Aspartic acid

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Publication in a peer reviewed journal. Guideline study with GLP.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

Principles of method if other than guideline:

N-acetyl-L-aspartic acid (NAA) and L-aspartic acid (ASP) were evaluated for mutagenicity in the micronucleus assay in accordance with OECD 474 guidelines.
The main target of the publication was the investigation of N-acetyl-L-aspartic acid (NAA) (CAS 997–55-7). ASP was tested additionally as a control substance as NAA is known to be metabolized to ASP.

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Aspartic acid

EC Number:

200-291-6

EC Name:

Aspartic acid

Cas Number:

56-84-8

Molecular formula:

C4H7NO4

IUPAC Name:

aspartic acid

Details on test material:

L-Aspartic acid (CAS 56–84-8) was obtained from Sigma–Aldrich Corporation (St. Louis, MO).
N-acetyl-L-aspartic acid (CAS 997–55-7) was also obtained from Sigma–Aldrich Corporation (St. Louis, MO).

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Crl:CD1 (ICR) mice were obtained from Charles River Laboratories
- Assigned to test groups randomly: yes, under basis of body weight
- Age at dosing: ca. 7 weeks
- Fasting period before study:
- Housing: stainless steel, wire-mesh cages suspended above cage boards
- Diet (e.g. ad libitum): Certified Rodent Diet #5002, PMI Feeds, Inc; ad libitum
- Water (e.g. ad libitum): tap water; ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Animals were clinically normal and free of antibody titers to pathogenic murine viruses and mycoplasma and free of pathogenic endo- and ectoparasites and bacteria.

After a quarantine period of approximately 5 days, mice that displayed adequate weight gain and freedom from clinical signs were divided by computerized, stratified randomization into three treatment groups of , with additional animals in the highest dose groups, so that there were no statistically significant differences among group body weight means.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Deionized water and aqueous methylcellulose (0.5%) in deionized water were used as the dosing vehicles for NAA and ASP, respective.
The dosing volume was 10 mL/kg.

Duration of treatment / exposure:

Single dosing.

Frequency of treatment:

Once.

Post exposure period:

Approximately 24 and 48 h following dosing, half of the animals in each group were sacrificed.
All animals in the positive control group were sacrificed 24 h post-dosing.

Doses / concentrations

Remarks:

Doses / Concentrations:
Doses: 333 (NAA), 500 (ASP), 1000, or 2000 mg/kg/body weight (bw).
Basis:
nominal conc.

No. of animals per sex per dose:

n = 5/sex for the low and middle dose groups and 7/sex for the highest dose.

Control animals:

yes, concurrent vehicle

Positive control(s):

Mice in the positive control group were given 30 mg/kg/body weight of cyclophosphamide dissolved in deionized water.

Examinations

Tissues and cell types examined:

Erythrocytes from bone marrow.

Details of tissue and slide preparation:

Following sacrifice, bone marrow was harvested from femurs of each animal by aspiration into a syringe containing fetal bovine serum (FBS). Erythrocytes were collected by centrifugation after transferring into a tube containing FBS. The sediment was re-suspended in FBS and one drop of the suspension was placed onto a clean microscope slide and smears were made using an Auto Prep blood smearing instrument (Genometric Data, Wayne, PA). At least three slides per mouse were prepared and air-dried, labeled with random numbers to blind the evaluation, fixed in absolute methanol and stained in acridine orange.

Evaluation criteria:

One thousand erythrocytes per animal were examined for signs of bone marrow toxicity to obtain the proportion of immature erythrocytes (polychromatic, PCE) to total (PCE + normochromatic; mature) and 2000 PCEs per subject were evaluated for presence of micronuclei to determine clastogenic effects. Low and intermediate dose groups were not analyzed at the 48 h time point.
Validity of the test was verified by the following; the mean frequency of MNPCEs in the vehicle group did not exceed 10/2000 PCES and were within the limits of the laboratory’s historical control range. Both biological and statistical significance were weighed for determining a positive response.

Statistics:

Data for the proportion of micronucleated PCEs among 2000 polychromatic erythrocytes and the proportion of PCEs among 1000 erythrocytes (MNPCE and PCE frequency, respectively) were analyzed statistically by using analysis of variance (ANOVA), Dunnett’s and Dunn’s test. The individual animal was considered the experimental unit. All data analyses were one-tailed and conducted at a significance level of 5%.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Neither NAA nor ASP caused an increase in the number of MNPCEs at any dose evaluated.
There was a significant increase in the frequency of MNPCEs in the positive control (cyclophosphamide) group compared to the negative control group.
As no toxicity was observed following exposure to NAA at the limit dose (2000 mg/kg of body weight) during the range finding study and no positive response was recorded at the 24 h time point, slides from the low and intermediate groups were not analyzed at the 48 h time point.
No clinical signs or differences in body weights were observed in mice during the course the study.
Based on these results, NAA and ASP were not considered clastogenic or aneugenic in the bone marrow micronucleus assay.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Based on the results, N-acetyl-L-aspartic acid (NAA) and L-aspartic acid (ASP) were not considered clastogenic or aneugenic in the bone marrow micronucleus assay.

Executive summary:

N-acetyl-L-aspartic acid (NAA) and L-aspartic acid (ASP) were evaluated for mutagenicity in the micronucleus assay in accordance with OECD 474 guidelines.

The main target of the publication was the investigation of N-acetyl-L-aspartic acid (NAA) (CAS 997–55-7). ASP was tested additionally as a control substance as NAA is known to be metabolized to ASP.

Based on the results, N-acetyl-L-aspartic acid (NAA) and L-aspartic acid (ASP) were not considered clastogenic or aneugenic in the bone marrow micronucleus assay.