Reaction products of 4'-amino-4-nitrodiphenylamine-2-sulfonic acid diazo, 4-nitroaniline diazo, aniline-2,5-disulfonic acid diazo and resorcinol, sodium salts

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From the 22th of April to the 11th of June, 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

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

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From the 22th of April to the 11th of June, 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across from supporting substance (structural analogue or surrogate)

Remarks:

There are no OECD and EEC guidelines and reccomendations available at the moment of the test.

Justification for type of information:

Justification for Read Across is detailed in the endpoint summary and it is further detailed in the report attached to the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline available

Principles of method if other than guideline:

There are no OECD and EC guidelines and reccomendations available at the moment of the test.

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Source: SAVO, med. Versuchstierzuchten GmbH- Weight at study initiation: ca. 160 - 180 g
Assigned to test groups randomly: Yes
Housing: single cage with granulated soft wood bedding
Diet (e.g. ad libitum): pelleted standard diet (ALTROMIN 1324, D-4937 Lage/Lippe, F.R.G.)
Water (e.g. ad libitum): tap, ad libitum
Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
Temperature (°C): 21 ± 3 °C
Humidity (%): 30 - 70 %
Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light (light from 6:00 a.m. to 6:00 p.m.)

Route of administration:

oral: unspecified

Vehicle:

- Vehicle(s)/solvent(s) used: Aqua bidest- Justification for choice of solvent/vehicle: The vehicle was chosen according to its relative nontoxicity for animals.- Amount of vehicle: 10 ml/kg bw

Frequency of treatment:

Single administration

Post exposure period:

4 hours16 hours

Remarks:

Doses / Concentrations:100 mg/kg bwBasis:nominal in diet

Remarks:

Doses / Concentrations:1000 mg/kg bwBasis:nominal in diet

No. of animals per sex per dose:

4 animals per sex per group

Control animals:

no

Positive control(s):

- Positive control substance: 2-acetylaminofluorene- Dissolved in: dimethyl sulphoxide/polyethylene glycol 400 (1 + 9)- Route of administration: Oral- Frequency of administration: Single administration- Volume administered: 10 ml/kg bw

Tissues and cell types examined:

Hepatocytes of rat liver were analyzed after treatment.

Details of tissue and slide preparation:

Isolation of the Primary Hepatocytes
The animals were sacrificed by liver perfusion. After anaesthetizing the rats with Nembutal the liver was perfused through the vena portae with Hank's balanced salt supplemented with collagenase (0.05 % w/v,) adjusted to pH 7.4 and maintained at 37 °C.The hepatocytes were isolated from the liver and washed twice with HESS.
The crude cell suspension was filtered through a 94 gm stainless steel mesh to yield a single cell suspension. The quality of the actual performed perfusion was determined by the trypan blue dye exclusion method. In addition, the number of the isolated cells was determined.
Cцlture Conditions
The washed hepatocytes were centrifuged and transferred into Williams medium E, supplemented with:Hepes (2.38 mg/l)Penicillin (100 units/ml)Streptomycin (0.10 mg/ml)Glutamin (0.29 mg/ml)Insulin (0.50 µg/ml)Fetal Calf Serum (100 µl/ml).
The medium without the cells was adjusted to pH 7.6.At least five cultures were established for each animal. Aliquots of 2.5 ml with freshly isolated hepatocytes in complete culture medium (1.0 x 10^5 cells/ml) were added to 35 mm six-well cluster dishes, containing one gelatinized 25 mm round plastic coverslip per well.After an attachment period of approximately 1.5 h in a 95 % air/ 5 % CO2 humidified incubator at 37 °C the culture medium was discarded. Then the cell layer was rinsed once with PBS to remove non-adherent cells.
Subsequently 3HTdR (5 µCi/m1, specific activity 20 Ci/mmol; New England Nuclear, D-6072 Dreieich, F.R.G.) in 2.0 ml culture medium (WME, 1% FCS) was added to the cultures. After a labelling time of 4 h the cells were washed twice with WME supplemented with 1 % FCS and 0.25 mM unlabelled thymidine. Cultures were incubated overnight using the same medium. To prepare for autoradiography the medium was replaced by a hypotonic solution of 1 % sodium citrate for 10 minutes to swell the nuclei for better grain quantification. The cells on the coverslips were then fixed by three changes of methanol: acetic acid (3+1 v/v) for 15 minutes each, rinsed with 96 % ethanol, and air dried.Autoradiographic Parocessin.
The cover slips were mounted the side carrying the cells up on glass slides and coated with ILFORD K-2 photographic emulsion in the dark.
The coated slides were stored in light-proof boxes in the presence of a drying agent for 12 -14 days at 4 °C. The photographic emulsion is then developed at room temperature, fixed in TETENAL and stained with hematoxylin/eosin.

Evaluation criteria:

QUANTIFICATION OF DOSE
valuation was performed microscopically on coded slides using NIKON microscopes with oil immersion objectives. The number of silver grains above the nucleus was counted automatically using the ARTEK 880 or 982 counter. In addition, the number of grain counts of one nuclear-sized cytoplasm adjacent to the nucleus was counted. At least two slides per animal and 50 cells per slide were evaluated. Heavily labelled S-phase cells were excluded from counting.Three animals per group were evaluated as described above. The remaining animal per test group would be evaluated if an animal dies spontaneously or in case of technical problems concerning the isolation of the hepatocytes.
EVALUATION OF RESULTS
The test article is classified as positive if it induces either a statistically significant dose-related increase. in radiolabel incorporation expressed as grains per nucleus or a reproducible and statistically significant positive response for at least one of the test points.A test article producing neither a statistically significant dose related increase in radiolabel incorporation expressed as grains per nucleus nor a statistically significant and reproducible positive response at any one of the test points is considered non-effective in this system. Statistical significance can be evaluated by means of the non-parametric Mann-Whitney test.However, both statistical and biological significance should be considered together.

Statistics:

A statistical evaluation of the results was not necessary to perform as the number of nuclear and net grain counts of the groups treated with the test article were in the range of the corresponding controls

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

No toxic reactions of the animals occurred at any of the treatment periods or dose groups. In addition, the viability of thehepatocytes was not dramatically affected due to the in vivo pre-treatment with the test article. The interindividual variationsobtained for the numbers and the viabilities of isolated hepatocytes are in the range of our historical laboratory control.

No dose level of the test article revealed UDS induction in thehepatocytes of the treated animals as compared to the currentnegative controls. Neither the nuclear grains nor the resultingnet grains were enhanced due to the in vivo treatment of the animals with the test article for 4 hours or 16 hours, respectively.

An appropriate referencemutagen (2-ARF, 100 mg/kgbw) was usedas positive control. In vivo treatment with 2-AAF revealed distinct increases in the number of nuclear and net grain counts.

Conclusions:

Negative.
The test article did non induce DNA-Damage leading to repair synthesis in the hepatocytes of the treated rats

Executive summary:

Method

The test article was assessed in the in vivo UDS assay for its potential to induce DNA Repair (UDS) in the hepatocytes of rats.

The test item was formulated in aqua bidist. This suspending agent vas used as negative control. The volume administered orally was 10 ml/kg bw. After a treatment period of 4 and 16 hours, respectively, the animals were narcotized and sacrificed by liver perfusion. Primary hepatocyte cultures were established and exposed for 4 hours to 3HTdR which is incorporated if UDS occurs.

The test article was tested at the following dose levels:

4 hours treatment period: 1000 mg/kg bw

16 hours treatment: 100 and 1000 mg/kg bw

For each dose level, including the controls, hepatocytes from three treated animals were assessed for the occurrence of UDS.

Observations

No toxic reactions of the animals occurred at any of the treatment periods or dose groups. In addition, the viability of thehepatocytes was not dramatically affected due to the in vivo pre-treatment with the test article. The interindividual variationsobtained for the numbers and the viabilities of isolated hepatocytes are in the range of our historical laboratory control.

No dose level of the test article revealed UDS induction in thehepatocytes of the treated animals as compared to the currentnegative controls. Neither the nuclear grains nor the resultingnet grains were enhanced due to the in vivo treatment of the animals with the test article for 4 hours or 16 hours, respectively.

An appropriate referencemutagen(2-ARF, 100 mg/kgbw)was usedas positive control. In vivo treatment with 2-AAF revealed distinct increases in the number of nuclear and net grain counts.

Conclusion

During the described study and under the experimental conditions, the test article did not induce DNA – damage leading to repair synthesis in the hepatocytes of the treated rats.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

There are no OECD and EC guidelines and reccomendations available at the moment of the test.

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

Test material

Test material information

Constituent 1

Reference substance name:

Similar substance 01 of Acid Brown 314:1

IUPAC Name:

Similar substance 01 of Acid Brown 314:1

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Source: SAVO, med. Versuchstierzuchten GmbH- Weight at study initiation: ca. 160 - 180 g
Assigned to test groups randomly: Yes
Housing: single cage with granulated soft wood bedding
Diet (e.g. ad libitum): pelleted standard diet (ALTROMIN 1324, D-4937 Lage/Lippe, F.R.G.)
Water (e.g. ad libitum): tap, ad libitum
Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
Temperature (°C): 21 ± 3 °C
Humidity (%): 30 - 70 %
Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light (light from 6:00 a.m. to 6:00 p.m.)

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

- Vehicle(s)/solvent(s) used: Aqua bidest- Justification for choice of solvent/vehicle: The vehicle was chosen according to its relative nontoxicity for animals.- Amount of vehicle: 10 ml/kg bw

Frequency of treatment:

Single administration

Post exposure period:

4 hours16 hours

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:100 mg/kg bwBasis:nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:1000 mg/kg bwBasis:nominal in diet

No. of animals per sex per dose:

4 animals per sex per group

Control animals:

no

Positive control(s):

- Positive control substance: 2-acetylaminofluorene- Dissolved in: dimethyl sulphoxide/polyethylene glycol 400 (1 + 9)- Route of administration: Oral- Frequency of administration: Single administration- Volume administered: 10 ml/kg bw

Examinations

Tissues and cell types examined:

Hepatocytes of rat liver were analyzed after treatment.

Details of tissue and slide preparation:

Isolation of the Primary Hepatocytes
The animals were sacrificed by liver perfusion. After anaesthetizing the rats with Nembutal the liver was perfused through the vena portae with Hank's balanced salt supplemented with collagenase (0.05 % w/v,) adjusted to pH 7.4 and maintained at 37 °C.The hepatocytes were isolated from the liver and washed twice with HESS.
The crude cell suspension was filtered through a 94 gm stainless steel mesh to yield a single cell suspension. The quality of the actual performed perfusion was determined by the trypan blue dye exclusion method. In addition, the number of the isolated cells was determined.
Cцlture Conditions
The washed hepatocytes were centrifuged and transferred into Williams medium E, supplemented with:Hepes (2.38 mg/l)Penicillin (100 units/ml)Streptomycin (0.10 mg/ml)Glutamin (0.29 mg/ml)Insulin (0.50 µg/ml)Fetal Calf Serum (100 µl/ml).
The medium without the cells was adjusted to pH 7.6.At least five cultures were established for each animal. Aliquots of 2.5 ml with freshly isolated hepatocytes in complete culture medium (1.0 x 10^5 cells/ml) were added to 35 mm six-well cluster dishes, containing one gelatinized 25 mm round plastic coverslip per well.After an attachment period of approximately 1.5 h in a 95 % air/ 5 % CO2 humidified incubator at 37 °C the culture medium was discarded. Then the cell layer was rinsed once with PBS to remove non-adherent cells.
Subsequently 3HTdR (5 µCi/m1, specific activity 20 Ci/mmol; New England Nuclear, D-6072 Dreieich, F.R.G.) in 2.0 ml culture medium (WME, 1% FCS) was added to the cultures. After a labelling time of 4 h the cells were washed twice with WME supplemented with 1 % FCS and 0.25 mM unlabelled thymidine. Cultures were incubated overnight using the same medium. To prepare for autoradiography the medium was replaced by a hypotonic solution of 1 % sodium citrate for 10 minutes to swell the nuclei for better grain quantification. The cells on the coverslips were then fixed by three changes of methanol: acetic acid (3+1 v/v) for 15 minutes each, rinsed with 96 % ethanol, and air dried.Autoradiographic Parocessin.
The cover slips were mounted the side carrying the cells up on glass slides and coated with ILFORD K-2 photographic emulsion in the dark.
The coated slides were stored in light-proof boxes in the presence of a drying agent for 12 -14 days at 4 °C. The photographic emulsion is then developed at room temperature, fixed in TETENAL and stained with hematoxylin/eosin.

Evaluation criteria:

QUANTIFICATION OF DOSE
valuation was performed microscopically on coded slides using NIKON microscopes with oil immersion objectives. The number of silver grains above the nucleus was counted automatically using the ARTEK 880 or 982 counter. In addition, the number of grain counts of one nuclear-sized cytoplasm adjacent to the nucleus was counted. At least two slides per animal and 50 cells per slide were evaluated. Heavily labelled S-phase cells were excluded from counting.Three animals per group were evaluated as described above. The remaining animal per test group would be evaluated if an animal dies spontaneously or in case of technical problems concerning the isolation of the hepatocytes.
EVALUATION OF RESULTS
The test article is classified as positive if it induces either a statistically significant dose-related increase. in radiolabel incorporation expressed as grains per nucleus or a reproducible and statistically significant positive response for at least one of the test points.A test article producing neither a statistically significant dose related increase in radiolabel incorporation expressed as grains per nucleus nor a statistically significant and reproducible positive response at any one of the test points is considered non-effective in this system. Statistical significance can be evaluated by means of the non-parametric Mann-Whitney test.However, both statistical and biological significance should be considered together.

Statistics:

A statistical evaluation of the results was not necessary to perform as the number of nuclear and net grain counts of the groups treated with the test article were in the range of the corresponding controls

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

No toxic reactions of the animals occurred at any of the treatment periods or dose groups. In addition, the viability of thehepatocytes was not dramatically affected due to the in vivo pre-treatment with the test article. The interindividual variationsobtained for the numbers and the viabilities of isolated hepatocytes are in the range of our historical laboratory control.

No dose level of the test article revealed UDS induction in thehepatocytes of the treated animals as compared to the currentnegative controls. Neither the nuclear grains nor the resultingnet grains were enhanced due to the in vivo treatment of the animals with the test article for 4 hours or 16 hours, respectively.

An appropriate referencemutagen (2-ARF, 100 mg/kgbw) was usedas positive control. In vivo treatment with 2-AAF revealed distinct increases in the number of nuclear and net grain counts.

Applicant's summary and conclusion

Conclusions:

Negative.
The test article did non induce DNA-Damage leading to repair synthesis in the hepatocytes of the treated rats

Executive summary:

Method

The Similar substance 01 was assessed in the in vivo UDS assay for its potential to induce DNA Repair (UDS) in the hepatocytes of rats.

The test item was formulated in aqua bidest. This suspending agent vas used as negative control. The volume administered orally was 10 ml/kg bw. After a treatment period of 4 and 16 hours, respectively, the animals were narcotized and sacrificed by liver perfusion. Primary hepatocyte cultures were established and exposed for 4 hours to 3HTdR which is incorporated if UDS occurs.

The test article was tested at the following dose levels:

4 hours treatment period: 1000 mg/kg bw

16 hours treatment: 100 and 1000 mg/kg bw

For each dose level, including the controls, hepatocytes from three treated animals were assessed for the occurrence of UDS.

Observations

No toxic reactions of the animals occurred at any of the treatment periods or dose groups. In addition, the viability of thehepatocytes was not dramatically affected due to the in vivo pre-treatment with the test article. The interindividual variationsobtained for the numbers and the viabilities of isolated hepatocytes are in the range of our historical laboratory control.

No dose level of the test article revealed UDS induction in thehepatocytes of the treated animals as compared to the currentnegative controls. Neither the nuclear grains nor the resultingnet grains were enhanced due to the in vivo treatment of the animals with the test article for 4 hours or 16 hours, respectively.

An appropriate referencemutagen(2-ARF, 100 mg/kgbw)was usedas positive control. In vivo treatment with 2-AAF revealed distinct increases in the number of nuclear and net grain counts.

Conclusion

During the described study and under the experimental conditions, the test article did not induce DNA – damage leading to repair synthesis in the hepatocytes of the treated rats.