Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

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.
Cross-reference
Reason / purpose:
reference to same study
Reference
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:
reference to same study
Qualifier:
equivalent or similar to
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.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): 4,4’-Oxydianiline
- Analytical purity: purity 98.9%
- Chemical source: Dupont (according to the paper)
- Molecular formula (if other than submission substance): Not specified
- Molecular weight (if other than submission substance): Not specified
- Smiles notation (if other than submission substance): Not specified
- InChl (if other than submission substance): Not specified
- Substance type: Not specified
- Physical state: Not specified
- Impurities (identity and concentrations): Not specified
- Composition of test material, percentage of components: Not specified
- Isomers composition: Not specified
- Purity test date: Not specified
- Lot/batch No.: Not specified
- Expiration date of the lot/batch: Not specified
- Radiochemical purity (if radiolabelling): Not applicable
- Specific activity (if radiolabelling): Not applicable
- Locations of the label (if radiolabelling): Not applicable
- Expiration date of radiochemical substance (if radiolabelling): Not applicable
- Stability under test conditions: Not applicable
- Storage condition of test material: Not applicable
Specific details on test material used for the study:
4,4'-oxydianiline
4,4'-diaminodiphenyl ether
CAS 101-80-4

Method

Species / strain
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.
Controls
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].

Results and discussion

Test results
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

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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