4,4'-oxydianiline

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Several data on this substance are available in literature.

A bacterial reverse mutation assay (Ames tests), a Chromosome aberrations test, a Sister Chromatid Exchange test and a Mouse Lymphoma assay have an acceptable reliability to allow the assessment of the substance.

All these tests showed clear positive results.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

Non-GLP literature study in 27 different chemicals. Methodology is detailed, as are results per substance, and well documented. Study addresses both chromosal aberrations and sister chromatid exchanges.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Deviations:

yes

Remarks:

See below for methodology

Principles of method if other than guideline:

Approximately 24 hours prior to cell treatment, 1x106 cells were seeded per 75 cm2 flask. A culture was established for each dose both with and without exogenous metabolic activation.
For assays without metabolic activation, the medium was replaced with fresh medium immediately before treatment with the test substance. Cells were treated with test or control substances for 2 hours to allow interaction with cells before the addition of bromodeoxyuridine (BrdUrd). BrdUrd was then added, and incubation was continued for an additional 24 hours. The medium was removed, and fresh medium containing BrdUrd and colcemid was added and incubation was continued for 2-3 hours. For assays with metabolic activation, the cells were rinsed twice, after which culture medium without fetal bovine serum (FBS) was added. Cells were incubated for 2 hours in the presence of the test or control substance and the S-9 reaction mixture. FBS was omitted to avoid the binding of serum proteins to short-lived, highly reactive intermediates.
After the 2 hour exposure period, cells were washed twice, and then complete medium containing 10% FBS and 10 pM BrdUrd was added was added. Cells were incubated for an additional 26 hours, with colcemid present for the final 2-3 hours of incubation.

Two to 3 hours after addition of colcemid, cells were harvested by mitotic shake-off. Prior to harvesting, the percent confluency in each flask was estimated using a widefield microscope. Harvested cells were treated for about 3 minutes at room temperature with hypotonic KCl, washed with fixative, dropped onto slides, air dried. Staining for the detection of SCE was accomplished by a modified fluorescence plus Giemsa (FPG) technique [Goto et al., 19781. Fifty seconddivision metaphase cells were scored per dose for the incidence of SCE. The number of chromosomes in each cell was also recorded. Any cell that had fewer than 19 or more than 23 chromosomes was excluded. All slides except for the high-dose positive controls were coded.

Repeat Tests
Positive results in initial tests were confirmed by additional tests. If both -S9 and +S9 studies gave a positive response and required confirmation, they were done sequentially (-S9 first). If the -S9 repeat was positive, the repeat +S9 study was not always performed.

GLP compliance:

no

Type of assay:

sister chromatid exchange assay in mammalian cells

Specific details on test material used for the study:

4,4'-oxydianiline
4,4'-diaminodiphenyl ether
CAS 101-80-4

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO cells, up to 15 passages since cloning, were used for all testing. Stock cells were obtained from Litton Bionetics (Kensington, MD) and stored in liquid nitrogen. At least once per year representative cells were sent to Flow Laboratories (McLean, VA) for mycoplasma testing using the Hoechst stain test followed by the Agar and Hyorhinis test. Results from all tests for mycoplasma contamination were negative. CHO cells were maintained in McCoy's 5A medium (modified) supplemented with L-glutamine (2 mM), antibiotics, and 10% fetal bovine serum (FBS). Pre-mixed culture medium and FBS were purchased from GIBCO Laboratories (Grand Island, NY). All stock and experimental cultures were maintained at 37°C in an atmo¬sphere of 5% C02 in air and 95% relative humidity.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Liver fraction (S9) prepared from Aroclor 1254-induced male Sprague Dawley rats. The final concentrations of the S9 fraction, NADP, and isocitric acid were 0.02 ml, 2.4 mg, and 4.5 mg, respectively, per milliliter culture medium

Test concentrations with justification for top dose:

Sister Chromatid Exchange without activation: 0, 5, 16, 50 μg/mL
Sister Chromatid Exchange with activation: 0, 160, 500, 1600, 2000, 3000, 4000, 5000 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Not specified; assumed to be based on historical use.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Conducted concurrently

True negative controls:

no

Positive controls:

yes

Remarks:

Conducted concurrently

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
Approximately 24 hours prior to cell treatment, 1x10E6 cells were seeded per 75 cm2 flask. A culture was established for each dose both with and without exogenous metabolic activation. For assays without metabolic activation, the medium was replaced with fresh medium immediately before treatment with the test substance. Cells were treated with test or control substances for 2 hours to allow interaction with cells before the addition of bromodeoxyuridine (BrdUrd). BrdUrd was then added, and incubation was continued for an additional 24 hours. The medium was removed, and fresh medium containing BrdUrd and colcemid was added and incubation was continued for 2-3 hours. For assays with exogenous metabolic activation, the cells were rinsed twice, after which culture medium without fetal bovine serum (FBS) was added. Cells were incubated for 2 hours in the presence of the test or control substance and the S-9 reaction mixture. After the 2 hour exposure period, cells were washed twice, and then complete medium was added. Cells were incubated for an additional 26 hours, with colcemid present for the final 2-3 hours of incubation.

Two to 3 hours after addition of colcemid, cells were harvested by mitotic shake-off. Prior to harvesting, the percent confluency in each flask was estimated. Harvested cells were treated for about 3 minutes at room temperature with hypotonic KCl, washed with fixative, dropped onto slides, air dried, and stained by a modified fluorescence pulse Giemsa (FPG) technique, described in Goto, K. et al. (1978). Chromosoma, 66:351-359. Fifty 2nd-division metaphase cells were scored per dose for the incidence of SCE. The number of chromosomes in each cell was also recorded. Any cell that had fewer than 19 or more than 23 chromosomes was excluded.

The standard time for obtaining 2nd-division metaphase cells in SCE studies was 26 hours after adding BrdUrd. If the test substance caused cell cycle delay, harvest times were extended, generally in 5-hour increments, with colcemid present for the last 2 hours.

Repeat Tests
Positive results in initial tests were confirmed by additional tests. If both -S9 and +S9 studies gave a positive response and required confirmation, they were done sequentially (-S9 first). If the -S9 repeat was positive, the repeat +S9 study was not always performed.

SELECTION AGENT (mutation assays): N/A
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): 6% Giemsa for 5-10 min

pH During Chemical Treatment
In instances when a change in the pH of the culture medium was noticed after addition of the test chemical and the overall response was negative, the test was considered sufficient. If, however, the overall response was positive, the experiment was repeated with the pH adjusted to 7.4.

Precipitation of the Test Compound
If the test chemical was not soluble at 5 mg/ml, it was tested up to doses at which precipitate was visible

NUMBER OF REPLICATIONS: Not specified

NUMBER OF CELLS EVALUATED: Fifty 2nd-division metaphase cells were scored per dose for the incidence of SCE

DETERMINATION OF CYTOTOXICITY
Not specified

OTHER EXAMINATIONS:
- Determination of polyploidy: Not specified
- Determination of endoreplication: Not specified
- Other: Not specified

OTHER: Not specified.

Evaluation criteria:

Dose Selection
Ten or eleven dose levels, at half-log intervals beginning at a high dose of 5 mg/ml (or as limited by solubility), were used for the first trial of the SCE study. Evaluation was conducted as detailed below under "Statistics".

Statistics:

Statistical analyses were conducted on the basis of the percentage of cells with aberrations was analyzed. Both the dose-response curve and individual dose points were statistically analyzed. A statistically significant (P < 0.003) trend test or a significantly elevated dose point (P < 0.05) was sufficient to indicate a chemical effect. A detailed discussion of these statistical methods is presented in Margolin et al. [1986], and their application in determining test conclusions is further explained in Galloway et al. [1987].

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

4,4'-Oxydianiline treatment caused a significant increase in the incidence of SCE in CHO cells both in the presence and absence of S9

The following results are taken from the appendices of the documented literature report.

Trial 1 of 2

Dose (pg/mL;-S9)	Total Chromosomes	Total SCE	SCE per Cell
0	1041	411	8.22
5	1040	426	8.52
16	1031	463	9.26
50	1039	632	12.64
Positive Control – CP
0.005	1047	1448	28.96

 

 Trial 2 of 2

Dose (pg/mL;-S9)	Total Chromosomes	Total SCE	SCE per Cell	Harvest Time
0	1030	371	7.42	26.50
50	1032	731	14.62	34.00
150	1042	1323	26.46	34.00
200	1034	1572	31.44	44.00
300	1041	1668	33.36	44.00
400	1041	1861	37.22	44.00
500	1041	1757	35.14	44.00
Positive Control – MMC
0.010	1019	2211	44.22	26.50

 

 Trial 1 of 2

Dose (pg/mL; +S9)	Total Chromosomes	Total SCE	SCE per Cell
0	1040	438	8.76
160	1039	440	8.80
500	1021	475	9.50
1600	1045	582	11.64
Positive Control – CP
1.5	1040	1369		27.38

 

 Trial 2 of 2

Dose (pg/mL; +S9)	Total Chromosomes	Total SCE	SCE per Cell
0	1051	380	7.60
500	1035	463	9.26
2000	1034	448	8.96
3000	1042	552	11.04
4000	1029	551	11.02
5000	560	479	17.74
Positive Control – CP
0.005	1047	949	18.96

 

Conclusions:

Conclusion:
positive with metabolic activation
positive without metabolic activation

4,4'-Oxydianiline treatment caused a significant increase in the incidence of SCE in CHO cells both in the presence and absence of S9

Executive summary:

The substance was tested in the Sister Chromatid Exchange test similar to the OECD guideline 479.

Significant increase in the numbers of SCE was noted both in the absence and in the presence of metabolic activation.

4,4'-Oxydianiline treatment caused a significant increase in the incidence of SCE in CHO cells both in the presence and absence of S9

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

Non GLP. The report does not detail a specific method; however it documents dose levels and responses in detail, so is deemed appropriate for use in the support of a formal registration. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

with metabolic activation only assessed; no negative control

Principles of method if other than guideline:

The ability of the test sample to revert four strains of Salmonella typhimurium from histidine dependence to histidine independence was determined. Strains TA 1535 and TA 100 were used to detect base-pair substitution mutations, whereas TA 1537 and TA 98 were used to detect frameshift mutations. "Ames, McCann and YamasakI, Mutation Res. 31: 347-364, 1975." is referenced within the scope of the report.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

4,4'-oxydianiline
4,4'-diaminodiphenyl ether
CAS 101-80-4

Target gene:

Histidine

Species / strain / cell type:

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

Metabolic activation:

with

Metabolic activation system:

Aroclor-induced rat liver S-9

Test concentrations with justification for top dose:

Trial 1: 250, 500, 1000, 2500, 5000 ug/plate
Trial 2: 10, 25, 50, 100, 250 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO; amounts not specified.
- Justification for choice of solvent/vehicle: Not specified, although standard solvent for this type of test.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar. One tenth ml of a solution of the test sample and approximately 10xE08 bacteria in 0.1 ml were added to 2 ml of top agar (0.6% agar, 0.6% NaCl, 0.05 mM L-histidine, 0.05 mM biotin).

One half ml of S-9 mix was added to the bacteria-test sample-top agar mixture. S9 is the 9,000 x g supernatant of liver homogenate from rats given 500 mg of Aroclor 1254/kg five days before sacrifice. The S9 mix contained per ml: 0.3 ml of S-9, 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP and 100 mM sodium phosphate
DURATION
- Preincubation period: Not specified
- Exposure duration: 48 hours
- Expression time (cells in growth medium): Not specified
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): Not specified.


SELECTION AGENT (mutation assays): Not specified.
SPINDLE INHIBITOR (cytogenetic assays): Not specified.
STAIN (for cytogenetic assays): Not specified.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: Not specified.

DETERMINATION OF CYTOTOXICITY
The cytotoxicity of the test sample in the presence of an activation system, as measured in strain TA 1535, was the basis for selecting concentrations to be used in the mutagenesis experiment. The protocol used to determine the cytotoxicity is identical to the mutagenesis protocol except that 10 E+03 rather than 10 E+08 bacteria are used per plate and a non-limlting concentration of histidfne was present. Concentrations of test sample that are nontoxic and, where possible, slightly toxic were selected for the mutagenesis assay.

OTHER EXAMINATIONS:
- Determination of polyploidy: Not evaluated.
- Determination of endoreplication: Not evaluated.
- Other:

OTHER:

Evaluation criteria:

Statistical evaluations of the data were used to classify a test sample as mutagenic or non-mutagenic. The total revertant colony number at each treatment condition was compared independently with the total revertant colony number for the appropriate negative control. Significance was judged at the 0.01 probability level.

The existence of a dose response was tested by the Spearman rank correlation. Significance was judged at the 0.05 probability level. A test sample was classified as mutagenic if both a statistically significant increase in total revertant colony number (p < 0.01) and a dose response (p < 0.05) are observed.
When a test sample was mutagenic, the highest single average number of revertants observed within each trial was expressed as a multiple of the control value for the sensitive strain(s). The number of revertants/nmole or ug of test sample is calculated from the slope of the line determined by linear regression analysis of the data before activity plateaus or decreases.

Statistics:

When the sum of control and treatment revertant numbers was less than or equal to eighty, the significance of the difference between the total counts from the two conditions was tested by referring to tables based on the Poisson distribution. When the sum of control and treatment revertant numbers was greater than eighty, the significance of the difference between the total counts from the two conditions was tested using a Z-test based on the normal approximation to the Poission distribution where

Z = N2 – N1 / [root (N2 + N1)]

N2 = Total revertant colony number at treatment condition; N1 = Total revertant colony number on control).

When Z>= 2.57, the treatment value is significantly different from the control value at the 0.01 probability level.

These statistical methods were chosen to judge significance because historical controls were found to follow a Poisson distribution.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not determined
- Effects of osmolality: Not determined
- Evaporation from medium: Not determined
- Water solubility: Not determined
- Precipitation: Concentrations >5000 ug/plate were not included in the mutagenesis experiment due to heavy precipitate on the plates.
- Other confounding effects: ot determined

RANGE-FINDING/SCREENING STUDIES: Not determined

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the initial cytotoxicity experiment with strain TA 1535, Aniline, 4,4'-oxydi- was not toxic at the concen¬trations tested (<10,000 ug/plate).

The mutagenesis data are listed in Table I for Trial I and In Table II for Trial 2 below.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1

 

Trial 1

 

Mutagenic Activity InSalmonella TyphimuriumStrainsTA 1535, TA 1537, TA 98, and TA 100with Metabolic Activation

 

 

Compound Added	ug/plate	TA 1535	TA 1537	TA 98	TA 100
DMSO		24.0	6.0	32.5	151.5
13,026
	250	50.0*	8.5	578.5*	1027.5*
	500	76.5*	11.5	511.5*	1818.5*
	1000	107.0*	12.5	757.0*	1981.5*
	2500	113.0*	11.5	-	-
	5000	78.5*	5.5	-	-
2AA	5				1384
	10	994	319	1092

 

Table 2

 

Trial 2

 

Mutagenic Activity InSalmonella TyphimuriumStrainsTA 1535, TA 1537, TA 98, and TA 100 with Metabolic Activation

 

 

Compound Added	ug/plate	TA 1535	TA 1537	TA 98	TA 100
DMSO		22.0	9.0	39.0	194.0
13,026
	10	16.0	8.5	63.0	384.0*
	25	22.5	10-0	127.0*	826.0*
	50	33.0	9.0	261.5*	1001.0*
	100	34.5	8.5	442.5*	1429.0*
	250	48.0*	10.0	672.0*	1558.5*
2AA	5				1957
	10	763	669	2554

 

 

DMSO             Dimethylsulfoxide (solvent control)

 

13,026            Aniline, 4,4'-oxydi-

 

2AA               2-Aminoanthracene (positive control)

 

*                     Treatment value significantly different from control value (p<_0.01)

Conclusions:

The substance is considered positive with metabolic activation.

The study demonstrated positive mutagenic results in three of the four test strains tested, and as such, is deemed to be potentially mutagenic based on this data.

Executive summary:

The ability of the test sample to revert four strains of Salmonella typhimurium from histidine dependence to histidine independence was determined according to Ames test and only in the presence of metabolic activation.

A significant increase of the mutation frequency was observed in the strains TA 1535, TA 98 and TA 100 in the presence of metabolic activation.

No significnat increase was noted in the strain TA 1537.

On the basis of the results observed, the test substance is considered to be mutagenic in Salmonella Typhimurium in the presence of metabolic activation.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

Non-GLP literature study in 27 different chemicals. Methodology is detailed, as are results per substance, and well documented. Study addresses both chromosal aberrations and sister chromatid exchanges.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

The protocol described by Galloway et al. [1985], with a few modifications, was used. A brief summary of the testing approach is provided below

Principles of method if other than guideline:

Approximately 24 hr prior to cell treatment, 1.2 x 106 cells were seeded per 75 cm2 flask. A culture was established for each dose both with and without metabolic activation. For assays without metabolic activation, the medium was replaced with fresh medium immediately before chemical treatment. Cells were treated with test or control substances cells were treated for about 10 hr. Colcemid was added 2-3 hr prior to cell harvest by mitotic shake-off.

For assays with metabolic activation, the cells were rinsed twice with phosphate buffered saline (PBS), after which culture medium without FBS was added. Cells were incubated for 2 hr in the presence of the test or control substances and the S9 reaction mixture. FBS was omitted to avoid the binding of serum proteins to short-lived, highly reactive intermediates. After the 2 hr exposure period, cells were washed twice with PBS, and then complete medium containing 10% FBS was added. Cells were incubated for an additional 26 hr, cells were harvested approximately 11 hr after removal of the S9 fraction. Colcemid was added 2 hr prior to harvest. Slides were stained in 6% Giemsa for 5-10 min. One hundred cells were scored for each dose in early studies and 200 cells per dose in later studies. All slides except high-dose positive controls were coded. Only metaphase cells in which the chromosome number was between 19 and 23 were scored.

The chromosome number was recorded for each cell and chromosome or chromatid type aberrations were classified into three categories: simple (breaks, fragments, double minutes), complex (interchanges, rearrangements), and other (pulverized, more than ten aberrations/cell).

Repeat Tests
Positive results in initial tests were confirmed by additional tests. If both -S9 and +S9 studies gave a positive response and required confirmation, they were done sequentially (-S9 first). If the -S9 repeat was positive, the repeat +S9 study was not always performed.

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

4,4'-oxydianiline
4,4'-diaminodiphenyl ether
CAS 101-80-4

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO cells, up to 15 passages since cloning, were used for all testing. Stock cells were obtained from Litton Bionetics (Kensington, MD) and stored in liquid nitrogen. At least once per year representative cells were sent to Flow Laboratories (McLean, VA) for mycoplasma testing using the Hoechst stain test followed by the Agar and Hyorhinis test. Results from all tests for mycoplasma contamination were negative. CHO cells were maintained in McCoy's 5A medium (modified) supplemented with L-glutamine (2 mM), antibiotics, and 10% fetal bovine serum (FBS). Pre-mixed culture medium and FBS were purchased from GIBCO Laboratories (Grand Island, NY). All stock and experimental cultures were maintained at 37°C in an atmo¬sphere of 5% C02 in air and 95% relative humidity.

Metabolic activation:

with and without

Metabolic activation system:

Liver fraction (S9) prepared from Aroclor 1254-induced male Sprague Dawley rats. The final concentrations of the S9 fraction, NADP, and isocitric acid were 0.02 ml, 2.4 mg, and 4.5 mg, respectively, per milliliter culture medium

Test concentrations with justification for top dose:

Chromosome Aberration without activation: 0, 50, 100, 160, 500, 1000, 1600, 2000, 3000 ug/mL
Chromosome Aberration with activation: 0, 160, 500, 1000, 1600, 2000, 3000, 4000, 5000 ug/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Not specified; assumed to be based on historical use.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Conducted concurrently

True negative controls:

no

Positive controls:

yes

Remarks:

Conducted concurrently

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION:in suspension

DURATION
Approximately 24 hr prior to cell treatment, 1.2 x 106 cells were seeded per 75 cm2 flask. A culture was estab¬lished for each dose both with and without metabolic activation. For assays without metabolic activation, the medium was replaced with fresh medium immediately before chemical treatment. Cells were treated with test or control substances cells were treated for about 10 hr and BrdUrd was omitted. Colcemid was added 2-3 hr prior to cell harvest by mitotic shake-off.

For assays with metabolic activation, the cells were rinsed twice with phosphate buffered saline (PBS), after which culture medium without FBS was added. Cells were incubated for 2 hr in the presence of the test or control substances and the S9 reaction mixture. FBS was omitted to avoid the binding of serum proteins to short-lived, highly reactive intermediates. After the 2 hr exposure period, cells were washed twice with PBS, and then complete medium containing 10% FBS was added. Cells were incubated for an additional 26 hr, cells were harvested approximately 11 hr after removal of the S9 fraction. Colcemid was added 2 hr prior to harvest
For chemicals that caused cell cycle delay, harvest times were extended, generally in 5 hr increments, with colcemid present for the last 2 hr. For ABS tests, harvest times were similarly extended based on the observation of cell cycle delay in the SCE trials.

SELECTION AGENT (mutation assays): N/A
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): 6% Giemsa for 5-10 min

pH During Chemical Treatment
In instances when a change in the pH of the culture medium was noticed after addition of the test chemical and the overall response was negative, the test was considered sufficient. If, however, the overall response was positive, the experiment was repeated with the pH adjusted to 7.4.

Precipitation of the Test Compound
If the test chemical was not soluble at 5 mg/ml, it was tested up to doses at which precipitate was visible

NUMBER OF REPLICATIONS: Not specified

NUMBER OF CELLS EVALUATED: 100 cells were scored for each dose in early studies and 200 cells per dose in later studies

DETERMINATION OF CYTOTOXICITY
Not specified

OTHER EXAMINATIONS:
- Determination of polyploidy: Not specified
- Determination of endoreplication: Not specified
- Other: Not specified

OTHER: Not specified.

Evaluation criteria:

Dose Selection
Ten or eleven dose levels, at half-log intervals beginning at a high dose of 5 mg/ml (or as limited by solubility), were used for the first trial of the SCE study. The dose levels for ABS studies were chosen based on the toxicity of the test chemical observed in the SCE studies. Evaluation was conducted as detailed below under "Statistics".

Statistics:

Statistical analyses were conducted on the basis of the percentage of cells with aberrations was analyzed. Both the dose-response curve and individual dose points were statistically analyzed. A statistically significant (P < 0.003) trend test or a significantly elevated dose point (P < 0.05) was sufficient to indicate a chemical effect. A detailed discussion of these statistical methods is presented in Margolin et al. [1986], and their application in determining test conclusions is further explained in Galloway et al. [1987].

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

4,4'-Oxydianiline treatment caused a significant increase in the incidence of the chromosome aberrations CHO cells both in the presence and absence of S9

The following results are taken from the appendices of the documented literature report.

Table 1 (without S9) / Trial 1 of 3 /  Harvest Time: 12 hours

Result: Ambiguous

dose	percent cells with aberrations
ug / ml	cells	total	simple	complex
0.0000	100.	1.00	1.00	0.00
50.0000	100.	2.00	2.00	0.00
160.0000	100.	9.00	2.00	6.00
500.0000	100.	3.00	2.00	1.00
1000.0000	100.	1.00	0.00	0.00
1600.0000	100.	6.00	1.00	3.00
2000.0000	100.	1.00	1.00	0.00
positive control: mmc
0.2500	100.	26.00	18.00	14.00

 

Table 2 (without S9) / Trial 2 of 3 / Harvest Time: 14 hours

Result: Positive

dose	percent cells with aberrations
ug / ml	cells	total	simple	complex
0.0000	100.	4.00	2.00	2.00
100.0000	100.	13.00	13.00	2.00
500.0000	100.	16.00	16.00	2.00
1000.0000	100.	24.00	22.00	6.00
2000.0000	100.	11.00	8.00	2.00
3000.0000	100.	17.00	8.00	10.00
positive control: MMC
0.5000	100.	68.00	44.00	52.00

 

Table 3 (without S9) / Trial 3 of 3 / Harvest Time: 17.5 hours

Result: Positive

dose	percent cells with aberrations
ug / ml	cells	total	simple	complex
0.0000	100.	4.00	2.00	2.00
100.0000	100.	12.00	7.00	5.00
500.0000	100.	32.00	18.00	16.00
1000.0000	100.	42.00	23.00	17.00
2000.0000	100.	24.00	11.00	17.00
3000.0000	100.	16.00	10.00	8.00
positive control: mmc
0.5000	100.	34.00	15.00	22.00

 

 

Table 4 (with S9) / Trial 1 of 3 / Harvest Time: 12 hours

Result: Positive

dose	percent cells with aberrations
ug / ml	cells	total	simple	complex
0.0000	100.	3.00	2.00	2.00
160.0000	100.	4.00	2.00	2.00
500.0000	100.	16.00	9.00	6.00
1600.0000	100.	18.00	10.00	13.00
5000.0000	100.	9.00	5.00	4.00
positive control: CP
50.0000	100.	34.00	15.00	22.00

 

Table 5 (with S9) / Trial 2 of 3 / Harvest Time: 13 hours

Result: Positive

dose	percent cells with aberrations
ug / ml	cells	total	simple	complex
0.0000	100.	1.00	0.00	1.00
500.0000	100.	7.00	4.00	3.00
1000.0000	100.	7.00	5.00	3.00
2000.0000	100.	13.00	7.00	8.00
3000.0000	100.	15.00	6.00	8.00
4000.0000	100.	16.00	9.00	7.00
5000.0000	100.	20.00	5.00	15.00
positive control: CP
50.0000	100.	38.00	44.00	52.00

 

 

Table 6 (with S9) / Trial 3 f 3 / Harvest Time: 17.5 hours

Result: Positive

dose	percent cells with aberrations
ug / ml	cells	total	simple	complex
0.0000	100.	1.00	0.00	1.00
500.0000	100.	4.00	3.00	2.00
2000.0000	100.	17.00	10.00	10.00
3000.0000	100.	10.00	8.00	4.00
4000.0000	100.	11.00	5.00	8.00
5000.0000	100.	20.00	4.00	18.00
positive control: CP
50.0000	100.	48.00	33.00	32.00

Conclusions:

Conclusion:
positive without metabolic activation
positive with metabolic activation

4,4'-Oxydianiline caused a significant increase in the incidence of chromosome aberations in CHO cells, both in the presence and absence of exogenous metabolic activation.

Executive summary:

The substance was tested in the chromosome aberrations test similar to the OECD guideline 473.

Significant increase in the numbers of cells with chromosome aberrations was noted both in the absence and in the presence of metabolic activation.

The substance is considered to cause a significant increase in the incidence of chromosome aberrations in CHO cells both in the presence and absence of metabolic activation.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

Non-GLP literature study in 72 different chemicals. Methodology is detailed, as are results per substance, and well documented.

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

see below methodology

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

yes

Remarks:

see below methodology

Principles of method if other than guideline:

Each experiment, other than the initial toxicity test, normally consisted of the following groups: vehicle control, 4 cultures; positive control, 2 cultures; and at least 5 test substance concentrations, 2 cultures/concentration. The initial experiment was a toxicity test in which cell population expansion was measured. Ten-fold differences in test substance concentrations were used in the toxicity test, the highest being 5 mg/mL unless a much lower concentration was indicated by the poor solubility of a test substance. This test was followed by at least 2 experiments in the absence of S-9 mix. Test substance concentrations were primarily 2-fold dilutions from the highest testable concentration, as estimated from the toxicity test. If a clear positive response was observed in these experiments, no further testing was performed either in the absence or presence of S-9.
Each exposed culture consisted of 6x10E+6 cells in a final volume of 10 mL in a screw-cap plastic tube. This tube was incubated for 4 hours on a rotating horizontal axis roller drum. At the end of the incubation, the cells were sedimented by centrifugation, washed, and resuspended in 20 mL. These cell suspensions (3x 10+5 cells/mL) were incubated for a 2-day expression period, the cell population density being adjusted back to 20 mL of 3x10+5 cells/mL after 24 hours. After 48 hours, the cell population densities were estimated and culture volumes containing 3x10+6 cells adjusted to 15 mL, giving a cell population density of 2x10+5 cells/mL.
A 0.1 mL sample of the cell suspension was withdrawn and diluted. Three 0.1 mL samples (200 cells) of the diluted cultures were transferred to tubes, mixed with cloning medium containing agar, and poured onto Petri plates. Three aliquots (each containing 10+6 cells) of the remaining culture were distributed to tubes, mixed with cloning medium containing agar and trifluorothymidine, and then poured onto Petri plates. The agar was gelled at 4°C for 5-10 minutes, then the plates were incubated for 11-14 days at 37°C. Colonies were counted using an automated colony counter. Toxicity was expressed as either a reduction of cell population growth in suspension during the expression period or a reduction in cloning efficiency. A measure of overall toxicity was relative total growth (RTG).
A test was considered positive when, out of 3 trials, a positive trial was reproducible. A test was considered negative when, out of 3 trials, a positive response or a positive dose was not reproducible. A test was considered questionable when, out of 3 trials, neither a positive nor a negative response was produced.

GLP compliance:

no

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

4,4'-oxydianiline
4,4'-diaminodiphenyl ether
CAS 101-80-4

Target gene:

mouse lymphoma L5178Y cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The tk+/tk- -3.7.2C heterozygote of L5178Y mouse lymphoma cells was obtained from Dr. D. Clive, Burroughs Wellcome Co., Research Triangle Park, NC 27709, and stored in liquid nitrogen. Thawed samples were cultured and used for up to 3 months, then discarded. Laboratory cultures were confimed as free from mycoplasma by cultivating or Hoechst staining techniques and maintained in Fischer's medium at 37 deg C on gyratory tables. Fischer's medium (designated Fo) was supplemented with 2 mM L-glutamine, sodium pyruvate, 110 pg/ml, 0.05% pluronic F68, antibiotics, and 10% heat-inactivated donor horse serum (v/v) (designated Flop). On a single occasion, within 1 week of the start of an experiment, cultures were purged of tk-/tk- mutants by exposure for 1 day to Flop containing THMG (thymidine, 6 pg/ml hypoxanthine, 5 pg/ml, glycine, 7.5 pg/d and methotrexate, 0.1 pg/ml), then for 3 days to Flop containing THG only, (i.e., THMG without methotrexate)

Metabolic activation:

without

Test concentrations with justification for top dose:

0, 15.625, 31.25, 50, 62.5, 100, 125, 150, 200, 250, 500 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Vehicle controls were the solvents used for the test chemicals, namely, medium without serum, distilled water, or
dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Not specified.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

medium without serum, distilled water, or dimethyl sulphoxide (DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

Each experiment, other than the initial toxicity test, normally consisted of the following groups: vehicle control, 4 cultures; positive control, 2 cultures; and at least 5 test substance concentrations, 2 cultures/concentration. The initial experiment was a toxicity test in which cell population expansion was measured. Ten-fold differences in test substance concentrations were used in the toxicity test, the highest being 5 mg/mL unless a much lower concentration was indicated by the poor solubility of a test substance. This test was followed by at least 2 experiments in the absence of S-9 mix. Test substance concentrations were primarily 2-fold dilutions from the highest testable concentration, as estimated from the toxicity test. If a clear positive response was observed in these experiments, no further testing was performed either in the absence or presence of S-9.

Each exposed culture consisted of 6x10E6 cells in a final volume of 10 mL in a screw-cap plastic tube. This tube was incubated for 4 hours on a rotating horizontal axis roller drum. At the end of the incubation, the cells were sedimented by centrifugation, washed, and resuspended in 20 mL. These cell suspensions (3x10E5 cells/mL) were incubated for a 2-day expression period, the cell population density being adjusted back to 20 mL of 3x10E5 cells/mL after 24 hours. After 48 hours, the cell population densities were estimated and culture volumes containing 3x10E6 cells adjusted to 15 mL, giving a cell population density of 2x10E5 cells/mL.

A 0.1 mL sample of the cell suspension was withdrawn and diluted. Three 0.1 mL samples (200 cells) of the diluted cultures were transferred to tubes, mixed with cloning medium containing agar, and poured onto Petri plates. Three aliquots (each containing 10E6 cells) of the remaining culture were distributed to tubes, mixed with cloning medium containing agar and trifluorothymidine, and then poured onto Petri plates. The agar was gelled at 4°C for 5-10 minutes, then the plates were incubated for 11-14 days at 37°C. Colonies were counted using an automated colony counter. Toxicity was expressed as either a reduction of cell population growth in suspension during the expression period or a reduction in cloning efficiency. A measure of overall toxicity was relative total growth (RTG).
A test was considered positive when, out of 3 trials, a positive trial was reproducible. A test was considered negative when, out of 3 trials, a positive response or a positive dose was not reproducible. A test was considered questionable when, out of 3 trials, neither a positive nor a negative response was produced.

Technical Quality Control Criteria

1. Solvent control
a) A solvent control count was rejected if the cloning efficiency was <50% or > 115%. Between 50 and 60%, judgment was used in accepting the result.

b) The average mutant fraction of the solvent controls had to be >15 mutants per 10E6 surviving cells and < 110 mutants per 106 surviving cells. The range was extended to >10-< 150 mutants per 10E6 surviving cells for experiments that were positive.

c) Unless at least two solvent control cultures were accepted, the experiment was rejected.

d) The experiment was rejected if a chi-square test for consistency of the acceptable mutant fractions showed P<5%.

2. Positive control
a) A positive control culture was rejected if the cloning efficiency was < 10% or > 115%.
b) A positive control culture was rejected if the relative total growth (RTG) was <1%.

3. Doses
a) A culture was rejected if the cloning efficiency was 115%.
b) A culture was rejected if the RTG was <1%.
c) A culture was rejected if the relative suspension growth for the second day of expression was <40%.

d) If the RTG was between 1 and 5% and/or the cloning efficiency was between 10 and 20%, then the mutant count was examined. If a significant increase in the mutant fraction was not supported by an increase in the mutant count, then the culture was rejected.

e) A culture was rejected if the day two count was <3 X 10E5 per ml.

f) A dose was rejected if the compound was not soluble at that dose.

g) No dose greater than 5 mg/ml was tested.

h) A dose set was rejected if the chi-square test for consistency of the acceptable mutant fractions within that dose set showed P<5%.

i) Each dose set had to contain two or more acceptable cultures.

j) If fewer than three dose sets were accepted, then the experiment was rejected unless the reason
for dose set rejection was precipitation and there was no mutagenic response.

Evaluation criteria:

Compliance with predetermined quality control criteria was required before the response of a cellular population to the test chemical was evaluated. The basis for this choice of criteria is described elsewhere [Caspary et al., 1988] and is the outcome of evaluation of historical data in two laboratories. The four response categories were defined as

Positive response ( + )
The dose-related trend and the response at one of the three highest acceptable doses were statistically significant.

Negative response (-)
Two categories were used. In both there was
a) no dose-related trend,
b) no statistically significant response at any dose,
c) an acceptable positive control response.

Nontoxic, negative response (=)
There was an RTG among the acceptable doses of >30% (approximately), higher toxicities being unattainable due to intrinsic properties of either the compound or the system.

Toxic, negative response (-)
There was either an RTG of <30% (approximately) at the maximum acceptable dose, or the lethal concentration was no greater than 1.5 X a lower concentration at which the RTG was >30%.

Inconclusive (i)
There was
a) no dose-related trend and a statistically significant dose was any other than one of the highest
b) a response which would have been negative, but the lowest RTG acceptable doses was >35%,
c) a response which would have been negative, but there were no acceptable positive controls.


Questionable (?)
There was either
a) no dose-related trend, but a statistically significant response occurred at one of the highest
b) a statistically significant dose-related trend, but none of the acceptable doses was statistically three doses, or significant on its own.

Statistics:

Primary judgments were made at the level of individual experiments, but judgment on the mutagenic potential of a chemical was made on a basis of concensus of all valid experimental results. The statistical analysis was based upon the mathematical model proposed for this system [Lee and Caspary, 1983] and consisted of a dose-trend test [Barlow et al., 1972] and a variance analysis of pair-wise comparisons of each dose against the vehicle control. Where a statistically significant response occurred, the lowest observed effective dose (LOED) was noted. There is a report that nonphysiological pH levels can produce increases in mutant fraction [Cifone et al., 1987]. Consequently, pH shifts observed from phenol red color changes to yellow or purple were noted and are recorded within the report.
According to Cifone et al. [1987], such changes impacted upon data evaluation only in the presence of S9 mix. However, studies at the testing laboratory [McGregor, unpublished] have failed to confirm this interaction of pH and S9 mix. It is possible that the lack of such an effect is due to the lower S9 concentration used in this laboratory. Osmotic pressure was not measured, but, again, osmotic effects were unlikely because the highest concentrations tested were never greater than 5 mg/ml.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed at 500 ug/ml in test 1 and 250 ug/ml in test 2

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The lowest tested concentration of 4,4'-oxydianiline, 50 pg/ml, induced statistically significant increases in the mutant fraction. A dose-related response was observed over three concentrations before toxicity became excessive. The RTG at the LOED was about 88%.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There is a report that nonphysiological pH levels can produce increases in mutant fraction [Cifone et al., 1987]. Consequently, pH shifts observed from phenol red color changes to yellow or purple were noted and are recorded within the report.
According to Cifone et al. [1987], such changes impacted upon data evaluation only in the presence of S9 mix. However, studies at the testing laboratory [McGregor, unpublished] have failed to confirm this interaction of pH and S9 mix. It is possible that the lack of such an effect is due to the lower S9 concentration used in this laboratory
- Effects of osmolality: Osmotic pressure was not measured, but, again, osmotic effects were unlikely because the highest concentrations tested were never greater than 5 mg/ml
- Evaporation from medium: Not specified.
- Water solubility:Not specified.
- Precipitation:Not specified.
- Other confounding effects:Not specified.

RANGE-FINDING/SCREENING STUDIES: Not specified.

COMPARISON WITH HISTORICAL CONTROL DATA: Not specified.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Not specified.

The following results were transcribed from the literature report.

Without S9 trial 1 (+)

Conc ug/ml	CE	RTG	MC	MF	AVG MF
DMSO	78	98	77	33	34
0	91	100	85	31
	86	98	92	36
	84	104	92	37
15.625	75	82	82	36	35
	94	98	97	34
31.25	75	93	46	20	31
	75	93	93	42
62.5	83	65	131	53	55
	89	60	156	58
125	68	28	184	90	100
	73	25	240	109
250	27	2	122	152	105
	93	7	164	59
500	Lethal
	Lethal
EMS 250 ug/ml	73	70	477	219	336
	28	27	579	454
MMS 15 ug/ml	32	23	138	143	156
	28	19	139	168

Precipitation observed at 500 ug/ml

Without S9 trial 2 (+)

Conc ug/ml	CE	RTG	MC	MF	AVG MF
DMSO	93	101	101	36	32
0	112	111	106	32
	94	88	94	33
	95	100	74	26
50	87	76	137	53	59
	80	102	157	66
100	73	39	393	181	179
	72	40	382	178
150	32	4	411	435	413
	35	7	406	390
200	8r	1	135	551	ND
	9r	1	119	467
250	Lethal
	Lethal
EMS 250 ug/ml	70	77	427	203	235
	51	56	405	268
MMS 15 ug/ml	51	26	200	130	154
	41	27	216	178

r = rejected when 50% > CE> 120% or RTC < 1%

Conclusions:

Conclusion: positive without metabolic activation

The lowest tested concentration of 4,4'-oxydianiline, 50 µg/ml, induced statistically significant increases in the mutant fraction in the absence of metabolic activation.

Executive summary:

The substance was tested in the Mouse Lymphoma Assay similar to the OECD guideline 490.

Significant increase in the mutant frequency was noted in the absence of metabolic activation. Therefore the test in presence of metabolic activation was not conducted.

The substance is considered to induce positive effects in the mouse lyphoma assay.

A dose-related response was observed over three concentrations before toxicity became excessive. The RTG at the LOED was about 88%.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Two in vivo studies are available:

- A mouse bone marrow micronucleus test (three daily exposures by intraperitoneal injection).

A statistically significant increase in the MN-PCE frequencies was observed.

- An Unscheduled DNA Synthesis test in rats (hepatocytes were collected and analyzed).

The results showed that the substance did not induce unscheduled DNA synthesis in rat hepatocytes

Link to relevant study records

Reference

Reference 1

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

Non-GLP literature study in 49 different chemicals. Methodology is detailed, as are results per substance, and well documented.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

See "principles of method" below

Principles of method if other than guideline:

The substance was tested in a mouse bone marrow micronucleus test that employed three daily exposures by intraperitoneal injection.
Bone marrow samples were obtained 24 hr following the final exposure.

Dose Determination Studies
Groups of 5 mice (aged 9-14 weeks, weighing 25-33 g) were administered 4,4’-oxydianiline (mixed in corn oil and suspended with a Tek-Mar Tissumizer) via i.p. injection at a volume of 0.4 mL per mouse on 3 consecutive days. Animals were monitored twice daily, and 48 hours after the third treatment, the surviving mice were euthanized. Bone marrow smears were prepared by a direct technique, fixed, and stained with acridine orange. Bone marrow smears from each animal were evaluated at 1000x magnification using epi-illuminated fluorescence microscopy for determination of the percentage of polychromatic erythrocytes (PCE) among 200 erythrocytes. Based on the results obtained, the maximum administered dose was estimated or additional dose determination experiments were conducted to more accurately estimate the maximum dose to be tested in the primary micronucleus (MN) test. The selection of the maximum dose to be tested for MN induction was based on mortality.

Main study
Groups of 5 mice were injected i.p. on 3 consecutive days with either the test substance (at 32.5, 75, or 150 mg/kg), the positive control chemical (12.5 mg/kg 7,12-dimethylbenzanthracene in corn oil), or the solvent (corn oil). Mice were euthanized 24 hours after the third treatment. Bone marrow smears were prepared, fixed, and stained with acridine orange. For each animal, slides were evaluated at 1000x magnification for the number of MNPCE among 2000 PCE and for the percentage of PCE among 200 erythrocytes.
A repeat test was performed since the results from the initial test suggested a possible positive effect.

GLP compliance:

no

Type of assay:

micronucleus assay

Specific details on test material used for the study:

4,4'-oxydianiline
4,4'-diaminodiphenyl ether
CAS 101-80-4

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: National Toxicology Program production facility at Taconic Farms
- Age at study initiation: 9 to 14 weeks
- Weight at study initiation: within a 2 g range of a mean weight between 25 and 33 g
- Assigned to test groups randomly: [no/yes, under following basis: Not specified
- Fasting period before study: Not specified
- Housing: Not specified
- Diet (e.g. ad libitum): Not specified
- Water (e.g. ad libitum): Not specified.
- Acclimation period: Not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not specified
- Humidity (%): Not specified
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): Not specified

IN-LIFE DATES: Not specified.

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: mixed in corn oil and suspended with a Tek-Mar Tissumizer
- Justification for choice of solvent/vehicle: Not specified
- Concentration of test material in vehicle: Variable, dependant on dose level (see below on details of exposure)
- Amount of vehicle (if gavage or dermal): Not applicable
- Type and concentration of dispersant aid (if powder): Not applicable
- Lot/batch no. (if required): Not specified
- Purity: Not specified.

Details on exposure:

Within each experiment, male B6C3F1 mice (obtained from the National Toxicology Program production facility at Taconic Farms) of a common age between 9 and 14 weeks and weighing within a 2 g range of a mean weight between 25 and 33 g were used. The substance was prepared in the appropriate solvent (corn oil for water-insoluble chemicals) and suspended using either a Tek-Mar Tissumizer for chemicals in corn oil All test chemicals were administered within 30 min of preparation. Suspended DMBA was stored at room temperature and dissolved MMC was stored at 0-5deg C between treatments within an experiment.

All treatments were by intraperitoneal (IP, injection at a volume of 0.4 ml per mouse. Identification numbers were randomly assigned to mice prior to euthanasia.

Dose Determination Studies
See below in the section "Any other information on materials and methods".

First main study
In the first main study, groups of 5-7 mice were injected IP on three consecutive days with either the test chemical (150, 75 and 37.5 mg/kg bw), a weakly active dose of the positive control chemical (Dimethylbenzanthracene or DMBA at 12.5 mgikg bw in corn oil) or the solvent (corn oil). Mice were euthanized with CO, 24 hr after the third treatment. Bone marrow smears (two slides mouse) were prepared, fixed in absolute methanol, and stained with acridine orange. For each animal. slides were evaluated at 1,000 X magnification for the number of MN-PCE among 2,000 PCE and for the percentage of PCE among 200 erythrocytes.

Repeated main study
A repeat test was conducted based on results of the first study, and according the same method, at the dose levels of 75 and 150 mg/kg bw.

Duration of treatment / exposure:

3 days

Frequency of treatment:

Once daily

Post exposure period:

24 hours after the 3rd treatment.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

First main test

Dose / conc.:

37.5 mg/kg bw/day (nominal)

Remarks:

First main test

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

First main test

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

First main test

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Repeat test

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

Repeat test

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Repeat test

No. of animals per sex per dose:

5 Males per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive controls used were Dimethylbenzanthracene and Mitomycin C
Route of administration: Interperitoneal
Dose: Dimethylbenzanthracene (12.5 mg / kg).

Tissues and cell types examined:

Bone marrow (two slides/tissue/mouse)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: toxicitylmortality observed in the dose determination studies (see below the section "Any other information on materials and methods")
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): As detailed above.
DETAILS OF SLIDE PREPARATION: Air dried. Fixed using absolute methanol and stained with acridine orange
METHOD OF ANALYSIS: 1000x magnification using epi-illuminated fluorescence microscopy for determination of the percentage of polychromatic erythrocytes (PCE) among 200 erythrocytes.

Statistics:

The data were analyzed using the Micronucleus Assay Data Management and Statistical software package (version 1.4), which was designed specifically for in vivo micronucleus data [ILS, 1990]. The level of significance was set at an alpha level of 0.05. To determine whether a specific treatment resulted in a significant increase in MN-PCE, the number of MN-PCE were pooled within each dose group and analyzed by a one-tailed trend test. In the software package used, the trend test incorporates a variance inflation factor to account for excess animal variability. In the event that the increase in the dose response curve is nonmonotonic, the software program allows for the data to be analyzed for a significant positive trend after data at the highest dose only has been excluded. However, in this event, the alpha level is adjusted to 0.01 to protect against false positives.
The %PCE data were analyzed by an analysis of variance (ANOVA) test based on pooled data. Pairwise comparisons between each group and the concurent solvent control group was by an unadjusted one-tailed Pearson chisquared test which incorporated the calculated variance inflation factor for the study.

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The initial test was negative by trend analysis but the MN-PCE frequencies in the low (37.5 mglkg) and middle (75 mg/kg) dose groups were markedly elevated.
A repeat test was therefore performed since the results from the initial test suggested a possible positive effect. The repeat test was positive by trend analysis with the high dose group elevated significantly above the control. Overall, these results were considered positive. Following decoding of the chemicals, a trend analysis was run on the initial test data after omitting the high dose group. This trend analysis gave a P = 0.001 adding support to the conclusion that this compound induces MN.

Results:

Test	Trend Pvalue	Dose (mg/kg)	MN-PCE/ l,000 (No. animals)	Pair-wise	Survival	% PCE
Initial test	0.095	0	1.70 ± 0.26 (5)		5/5	58.2
	(0.001)	37.5	3.30 ± 0.46 (5)	0.0117	5/5	46.3
		75	4.20 ± 0.89 (5)	<0.001	5/5	56.0
		150	2.90 ± 0.40 (5)	0.0383	5/5	50.1
Repeat test	0.013	0	1.20 ± 0.41 (5)		5/5	50.8
		75	1.70 ± 0.34 (5)	0.1764	5/5	62.8
		150	2.63 ± 0.24 (4)	0.0132	4/5	61.4

Conclusions:

Conclusion: positive
The initial test was negative by trend analysis but the MN-PCE frequencies in the low (37.5 mg / kg) and middle (75 mg/kg) dose groups were markedly elevated. The repeat test was positive by trend analysis with the high dose group elevated significantly above the control. Overall, these results were considered positive. Following decoding of the chemicals, a trend analysis was run on the initial test data after omitting the high dose group. This trend analysis gave a P = 0.001 adding support to the conclusion that this compound induces MN.

Executive summary:

The substance was tested in a mouse bone marrow micronucleus test that employed three daily exposures by intraperitoneal injection.

Bone marrow samples were obtained 24 hr following the final exposure.

A statistically significant increase in the MN-PCE frequencies was observed.

According to these results, the substance is considered to induce micronuclei in the mouse bone marrow.

Reference 2

Endpoint:

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

Remarks:

Measurement of Unscheduled DNA Synthesis

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

Non-GLP literature study in 24 different chemicals. Methodology is detailed, as are results per substance, and well documented.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

yes

Remarks:

See below

Principles of method if other than guideline:

Treatment of animals:
Male rats (180-300 g) were administered 4,4’-oxydianiline via intragastric intubation as a single bolus dissolved in corn oil. The S-Phase Synthesis was examined at 48 hr post-treatment.

Hepatocyte Isolation and Culture:
Primary hepatocyte cultures were prepared from rats as described in Mitchell, A. D. and J. C. Mirsalis (1984).
Livers were perfused in situ. A single-cell suspension of hepatocytes was obtained. Cells were collected by centrifugation.
Approximately 6x10E+05 cells were seeded into each well of a 6-well culture plate with appropriate medium. After 1.5-2.0 hours incubation in a humidified atmosphere at 37ºC, 5% CO2, the cultures were washed to remove non viable cells .

Cell Culture, Fixation, and Staining:
Cultures were incubated with 10 µCi/rnl 3H-(meth yl)thymidine for 4 hours at 37ºC and 5% CO2, followed by 14-18 hours with unlabeled thymidine.
The cultures were then washed, followed by hypotonic treatment with sodium citrate to swell the cells, fixed and washed 3-6 times with deionized water. The dried coverslips were mounted to glass slides. The slides were dipped in nuclear track emulsion diluted with deionized water, and exposed at -20ºC for 7-14 days and then developed and stained as described in Mitchell and Mirsalis, 1984.

Measurement of UDS:
Quantitative autoradiographic grain counting was accomplished as previously described [Mitchell and Mirsalis, 19841.
Slides were coded prior to scoring to preclude scorer bias. An area of a slide was randomly selected, and 50 morphologically unaltered cells were counted. The highest of two
nuclear-sized areas over the cytoplasm and adjacent to the nucleus was subtracted from the nuclear count to determine the net grains/nucleus (NG).
The percentage of cells undergoing repair (%IR) was determined as the percent of those cells exhibiting 5 or more NG.

Measurement of S-Phase Synthesis:
The number of S-phase cells and non-S-phase cells were counted beginning with a randomly selected patch of a slide.

GLP compliance:

no

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

4,4'-oxydianiline
4,4'-diaminodiphenyl ether
CAS 101-80-4

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories (Gilroy, CA) and Hilltop Laboratory Animals (Chatsworth, CA)
- Age at study initiation: Not specified
- Weight at study initiation: 180 to 300 g
- Assigned to test groups randomly: Not specified.
- Fasting period before study: None
- Housing: Polypropylene cages with hardwood-chip bedding
- Diet (e.g. ad libitum): Purina Rodent Chow #5001 (Ralston Purina Co., St. Louis), ad libitum
- Water (e.g. ad libitum): deionized, 0.5 um charcoal filtered tap water ad libitum
- Acclimation period: Not specified.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not specified.
- Humidity (%): Not specified.
- Air changes (per hr): Not specified.
- Photoperiod (hrs dark / hrs light): 12 hr light: 12 hr dark cycle

IN-LIFE DATES: Not specified.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil
- Justification for choice of solvent/vehicle: solubility and historical usage.
- Concentration of test material in vehicle: As per dose criteria selected as 80%, 40%, and 10% of the LD50
- Amount of vehicle (if gavage or dermal): Not specified
- Type and concentration of dispersant aid (if powder): Not determined
- Lot/batch no. (if required): Not specified
- Purity: Not specified; report details "technical grade" used.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Animals were administered the compound by oral gavage as a single bolus dissolved or suspended in corn oil or water.
The doses selected were 40, 180 and 725 mg/kg (based approximately on the LD50).

DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable.

Duration of treatment / exposure:

Not applicable

Frequency of treatment:

Single oral gavage

Post exposure period:

The S-phase synthesis (SPS) was examined at 48 hr post treatment.

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

180 mg/kg bw/day (nominal)

Dose / conc.:

725 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Male/3 per dose level

Control animals:

yes, concurrent vehicle

Positive control(s):

2-acetylaminofluorene; N-dimethylnitrosamine; 11-Aminoundecanoic acid
- Justification for choice of positive control(s): Not specified; assumed historical usage.
- Route of administration: Oral Gavage
- Doses / concentrations:

Dimethylnitrosamine 10 mg/kg
2-Acetylaminofluorene 50 mg/kg
11-Aminoundecanoic acid 50 mg/kg

Tissues and cell types examined:

hepatocytes

Details of tissue and slide preparation:

Primary hepatocyte cultures were prepared from rats as described in Mitchell, A. D. and J. C. Mirsalis (1984). Single Cell Mutation Monitoring System: ethodologies and Applications, Ansari, A. A. and F. de Serres (eds.), pp. 165-216, Plenum Pub. Corp., New York, and Mirsalis, J. C. et al. (1985). Carcinogenesis, 6:1521-1524.
Livers were perfused in situ with a solution of ethyleneglycolbis (ß-amino ethyl ether)N,N’-tetraacetic acid (EGTA) in Hanks’ balanced salt solution without Ca+2 or Mg2+, followed by a 37ºC solution of Type I collagenase in Williams’ Medium E.
A single-cell suspension of hepatocytes was obtained by combing out cells from the perfused liver into a petri dish containing 37ºC collagenase solution. Cells were collected by centrifugation, resuspended in cold medium, and filtered through sterile gauze. Viability was determined using Trypan blue exclusion. In general, hepatocyte viability was not adversely affected by test substance treatment, i.e. viability generally exceeded 70%, and attachment to the coverslips in the culture plate wells did not vary.
Approximately 6x10E+05 cells were seeded into each well of a 6-well culture plate. Each well contained a coverslip in Williams’ medium E (WE) supplemented with l-glutamine, gentamycin sulfate, and fetal bovine serum. After 1.5-2.0 hours incubation in a humidified atmosphere at 37ºC, 5% CO2, the cultures were washed to remove nonviable cells (those not attached to the coverslips).
Cultures were incubated in WE containing 3H-(methyl)thymidine for 4 hours at 37ºC and 5% CO2, followed by 14-18 hours in WE containing unlabeled thymidine. The cultures were then washed twice with WE, followed by hypotonic treatment with sodium citrate to swell the cells, fixed in glacial acetic acid:ethanol, and washed 3-6 times with deionized water. The dried coverslips were mounted to glass slides. The slides were dipped in nuclear track emulsion diluted with deionized water, and exposed at -20ºC for 7-14 days and then developed and stained as described in Mitchell and Mirsalis, 1984.

Measurement of UDS
Quantitative autoradiographic grain counting was accomplished as described [Mitchell and Mirsalis, 19841. Slides were coded prior to scoring to preclude scorer bias. An area of a slide was randomly selected, and 50 morphologically unaltered cells were counted using a colony counter The highest of two nuclear-sized areas over the cytoplasm and adjacent to the nucleus was subtracted from the nuclear count to determine the net grains/nucleus (NG). The percentage of cells undergoing repair (%IR) was determined as the percent of those cells exhibiting 5 or more NG.
Three slides were scored for each animal or concentration for a total of 150 cells per animal.

Measurement of S-Phase Synthesis
The number of cells in S-phase is easily distinguished in autoradiographic preparations. Slides were coded prior to scoring to preclude scorer bias. The number of S-phase cells and non-S-phase cells were counted beginning with a randomly selected patch of a slide. From 1,000 to 2,000 cells were scored from each of three slides per animal for a total of 3,000-6,000 cells per animal.

Evaluation criteria:

Measurement of UDS
The test substance was considered negative if the NG of all dose groups was a negative number and the %IR was less than 10%.
The test substance was considered positive f the average NG of any dose group exceeded 0 NG.
Test substances with negative NG values, but %IR values greater than 10% were considered equivocal

Measurement of S-Phase Synthesis
SPS responses less than 0.5% were considered negative.
Responses of 0.5-to 1.0% were considered to be equivocal.
Responses greater than 1.0% (when concurrent controls were <0.2%) were considered positive.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

4,4’-Oxydianiline failed to induce unscheduled DNA synthesis in rat hepatocytes following in vivo treatment.

Taken from TABLE 1 of the literature report - Measurement of UDS in Male Rat Hepatocytes Following In Vivo Treatment

 

	Dose (mg/kg)	Time (hr)	NG	Male rat (n)	%IR
4,4'-Oxydianiline	40	2	-3.3 ± 0.4	(3)	2 ± 1
		12	-2.5 ± 0.3	(3)	5 ± 2
	180	2	-3.0 ± 0.6	(3)	1 ± 0
		12	-4.4 ± 1.7	(3)	4 ± 4
	725	2	-2.9 ± 0.3	(3)	2 ± 1
		12	-5.0 ± 1.4	(3)	5 ± 3
Control/corn oil	-	2	-6.4 ± 2.9	(2)	1 ± 0
	-	12	-5.6 ± 0.4	(52)	2 ± 0

NG is the net grains / nucleus +/- the standard error of the mean (S.E.);

%IR is the percentage of cells with at least 5 NG +/- the standard error of the mean;

(n) is the number of treated animals.

Conclusions:

Conclusion: negative
4,4’-Oxydianiline failed to induce unscheduled DNA synthesis in rat hepatocytes following in vivo treatment.

Executive summary:

The substance was tested in the Unscheduled DNA Synthesis test. This study was conducted on males rats at the dose levels of 40, 180 and 725 mg/kg bw. The substance was administrated orally by a single bolus. Hepatocytes were collected and analyzed for DNA synthesis.

The results showed that the substance did not induce unscheduled DNA synthesis in rat hepatocytes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Justification for classification or non-classification

According to the data obtained in vitro and in vivo studies and the criteria in the CLP regulation, the substance is classified mutagen, category 1B (H340).

This classification is compliant with the harmonized European classification listed in the Annex VI of the CLP regulation.