Aspartic acid

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Recently published guideline study.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

Some additional investigations were performed. Only one dose was tested.

Principles of method if other than guideline:

The focus of the investigation was on N-acetyl-L-aspartic acid.
L-aspartic acid served as a control for comparison.

GLP compliance:

yes

Limit test:

yes

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:

Name: L-aspartic acid; ASP
Supplier: VWR International, LLC, Catalog No. AS125.
Lot: Multiple vials of ASP from different lots were used in the study.
Purity: of the lots ranged from 99.3% to 100.4%, according to the manufacturer’s certificates of analysis.

N-acetyl-L-aspartic acid; NAA; 100% pure was obtained from the Sigma–Aldrich Co., Catalog No. 00920

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Supplier: Charles River Laboratories Inc. (Portage, MI)
- Age: at least 45 days old at receipt
- Identification: Monel_ self-piercing ear tags.
- Acclimation: at least six days.
- Assignment to groups: by randomisation.
- Housing: The rats were housed individually throughout most phases of the study in stainless steel wire bottomed cages.
Adult male and female rats in the P and F1 generations cohabited in the male rat’s cage until mating was confirmed or up to a maximum of 21 days.
F1 and F2 generation pups cohabited with their respective dams in nesting boxes layered with bedding until weaning.
All cage sizes and housing conditions were in compliance with the Guide for the Care and Use of Laboratory Animals.
- Diet (e.g. ad libitum): Diets were available ad libitum from individual feeders except the evening prior to sacrifice.
- Water (e.g. ad libitum): Water was available to the rats ad libitum both from an automatic watering system and individual water bottles attached to the cages.

ENVIRONMENTAL CONDITIONS
- Temperature (°C), Humidity (%): Room temperature and relative humidity were monitored constantly and were maintained at 19 to 25 °C and at 30–70%, respectively.
- Air changes (per hr): The study room was maintained under conditions of positive airflow relative to a hallway and independently supplied with a minimum of 10 changes per hour of 100% HEPA-filtered fresh air.
Photo period: 12-h light/dark cycle.

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

A commercially sourced rodent laboratory diet in meal form (Certified Rodent LabDiet_ #5002, PMI_ Nutritional International, St. Louis, MO) served as carrier.
A carrier control group received diet with no added NAA, and a comparative control group was administered diet containing the parent amino acid (ASP) at a target dose of 500 mg/kg of body weight/day. NAA was administered in the diet at three target doses; 100, 250 and 500 mg/kg of body weight/day.
The diets were prepared weekly and stored at room temperature in light-proof containers.

Details on mating procedure:

After 10 weeks of exposure, each female was cohabited with one male from the same exposure group until pregnancy was verified or 2 weeks had elapsed. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be at day 0 of presumed gestation and assigned to individual housing (in nesting boxes). Female rats not mated within the first 14 days of cohabitation were assigned alternate male rats from the same dosage group that had mated and remained in cohabitation for a maximum of seven additional days. Female rats not mated after completion of 21-day cohabitation were considered to be at day 0 of presumed gestation on the last day of cohabitation and assigned individual housing (in nesting boxes).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of NAA and ASP in the diets covering the range of concentrations used in this study was determined in a subchronic toxicity study conducted concurrently at the same test facility with the same test substance and doses. The test substance was determined to be stable in the formulations after storage for at least 21 days at room temperature.

Duration of treatment / exposure:

From 10 weeks before mating the P-generation to 80 days postnatal of the F2-generation.

Frequency of treatment:

Continuously.

Details on study schedule:

The day of birth was defined as day 1 of lactation (DL1 postpartum). To avoid potential biases in pup viabilities and body weight gains, F1 and F2 generation litters were not culled during the lactation period. All F1 and F2 generation pups were weaned at postnatal day (PND) 22 through PND25. The surviving F1 and F2 generation male and female pups from each litter were assigned to one of four subsets (1 pup/sex/litter/subset, when possible).
Types of evaluations conducted for each subset are indicated in Table 1. F1 and F2 generation pups (Subsets 1, 2, 5 and 6) were not individually identified during the lactation period; all response variables were evaluated in terms of the litter.
At weaning, F1 generation (Subsets 3 and 4) and F2 generation (Subsets 7 and 8) pups selected for continued observation were identified using tail tattoo or Monel self-piercing ear tags and housed individually during the rest of the study.
At sacrifice of the F1 and F2 generation pups and adult rats (Subsets 1 [pups] and 3 [adults], and Subset 5 [pups] and 7 [adults], respectively), brain weights were recorded and a neurohistological examination was performed on brain tissues from pups or adult rats selected randomly (total of 10 rats/sex/group).
F1 generation Subset 4 rats were randomly assigned to cohabitation (one male rat per female rat) by use of random units. In the event that random assignment resulted in the pairing of F1 siblings, an alternate assignment was made. The cohabitation period lasted a maximum of 21 days. Alternate male rats that mated were assigned to female rats which did not mate within the first 14 days of cohabitation and the beginning of presumed gestation was determined as described above for P generation female rats. See also the attachment.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
500 mg L-aspartic acid/kg bw/d. The actual average consumed doses of ASP in male and female rats across generations were 462.9 and 629.8 mg ASP/kg of body weight/day for the target dose of 500 mg ASP/kg of body weight/day, respectively.
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
100; 250 and 500 mg N-acetyl-L-aspartic acid/kg bw/d. The actual doses in male and female rats across generations were 93, 232 and 471 mg NAA/kg bw/day, and 120, 307 and 633 mg NAA/kg bw/day for target doses of 100, 250 and 500 mg NAA/kg bw/d.
Basis:
nominal in diet

No. of animals per sex per dose:

25.
Five groups were used: negative control; comparative control (L-aspartic acid); 3 groups with NAA.

Control animals:

yes, concurrent no treatment

Positive control:

No.

Examinations

Parental animals: Observations and examinations:

Clinical observations and body weight and feed consumption measurements were conducted according to OECD Guideline 416 (2001).

Oestrous cyclicity (parental animals):

For P generation and F1 generation dams, estrous cyclicity was evaluated by examination of vaginal cytology for 14 days before the scheduled cohabitation period and then until spermatozoa were observed in a smear of the vaginal contents and/or a copulatory plug was observed in situ during the cohabitation period. Male and female rats from F1 generation Subset 3, and F2 generation Subsets 7 and 8 were observed for the age at which sexual maturation begins. Female rats were evaluated for the age of vaginal patency (VP), beginning on day 28 postpartum. Male rats were evaluated for the age of preputial separation (PS), beginning on day 39 postpartum. The body weight was recorded for each rat on the day VP or PS was observed.

Litter observations:

Behavioural and developmental assessment of progeny (F1 and F2 generations; this assessment included motor activity, passive avoidance and water maze performance).

Postmortem examinations (parental animals):

Anatomic pathology including organ weights, histopathological evaluation of reproductive organs and organs related to reproduction and neurohistopathological evaluation.

Postmortem examinations (offspring):

Anatomic pathology including organ weights, histopathological evaluation of reproductive organs and organs related to reproduction and neurohistopathological evaluation.
At sacrifice of the F1 and F2 generation pups and adult rats (Subsets 1 [pups] and 3 [adults], and Subset 5 [pups] and 7 [adults], respectively), brain weights were recorded and a neurohistological examination was performed on brain tissues from pups or adult rats selected randomly (total of 10 rats/sex/group).

Statistics:

With the exception of the concentrations of NAA and ASP in brain and plasma, statistical analyses were carried out using the SAS System.
Individual rat and litter values were used as the unit measured in evaluation of adult and pup data, respectively.
First, the carrier control group was compared with each of the NAA exposure groups. When statistical significance was identified in that analysis for a particular response variable (e.g., mean body weight value on the day of preputial separation in 500 mg of NAA/kg of body weight/day group), the same scheme was used to compare the comparative control group to each of the NAA exposure groups.

For the statistical analysis of concentrations of NAA and ASP in brain and plasma collected from P generation rats and rats from certain age-matching subsets of F1 and F2 generations, a two-way Analysis of Variance was performed first, followed by comparison with tolerance intervals which were calculated to capture 99% of the values of the control population with a 95% confidence level.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

Including ophthalmology.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

No treatment-related differences in mean estrous stages per 14 days, lengths of durations of dioestrus or estrus, natural delivery or litter observations were observed in female rats from any of the NAA exposure groups compared with the female rats from the carrier or comparative control groups in either the P or F1 generations.
There were no differences in any of the mating or fertility parameters in male or female rats from any of the NAA exposure groups compared with the carrier or comparative control group values in the P generation.
No biologically important or test substance-related differences were observed in terminal body weights, organ weights, ratio (%) of organ weights to terminal body weights, or ratio (%) of organ weights to brain weights in male or female rats from any of the NAA exposure groups compared with the carrier control group or the comparative control group values in the P, F1 or F2 generations.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: A nominal dose of 500 mg/kg bw/d corresponds to an actual average consumed doses of ASP in male and female rats across generations of 462.9 and 629.8 mg ASP/kg of body weight/day, respectively.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

The mean age at which vaginal patency or preputial separation were observed and body weights when sexual maturation was observed were comparable across all groups with one exception, a not relevant
No biologically important or test substance-related differences were observed in terminal body weights, organ weights, ratio (%) of organ weights to terminal body weights, or ratio (%) of organ weights to brain weights in male or female rats from any of the NAA exposure groups compared with the carrier control group or the comparative control group values in the P, F1 or F2 generations. In the P generation, lower mean absolute pituitary weight (p < 0.05) and a lower mean ratio (%) of the pituitary weight to terminal body weight values (p < 0.05) were observed in male rats from the comparative control group and the 100 and 500 mg NAA/kg of body weight/day exposure groups compared with the carrier control group. These observations were not test substance related because similar differences were not observed in females and differences were not dose-dependent. Additionally, the individual data values were within the range of historical control data recorded at the testing facility.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: A nominal dose of 500 mg/kg bw/d corresponds to an actual average consumed doses of ASP in male and female rats across generations of 462.9 and 629.8 mg ASP/kg of body weight/day, respectively.

Results: F2 generation

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: A nominal dose of 500 mg/kg bw/d corresponds to an actual average consumed doses of ASP in male and female rats across generations of 462.9 and 629.8 mg ASP/kg of body weight/day, respectively.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The target dose of 500 mg L-aspartic acid/kg of body weight/day represents the no-observed-adverse-effect-level (NOAEL) for systemic toxicity and reproductive toxicity from dietary exposure for two generations for male and female Sprague–Dawley rats.

Executive summary:

A 2-generation reproduction toxicity test according to OECD 416 was performed in rats with the main goal to investigate N-acetyl-L-aspartic acid. L-aspartic acid was used as a comparative control at 500 mg/kg bw/d, mixed to the diet.

The no-observed-adverse-effect-level (NOAEL) for systemic toxicity and reproductive toxicity was 500 mg L-aspartic acid/kg of body weight/day (and also 500 mg N-acetyl-L-aspartic acid/kg of body weight/day).