N-(1,1-dimethyl-3-oxobutyl)acrylamide

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:

1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study with GLP and equivalent to OECD guideline 486 that was not available at the time of performance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Remarks:

: no relevant deviations from the guideline that appeared 6 years later

Principles of method if other than guideline:

This test provides a method for investigating genotoxic effects of chemicals in liver cells. The end-point measured is indicative of DNA damage and subsequent repair. The liver is usually the major site of metabolism of absorbed compounds. It is thus an appropriate site to measure DNA damage in vivo. The UDS test with mammalian liver cells in vivo indicates DNA repair synthesis after excision and removal of a stretch of DNA containing a region of damage induced by chemical substances. The test is based on the incorporation of tritium labelled thymidine (3H-TdR) into the DNA of liver cells which have a low frequency of cells in the S-phase of the cell cycle. The uptake of 3H-TdR is determined by autoradiography.

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Test material

Test material information

Constituent 1

Reference substance name:

diacetone acrylamide

IUPAC Name:

diacetone acrylamide

Details on test material:

Appearance: White solid. Batch No.: A-46318Conditions of storage: room temperatureStability in aqueous solution: 10 hPurity: 99.2 %

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Savo-Ivanovas, 7964 Kisslegg, Germany- Weight at study initiation: 160 - 180 g- Assigned to test groups randomly: yes- Housing: single, Makrolon, type I- Diet (e.g. ad libitum): pelleted Altromin 1324The animals were starved overnight (4 h treatment) or ca. 6 h (16 h treatment) before dosing.- Water ad libitum: tap water- Acclimation period: >= 5 dENVIRONMENTAL CONDITIONS- Temperature (°C): 21 +- 3- Humidity (%): 30 - 70- Photoperiod (hrs dark / hrs light): light from 6:00 - 18:00

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Aqua bidest.

Duration of treatment / exposure:

4 h and 16 h

Frequency of treatment:

Once

Post exposure period:

Rats were sacrificed after the exposure, the liver was perfused. Hepatocyte cultures were prepared and exposed to 3H-TdR for 4 h.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:1000 mg/kg bwBasis:nominal conc.for the 4 h treatment period

Doses / concentrations 2

Remarks:

Doses / Concentrations:1000 mg/kg bwBasis:nominal conc.for the high dose in the 16 h treatment period

Doses / concentrations 3

Remarks:

Doses / Concentrations:100 mg/kg bwBasis:nominal conc.for the low dose in the 16 h treatment period

No. of animals per sex per dose:

5 male rats per group.5 hepatocyte cultures were established for each rat.3 of the cultures from each animal were used for the UDS assay.

Control animals:

yes, concurrent vehicle

Positive control(s):

2-acetylaminofluorene; dissolved in DMSO/polyethylene glycol 400 (1 + 9)Route of administration: orallyDoses / concentrations: 100 mg/kg bw

Examinations

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:Based on a range finding study at 1000 mg/kg bw. Result: no sign of toxicity.DETAILS on culture and slide PREPARATION:The washed hepatocytes were centrifuged and transferred into Williams medium E. Aliquots of freshly isolated hepatocytes in complete culture medium were added to 35 mm six-well cluster dishes. After an attachment period of ca. 1.5 h the medium was discarded. 3H-TdR was added for 4 h. After washing again the cultures were incubated overnight, followed by hypotonic treatment. The cells on coverslips were fixed and air dried. Autoradiographic processing:The cover slips were coated with Ilford K-2 photographic emulsion in the dark for 12 - 14 d at 4 °C.METHOD OF ANALYSIS:OTHER:

Evaluation criteria:

Evaluation was performed microscopically on coded slides. The number of silver grains above the nucleus was counted automatically. In addition, the number of grain counts of one nuclear-sized cytoplasm adjacent to the nucleus was counted. At least 2 slides per rat and 50 cells / slide were evaluated.A test substance 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 significant positive response for at least one of the test points.

Statistics:

Significance was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No toxic reaction occurred in any group. Viability and intro attachment of the hepatocytes was not affected to a relevant degree by the pre-treatment with the test substance.Neither the nuclear grains nor the resulting net grains were enhanced due to the in vivo treatment of the rats with the test substance for 4 h or 16 h.The positive control substance induced a distinct increase of the DNA repair.For the grain counts, see the attachment.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negativeThe test substance did not induce DNA-damage leading to repair synthesis.

Executive summary:

The test substance was assessed in an in vivo / in vitro UDS assay, to detect a possible induced DNA repair in hepatocytes. The treatment periods were 4 h and 16 h. The highest dose level was 1000 mg / kg body weight. DNA repair was followed by autoradiography after incorporation of tritiated thymidine.

No toxic reaction occurred in any group. Viability and intro attachment of the hepatocytes was not affected to a relevant degree by the pre-treatment with the test substance.

Neither the nuclear grains nor the resulting net grains were enhanced due to the in vivo treatment of the rats with the test substance for 4 h or 16 h. The test substance did not induce DNA-damage leading to repair synthesis.

The positive control substance induced a distinct increase of the DNA repair.