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Effects on fertility

Link to relevant study records
Reference
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.
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
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.
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.
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
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.
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.
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: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
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.
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.
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
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.
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.
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.
Reproductive effects observed:
not specified
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).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Klimisch 1.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information
Short description of key information:
A 2-generation reproduction toxicity test according to OECD 416 was performed in rats at a single dose of 500 mg/kg bw/d, with L-aspartic acid mixed to the diet. No toxic effects were observed at this dose.
It can be anticipated that no different toxic effects will be produced by the other routes because L-aspartic acid will be metabolised in each cell where it enters the Krebs cycle and the urea cycle and is rapidly metabolised to CO2.

Justification for selection of Effect on fertility via oral route:
A Klimisch 1 study.

Effects on developmental toxicity

Description of key information
Justifications for not to perform a developmental toxicity test were  based on scientific arguments, exposure arguments, existing data, previous assessments by authorities and avoidance of animal experiments.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Abnormalities:
not specified
Developmental effects observed:
not specified
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Justification based on scientific arguments:

L-aspartic acid is a naturally occurring substance. It is a basic component of proteins (together with L-glutamic acid they account for approximately 20-25 % of the total amino acid composition of dietary protein, including those found in human breast milk (Stegink LD., J Toxicol Environ Health 2 (1); 215-242; 1976). L-aspartic acid enters the citric acid cycle and the urea cycle and is metabolised there.

Protein digestion results mainly in free amino acids and small peptides in the intestinal lumen which were absorbed leading finally to blood plasma levels of L-aspartic acid between 1-25 µmol/L (Slocum and Cummings, 1991). The structurally closely related amino acid glutamic acid has blood plasma levels of 10-131 µmol/L (Slocum and Cummings, 1991). Cells contain a considerable quantity of free L-aspartate and L-glutamate. Considerable quantities of these amino acids are also found in human brain and liver.

It is obvious that human cells handle large quantities of L-aspartate / L-aspartic acid and that this amino acid is an essential component of normal cellular function.

It is therefore highly improbable that L-aspartic acid possesses any developmental toxicity activity and therefore further testing of L-aspartic acid is not justified.

Justification based on exposure arguments:

L-aspartic acid which is absorbed in the maternal organism is metabolised and does not reach the fetal system in a comparably high concentration as in the maternal system. Stegink (Journal of Toxicology and Environmental Health 2:215-242, 1976) investigated this issue and pointed out "that a maternal intake in excess of 1 g/kg would be required in order to significantly raise the fetal plasma level, a highly unlikely possibility. Thus, the human fetus, even if susceptable, is at little risk from maternal dicarboxylic amino acid [aspartate or glutamate] ingestion." A significant increase of the plasma level in the fetus is a prerequisite for a toxic effect in the fetus.

Justification based on existing data:

- Stegink 1976, see section 7.8.2, has investigated and reviewed the risk to the fetus and neonate from maternal ingestion of the 2 dicarboxlic amino acids, L-aspartic acid and glutamic acid, and came to the conclusion "Thus, the human fetus, even if susceptable, is at little risk from maternal dicarboxylic amino acid ingestion.

- Zawoiski 1977 investigated the influence of L-aspartic acid in the diet of mice at a very large dose of ca. 10000 mg/kg bw/d, from Day 2 to Day 17 of gestation, after inducing malformations with trypan blue. No teratogenic nor antiteratogenic effect of L-aspartic acid was found. L-aspartic acid had a protective effect against embryolethality.

- In a 2-generation study with rats (Karaman 2011, see section 7.8.1) no adverse effects to the unborn or the newborn were detected.

Justification based on previous assessments by authorities:

L-aspartic acid (and L-aspartate) is regarded as “inherently safe” by the US FDA and can be added to the diet by the food industry.

The statement in the WHO Technical Report Series 928, Evaluation of certain food additives, Sixty-third report of the Joint FAO/WHO Expert Committee on Food Additives, WHO 2005, reads:

"The Committee was of the opinion that the use of the Procedure for the Safety Evaluation of Flavouring Agents (...) was inappropriate for 12 members of this group, namely, the eleven l-form a-amino acids (...; l-glutamic acid, No. 1420; ...; l-aspartic acid, No. 1429; ...) and the one alpha-imino acid (l-proline, No. 1425). These substances are macronutrients and normal components of protein and, as such, human exposure through food is orders of magnitude higher than the anticipated level of exposure from use as flavouring agents."

Justification based on avoidance of animal experiments:

The performance of a new developmental toxicity study with L-aspartic acid will not provide new insight or new results in the possible developmental toxicity. It has therefore to be avoided.


Justification for selection of Effect on developmental toxicity: via oral route:
Not relevant.

Justification for classification or non-classification

No evidence was obtained to justify a classification of L-aspartic acid.

Additional information

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