N2-acetyl-N-benzyl-O-methyl-L-serinamide

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 May 2000 to 25 July 2000

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

Test material

Test material information

Constituent 1

Reference substance name:

(R)-2-acetamido-N-benzyl-3-methoxypropionamide

EC Number:

605-756-0

Cas Number:

175481-36-4

Molecular formula:

C13H18N2O3

IUPAC Name:

(R)-2-acetamido-N-benzyl-3-methoxypropionamide

Test material form:

other: Crystalline powder

Specific details on test material used for the study:

batch number KK 02457

Test animals

Species:

rat

Strain:

other: Fischer (Iffa-Credo)

Details on species / strain selection:

Male rats are commonly used in this test and are recommended by the OECD guideline

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: IFFA CREDO origin, Saint-Germain-sur-l'Arbresle FRANCE)
- Age at study initiation: 5 to 6 weeks
- Weight at study initiation: 153 to 208 g
- Assigned to test groups randomly: yes, randomly assigned to the negative, positive control and treatment groups, 3 animals per group.
- Fasting period before study: no
- Housing: polypropylene cages covered by stainless steel netted lid
- Diet (e.g. ad libitum): not stated
- Water :ad libitum):
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 2'C
- Humidity (%): 55+/- 15%
- Air changes (per hr): 12 per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16 May 2000 To: 25 July 2000

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

0.5% carboxymethyl cellulose dosed at 10 ml/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test compound was dissolved in 0.5% carboxymethyl cellulose at 32 mg/mL (preliminary test) or 20 mg/mL (genotoxicity test) and used at a dose volume of 10 ml/kg

Duration of treatment / exposure:

single dose with expression times of either 2-4 hours or 12-16 hours after dosing

Frequency of treatment:

once

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

2-acetamidofluorene (positive control)

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Dimethylhydrazine (positive control)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

High dose

Doses / concentrations 4

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low dose

Doses / concentrations 5

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

4 treated and 3 used for hepatocyte culture per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

2-acetylaminofluorene; Dimethylhydrazine
- Justification for choice of positive control(s):
- Route of administration: oral
- Doses / concentrations: 25 and 10 respectively (single doses)

Examinations

Evaluation criteria:

For each slide and dose, the following parameters are calculated and presented in tables:
- the average NNG and standard deviation (SD),
- the percent of cells in repair and standard deviation (NNG 5),
- the population average cytoplasmic and nuclear grain count,
- the number of cells in S-phase.
The study would be considered valid if the negative control mean had zero NNG counts or less (i.e. a negative value, within the historical range). The positive control treatments should have va­lues of 5 or more NNG with 50 % or more cells having NNG counts of 5 or greater.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Investigation at two expression times (2-4 hours and 12-16 hours) in the in vivo Unscheduled DNA Synthesis (UDS) test in hepatocytes from male Fischer rats, treated orally once at 100 and 200 mg/kg was negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The test compound was investiga­ted at two expression times (2-4 hours and 12-16 hours) in the in vivo Unscheduled DNA Synthesis (UDS) test in hepatocytes from male Fischer rats, treated orally once at 100 and 200 mg/kg. As a conclusion, the test item did not reveal any genotoxic activity un­der these test conditions.

Executive summary:

The potential genotoxic activity of the test item was tested using the In Vivo Unscheduled DNA Synthesis (UDS) test in rat hepatocytes in compliance with the OECD guideline no. 486.

Preliminary toxicity assay demonstrated that under the test conditions, the maximum tolerated dose of the test compound was 320 mg/kg by oral route, this dose was not retained as the top dose due to the severity of symptoms. Therefore, 200 mg/kg was retained as the top dose for the UDS test. A lower dose of 100 mg/kg was also tested. No mortality was noted in the animals treated at any doses during the UDS assay.

Negative control animals gave a group mean NNG value of less than zero with a percentage of cells in repair comparable with historical control data. In positive controls, group mean net nu­clear grain count (NNG) values as well as percentage of cells in repair obtained were within the range of historical control. These values showed the sensitivity of the animal strain used to two DNA damaging agents requiring metabolism for their action, dimethylhydrazine and 2-ace­tamidofluorene:' Therefore, the validity criteria for the test were fulfilled.

Over the two experiments, group mean net nuclear grain count (NNG) values at the two doses tested (100 or 200 mg/kg) were less than zero (-3.43 to -4.07 for 2-4 hour expres­sion time and -2.86 to -2.18 for 12-16 hour expression time), that is to say below the threshold value 0 NNG for a positive response.

Furthermore, no significant increase in the percentage of cells in repair at any doses tested when compared with the respective controls (2.87 % at 100 mg/kg and 2.42 % at 200 mg/kg vs 2.30 % in control in the 2-4 hour assay and 3.44 % at 100 mg/kg and 5.34 % at 200 mg/kg vs 4.16% in control in the 12-16 hour assay, respectively). In addition, in cells in re­pair,· group mean net nuclear grain count (NNG;::::5) ·values were comparable with the solvent controls  (7.37 at 100 mg/kg to 10.27 at 200 mg/kg vs 6.46 in control  in the 2-4 hour assay and 6.65 at 100 mg/kg to 7.38 at 200 mg/kg vs 6.81 in control in the 12-16 hour assay).

Moreover, the frequency of cells in S-phase was low from 0 to 0.1% in animals treated with 100 and 200 mg/kg of the test item in the 12-16 hour assay and from 0.6 to 1.3% in animals treated with 100 and 200 mg/kg in the 2-4 hour assay. It was concluded that the test item did not induce a proliferative effect in rat livers under the experimental conditions employed.

In conclusion, the test compound was investiga­ted at two expression times (2-4 hours and 12-16 hours) in the in vivo Unscheduled DNA Synthesis (UDS) test in hepatocytes from male Fischer rats, treated orally once at 100 and 200 mg/kg. As a conclusion, the test item did not reveal any genotoxic activity un­der these test conditions.