

Peripheral blood preparations were made just before treatment and 24, 48, and 72 h after the second treatment, using a supravital acridine orange staining method.

Evaluation criteria:

not reported

Statistics:

Kastenbaum and Boman (1970). Tables for determining the statistical significance of mutation frequencies. Muta. Res. , 9; 527-549.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

one highest dose (3200 mg/kg) male died after 72 hrs

Reference 8

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

GLP compliance:

no

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan (Indianapolis, Indiana, USA)
- Age at study initiation: 21 days
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22–25
- Humidity (%): 50–54%,
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Duration of treatment / exposure:

once per day

Frequency of treatment:

5 days

Post exposure period:

24 hr after the last exposure

Dose / conc.:

1 500 mg/kg bw/day (actual dose received)

Dose / conc.:

2 500 mg/kg bw/day (actual dose received)

Dose / conc.:

3 500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 per group

Control animals:

yes

Positive control(s):

vinblastine sulphate (ip injection)

Tissues and cell types examined:

hepatocytes and bone marrow erythrocytes

Evaluation criteria:

The frequency of micronucleated eryhtrocytes among total erythrocytes were (2000/animal) was determined from the CREST stained bone-marrow slide. The proliferation-index was calculated as the number of nuclei incorporating BrdU divided by the total nuclei counted. Dose-related increases in the frequency of micronucleated eryhtrocytes and hepatocytes induced by the test chemicals were determened using ANOVA or regression analysis on the transformated data.

Statistics:

Following a positive result, Fisher's protected least significant difference (PLSD) was used as a post-hoc test.

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Significant dose-related increases in micronuclei were seen in both the liver and the bone-marrow with significant increases being detected at all the tested doses in the bone marrow and at the 2500 and 3500 mg/kg doses in the liver. Using CREST staining or pancentromeric FISH to determine the origin of the induced micronuclei, it was determined that 80-90% of the micronuclei in both tissues originated from chromosomal breakage. Small increases in centromere-containing micronuclei were also seen in the hepatocytes. Decreases in hepatocyte proliferation as well as in the ratio of bone marrow PCE:NCE were also observed.

Reference 9

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

GLP compliance:

no

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

other: C57BL6 and CBA

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8-12 weeks
no further data

ENVIRONMENTAL CONDITIONS
no data

Route of administration:

oral: gavage

Vehicle:

distilled water

Duration of treatment / exposure:

once

Frequency of treatment:

once

Post exposure period:

24 hours

Dose / conc.:

1 800 mg/kg bw (total dose)

Dose / conc.:

3 600 mg/kg bw (total dose)

No. of animals per sex per dose:

CBA mice: 1800 mg/kg: 12 animals
C57BL6: 3600 mg/kg: 4 animals

Control animals:

yes

Positive control(s):

cyclophosphamide

Tissues and cell types examined:

bone marrow erythrocytes

Details of tissue and slide preparation:

Bone marrow smears were prepared as described previously (Tinwell and Ashby, 1989). As the present studies were performed during our transition from the use of Giemsa to acridine orange (AO) as our stain of choice for bone marrow smears, one study was analysed using Giemsa stained slides.

Evaluation criteria:

The incidence of micro nucleated polychromatic erythrocytes (MPE) was determined among 2000 PE for each animal . The ratio of PEs to normocytes (PE/NE) was based on 1000 erythrocytes per smear .

Statistics:

one-sided Students t-test

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

ratio PCE/NCE was decreased in CBA mice

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Reference 10

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

Method: according to Hayashi et al., 1990 (periphal blood), Igarashi, Shimada, 1994 (liver)

GLP compliance:

no

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Japan, Inc . (T'sukuba, Japan),
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 30.3-38.5 g)
- Assigned to test groups randomly: five animals were randomly allocated to each group except for the positive control group, which had four animals
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
no data

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: physiol. saline

Duration of treatment / exposure:

once

Frequency of treatment:

once

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

2 000 mg/kg bw (total dose)

Dose / conc.:

3 000 mg/kg bw (total dose)

No. of animals per sex per dose:

5/dose

Control animals:

yes

Positive control(s):

mitomycin C

Tissues and cell types examined:

peripheral blood and liver

Evaluation criteria:

The selection and scoring of hepatocytes for micronucleated hepatocytes was according to published criteria [Braithwaite and Ashby,1988; Cliet et al ., 1989]. For the peripheral blood micronucleus assay the results were analyzed with Kastenbaum and Bowman's [1970] table.

Statistics:

Not reported.

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: liver

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: peripheral blood

Additional information on results:

The blood micronucleus assay was negative. The mouse liver micronucleus assay, on the other hand, was positive.

Table 1 Liver MN-test

Dose (mg/kg bw)	Hepatocytes with MN/2000 hepatocytes
0	0.43 +/- 0.14
1000	0.49 +/- 0.19
2000	1.02 +/- 1.04*
3000	1.24 +/- 0.33*
Mitomycin C	2.65 +/- 1.80*

(*p < 0.01)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro studies

Bacterial reverse mutation assay (Ames test)

There are four valid Ames tests available with 1,4-dioxane that are considered reliable and sufficient for evaluation (BASF AG, 1997a, b, c; Morita 1998). Table 1 summarises respective test conditions and results.

Table 1. Summary of Ames tests with 1,4 -dioxane

Strains tested	Concentrations tested	Vehicle	Metabolic activation (S9)	Outcome	Source
S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 1538	4, 20, 100, 500, 2500 µg/plate	Aqua. Dest.	With and without	Negative	BASF AG, 1997a
S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 1538	4, 20, 100, 500, 2500 µg/plate	Aqua. Dest.	With and without	Negative	BASF AG, 1997b
S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 1538	4, 20, 100, 500, 2500 µg/plate	Aqua. Dest.	With and without	Negative	BASF AG, 1997c
S. typhimurium TA 1535, TA 1537, TA 98, TA 100,   E. coli WP2,  WP2 uvr A pKM 101	0, 156, 313, 625, 1250, 2500, 5000 µg/plate	Water	With and without	Negative	Morita, 1998

 

In summary, all four bacterial reverse mutation tests revealed negative results.

Gene mutation in mammalian cells (HPRT)

A test on gene mutation in mammalian cells according to OECD TG 476 was conducted with 1,4-dioxane applying concentrations ranging from 0.05 to 10 mg/mL with and without metabolic activation. Under the conditions of this assay, 1,4-dioxane was found to be non-mutagenic.

Chromosome aberration

1,4-dioxane was negative in an in vitro chromosome aberration assay conducted in Chinese hamster ovary cells with and without metabolic activation system at concentrations ranging from 1050 to 10500 µg/mL (Galloway, 1987).

 

In vivo

A rodent dominant lethal test in mice was conducted with 1,4-dioxane applying a single dose of 2575 mg/kg bw via intraperitoneal injection (BASF AG, 1991). Under the conditions of this study no germ cell mutagenicity could be detected.

An unscheduled DNA synthesis test in male rats administered 1,4-dioxane orally by gavage with dose levels ranging from 10 to 1000 mg/kg bw/d and additionally exposed via drinking water at a concentration of 1 % revealed no positive findings in both liver hepatocytes and nasal epithelial cells (Goldsworthy et al., 1991).

In a transgenic rodent mutagenicity assay somatic gene mutation activity of 1,4-dioxane was investigated (Gi et al., 2018). Male gpt delta transgenic rats were administered 1,4-dioxane at doses ranging from 0.2 to 5000 ppm in drinking water for 16 weeks. Under the conditions of this study indications of mutagenicity were observed at doses levels of 2000 and 5000 ppm in drinking water.

Mutation of Pig-A gene in peripheral blood of male Fischer 344 rats was investigated after single oral exposure with 1,4-dioxane at doses of 1000, 2000, 3000 mg/kg bw/d (Itoh &Hattori, 2019). Under the conditions of this study, no indications for mutagenicity were observed.

 

Micronucleus tests

There are numerous in vivo micruncleus tests (MNT) available conducted with 1,4-dioxane. Only relevant and reliable studies are included and discussed in this dossier. MNT test conditions and results are summarized in table 2.

Table 2. Summary of in vivo Micronucleus Tests (MNT) conditions and results.

Species	Dose level (mg/kg bw)	Vehicle	Route & frequency of exposure	Outcome (tissue examined)	Source
Rat (Fischer 344)	1000, 2000, 3000	water	Oral (once)	Positive (liver)   Negative (bone marrow)	Itoh & Hattori, 2019
Mouse (B6C3F1)	500, 1000, 2000, 3000, 4000	Saline (PBS)	i.p. (once or daily for 3 days)	Negative (bone marrow)	McFee et al., 1994
Mouse (C57BL6)	450, 900, 1800, 3600, 5000	Dist. water	Oral (once)	Positive (bone marrow)	Mirkova, 1994
Mouse (BALB/c)	450, 900, 1800, 3600, 5000	Dist. water	Oral (once)	Negative (bone marrow)
Mouse (C57BL6 & CBA)	1800, 3600	Dist. water	Oral (once)	Negative (bone marrow)	Tinwell & Ashby, 1994
Mouse (CD-1)	500, 1000, 2000, 3200	saline	i.p. (daily for 2 days)	Negative (peripheral blood)	Morita, 1994
Mouse (CD-1)	1500, 2500, 3500	n. a.	Oral (daily for 5 days)	Positive (bone marrow)   Positive (hepatocytes)	Roy et al., 2005
Mouse (CD-1)	1000, 2000, 3000	saline	Oral (once)	Negative (peripheral blood)   Positive (liver)	Morita, 1998

 

Discussion

Gi et al. (2018) reported indications for a mutagenic potential of 1,4-dioxane under the conditions of the TRM study conducted. It is noted that the treatment period of 16 weeks applied in this study is unusually long for this type of study and it raises concerns of false positive findings. In particular, according to the validated OECD test guideline 488 (2013) treatment periods longer than 8 weeks in transgenic rodent models “may produce an apparent increase in mutant frequency through clonal expansion” (OECD TG 488, 2013; Thybaud et al., 2003).
Further, it is noteworthy that the two highest doses applied at which positive findings were obtained only, i. e. 2000 and 5000 ppm (equivalent to approx. 176 and 440 mg/kg bw/d) exceeded the linear toxicokinetic range of 1,4-dioxane in rats by far (please also refer to IUCLID section 7.1). The OECD TG 488 states that “chemicals which exhibit saturation of toxicokinetic properties may be exceptions to the dose-setting criteria and should be evaluated on a case-by-case basis”. Therefore, positive results obtained above the toxicokinetic linearity must be treated with caution.

Itoh & Hattori (2019) reported positive findings in the liver micronucleus test conducted in rats applying the juvenile rat method as well as two different partial hepatectomy methods. As already discussed above, partial hepatectomy may display an additional primary event cofounding genotoxicity findings by means of induction of regenerative cell proliferation. Hepatectomy is thus considered an abnormal physiological condition which might lower the informative value of the study´s results.
Further, it is noted that again all three study designs applied doses being far beyond the toxicokinetic linear range. Though it is recognized that only single or two time treatments were conducted, the dose levels administered highly likely led to over proportional high internal doses. This in turn, questions the value of the resulting findings at the dose levels tested.

Mirkova (1994) reported increased micronuclei findings in bone marrow of one (C57BL6) but not in another (BALB/c) mouse strain exposed to the same dose levels of 1,4-dioxane and tested under the same conditions. Tinwell & Ashby (1994) could not re-produce this finding when testing 1,4-dioxane in the same mouse strain at same dose levels in a bone marrow micronucleus test applying same sampling times. Against this background, the positive finding reported by Mirkova is regarded as rather equivocal.

Regarding the results of Roy et al. (2005) in CD-1 mice, it should be recognized that particularly high doses were applied (1500, 2500, and 3500 mg/kg bw/d) for five consecutive days exceeding the normally used maximum dose of 2000 mg/kg bw for these type of studies. Furthermore, the effect appeared to be stronger in the study due to the low background of very young mice used (only 3 weeks old). Therefore, the result of this study is questionable.

In the oral MNT study by Morita (1998), negative effects were observed in blood, while positive effects were observed in the liver. As partial hepatectomy was performed, the positive results in the liver may have been caused by another primary stimulus. Partial hepatectomy will cause proliferation in the liver and is considered an abnormal physiological condition. The overall test result of the Morita study therefore has to be considered as negative.

Overall, it is noteworthy that in vivo micronucleus studies using the i. p. route (McFee et al., 1994; Morita, 1994 (B6C3F1 and CD-1 mice)) showed clearly negative results.  

Conclusion

Based on in vitro data, there are no indications for a genotoxic potential of 1,4-dioxane. In vivo studies revealed both positive and negative results in regards to point mutation as well as cytogenicity.

Where the negative results obtained in in vivo studies are consistent with in vitro results, it is noteworthy that positive results, were found at high dose levels only. Considering that 1,4-dioxane is known to follow a non-linear toxicokinetic and its elimination becomes saturated at doses of approx. 9.6 – 42 mg/kg bw/d in rats and 57 – 66 mg/kg bw/d in mice (Dourson et al., 2017; IUCLID Section 7.1 & 7.12), any effects observed beyond this kinetic MTD must be regarded to be of questionable relevance for the assessment of 1,4-dioxane. Moreover, respective study designs did not always follow generally agreed methods, i. e. exposure duration or dose levels were exceeded where positive findings were obtained.

It can be concluded that the weight of scientific evidence supports that 1,4-dioxane is not mutagenic. It is noted that exposure to 1,4-dioxane may elicit clastogenicity in vivo at high doses exceeding the linear range of toxicokinetics.

 

References not provided as RSS in this dossier:

- OECD TG 488, OECD Guidelines for the testing of chemicals: Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays, 2013

- Thybaud, V., S. Dean, T. Nohmi, J. de Boer, G.R. Douglas, B.W. Glickman, N.J. Gorelick, J.A. Heddle, R.H. Heflich, I. Lambert, H.-J. Martus, J.C. Mirsalis, T. Suzuki and N. Yajima (2003), “In vivo Transgenic Mutation Assays”, Mutation Res., 540: 141-151.

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. Based on the total weight of evidence of all available data, classification as a genotoxic substance is not triggered according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.