Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

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.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material: p,p'-oxydianiline or 4,4'-diaminodiphenyl ether or 4,4'-oxidianilline
- 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.
- Structural formula attached as image file (if other than submission substance): Included within the literature report; confirms the identity.
- Substance type: Not specified.
- Physical state: Not specified.
- Analytical purity: 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

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male
Details on test animals 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.

Administration / exposure

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.
Doses / concentrationsopen allclose all
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).

Examinations

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.

Results and discussion

Test results
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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.