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Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

The study from Matsueda and Niiyama on effects of dietary excess of phenylalanine on maintenance of pregnancy and reproductive performance, including fetal growth in rats showed that pregnancy was maintained in 100% of the rats on diets with the excess of phenylalanine; the weights of conception products were similar to those of the corresponding pair-fed control; the weights of fetuses were significantly lower than those of the control; the litter size was normal in all; did not result in appreciable alterations in plasma or brain
amino acid concentrations. It has to be considered, that this scientific study was not performed according to common guidelines and the feeding conditions in this study are very artificial which by itself have significant influence on fetal growth. It is well known that restricted maternal protein supply perturbs fetal growth (e.g. Rees et al 1999). Offspring of pregnant rats fed on protein-deficient diets (like it is the case in the Matsueda and Niiyama study) are not only slightly smaller than controls fed on an adequate diet but also have permanent changes in the structure and function of their organs which result in reduced glucose tolerance and hypertension in adult life. Therefore, seen effects may be caused only by this extremely artificial situation of protein-restriction in combination with amino acid excess. Such situations are very artificial and unlikely for humans to occur.


The two-generation reproductive toxicity study of the read-across substance aspartame showed similar effects. All parameters were comparable between the control and test groups with the exception of body weight suppression at weaning among the high dose pups which was evident during both the first (F1) and
second (F2) generation litters. Statistical analysis performed on the F1 and F2 litters revealed the body weights at the end of weaning of both sexes at the high dose level to be statistically significantly lower. The high dose pups
were noted to be generally smaller than the control and lowdose pups. 


It is considered that the depression in pup body weight in the high dose groups might be accounted for by the effect of high exposure to phenylalanine from metabolism of aspartame and it should be regarded as an (indirect) effect related to the test compound. This might provide a plausible explanation for the effects of aspartame on pup body weight at weaning.


The studies do not indicate any particular cause for concern and do not provide a suitable point of departure for derivation of a toxicological reference value.


A significant amount of L-phenylalanine is usually taken up via the food. In usual diet, most amino acids are supplied as constituents of protein and not as free amino acid. Phenylalanine is an essential amino acid and therefore must be supplied by the diet. The recommended aromatic amino acid requirement (phenylalanine) is set at 25 mg/kg bw/day (WHO, 2007).
In the US, the Institute of Medicine sets estimated average requirement (EAR) for amino acids; phenylalanine EAR for adults are estimated at 27 mg/kg bw/day (IOM, 2005). The mean daily consumption of phenylalanine is estimated in the US at 3.4 g/day (IOM, 2005). The levels of phenylalanine content per food category range up to 31.05 mg/g. These levels linked to the EFSA Comprehensive Database allow calculation of phenylalanine intake from the diet. These intakes range up to 4.1 g/day (corresponding to 58.7 mg/kg bw/day for adults) at the mean within the EU population.
Taking the molecular weight of phenylalanine into account, the factor for aspartame exposure to phenylalanine exposure is 56 %. Protein intake clearly modifies plasma amino acid levels. However, amino acid concentrations are subject to homeostasis and the plasma concentrations vary within fixed limits and are tightly regulated.


Exposure with L-phenylalanine from uses which are covered by this registration would only marginally increase the total daily L-phenylalanine dose which is taken up via the food. Even if the plasma amino acid concentration would increase/vary by any use such fluctuations are physiological and subject to homeostasis. Therefore it is highly unlikely that L-phenylalanine taken up via any use covered by this registration would result in systemic effects.


Reference:


Rees WD, Hay SM, Buchan V, Antipatis C, Palmer RM. (1999).The effects of maternal protein restriction on the growth of the rat fetus and its amino acid supply.Br J Nutr.; 81(3):243-50.

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
Lack of information/clarity on dosing – which seems to have stopped at mating, rather than continuing to weaning · No information on stability and homogeneity of diet mixes, or whether any analyses of concentrations were conducted. No calculations of actual doses given. No other internal inconsistencies within the report were noted, although there is very little data present.
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
please see attached document
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
no
Remarks:
Study was reported before any GLPs extant.
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Route of administration:
oral: feed
Duration of treatment / exposure:
Start of dosing: Nine weeks before start of mating. Duration: Two parental generations, Two one-litter filial
generations
Frequency of treatment:
daily, 7 days each week
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
900 mg/kg bw/day (nominal)
Dose / conc.:
1 800 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 Males 24 Females
Control animals:
yes, concurrent no treatment
Mortality:
no mortality observed
Description (incidence):
All P males and females survived their respective phases of study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights during pre-mating periods were generally comparable between groups.
Parental terminal body weights were generally comparable between groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Generally comparable between control and test groups.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental observations among a few control and test animals during one or both generations:
· Bloody crust on eyes
· Red eyelids
· Lacrimation
· Wheezing
· Hunched appearance
· Rough fur
· Alopecia
· Soft faeces
Reproductive performance:
no effects observed
Description (incidence and severity):
No indication of a compound-related effect on indices of:
· Fertility
· Gestation
· Live births
· Lactation
Dose descriptor:
NOAEL
Effect level:
> 1 800 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Critical effects observed:
no
Body weight and weight changes:
effects observed, treatment-related
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental findings among pups from a few control and test litters:
· Weak/thin appearance
· Hunching
· Wheezing
· Laboured respiration
· Squinting
Dose descriptor:
NOAEL
Generation:
F1a
Effect level:
>= 900 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Critical effects observed:
no
Mortality / viability:
mortality observed, non-treatment-related
Haematological findings:
no effects observed
Description (incidence and severity):
Haematology values:
· Haematocrit, haemoglobin and erythrocyte count were similar between groups.
o Slightly lower than in older animals; thought to be reflective of the age of the animals
· There was a general reduction in total leukocyte counts among treated groups at pnd 15 and 21
o Significant in males on pnd 15 and females on pnd 21
Blood chemistry values comparable between control and test groups.
· ALP values differed between 15 and 21 days postpartum
o This is attributed to normal variation for animals of this age.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross pathology observed in control or test pups killed by design 24 hr post partum or on pnd 5, 15 or 21.
Histopathological findings:
effects observed, non-treatment-related
Other effects:
effects observed, non-treatment-related
Dose descriptor:
NOAEL
Generation:
F2a
Effect level:
>= 900 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Reproductive effects observed:
no
Conclusions:
The NOAEL from these studies was determined to be 2000 mg/kg bw/day aspartame, based on the lower pup weights at weaning in both generations. This corresponds to a equimolar dose of L-Pheylalanine of 900 mg/kg bw/d.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed

Effects on developmental toxicity

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Scientific study not performed according to common guidelines. The feeding conditions: fed with phenylalanine-excess diet in this study. Dosing of females from day 1-21 of pregnancy.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Scientific study in which the effects of dietary excess of phenylalanine on maintenance of pregnancy
and reproductive performance, including fetal growth in rats was examined.
GLP compliance:
not specified
Species:
rat
Strain:
Sprague-Dawley
Route of administration:
oral: feed
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
from day 1 to day 14 or 21 of pregnancy
Dose / conc.:
5 other: %
No. of animals per sex per dose:
8 rats in the 5% Phenylalanine group
7 rats in the ad libitum control group
5 rats in the pair-fed control
Control animals:
yes
Details on study design:
Two experiments were carried out:
Experiment 1: the effect of amino acid-excess diets on maintenance of pregnancy was examined. For this purpose pregnant animals receiving the experimental diets were killed on day 14 of pregnancy and the products of conception (uterus, amniotic fluid, placenta, and fetuses) were weighed, because nutritionally induced fetal resorption begins on about day 10 of pregnancy.
Experiment 2: the effects of excess of phenylalanine on the nitrogen balance of the dams, the free
amino acid concentrations in maternal plasma and fetal brain and fetal growth were examined.
Maternal examinations:
BODY WEIGHT: Yes, daily
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, daily
Fetal examinations:
Fetuses weights,
Litter size

Reproductive indices
Weight of products of conception,
placenta weight,
fetuses weights,
fetal resorptions
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
During pregnancy, daily food intakes initially increased gradually, but near term they decreased app
reciably in all pregnant rats.
Number of abortions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Pregnancy was maintained in 100% of the rats on diets with an excess of phenylalanine.
The weights of conception products were similar to those of the corresponding pair-fed control.
The litter size was normal in all.
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Given excess phenylalanine, the weights of fetuses were significantly lower than those of the
control.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The litter size was normal in all.
Other effects:
no effects observed
Description (incidence and severity):
No changes in maternal plasma or fetal brain amino acid concentrations were observed in rats given phenylalanine-excess diet.
Fetal brain on diets with excess of phenylalanine were lower than the controls. This finding indicates that prenatal brain growth is severely and specifically affected by excess of aromatic amino acids.
Conclusions:
This study on effects of dietary excess of phenylalanine on maintenance of pregnancy and reproductive performance, including fetal growth in rats showed that pregnancy was maintained in 100% of the rats on diets with the excess of phenylalanine; the weights of conception products were similar to those of the corresponding pair-fed control; the weights of fetuses were significantly lower than those of the control; the litter size was normal in all; did not result in appreciable alterations in plasma or brain amino acid concentrations.
Executive summary:

The effects of diet with an excess of phenylalanine on the maintenance of pregnancy and fetal growth in rat were investigated in this study. Female Sprague Dawley rats weighing about 190 g were fed on 6 % casein diet (a low protein diet) containing 5% Phenylalanine from day 1 to 14 or 21 of pregnancy (also the day of autopsy). Pregnancy was maintained in 100 % of the rats on diets with an excess of phenylalanine. During pregnancy, daily food intakes initially increased gradually but near term they decreased appreciably in all pregnant rats. With regard to phenylalane in excess, the body weight gain during pregnancy for the groups receiving phenylalane were almost comparable to that of the pair-fed control. In all animals given excess amino acid phenylalnine the weights of conception products were similar to those of the corresponding pair-fed controls. However, in most groups given excess amino acid phenylalanine, the weight of foetuses was significantly lower than those of the respective pair-fed controls. The average weight of the foetuses of the groups receiving 5% excess phenylalanine was 3.24 ± 0.29 g compared to 4.11 ± 0.22 g for their pair-fed control. The litter size was almost normal in all rats on excess amino acid diets compared to the controls. The concentrations of phenylalanine in the maternal plasma were not increased appreciably by excess amounts of phenylalanine in the diet. Ingestion of diet containing excess phenylalanine did not result in appreciable alterations in plasma or brain amino acid concentrations.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Quality of whole database:
The artifically low protein diet may significantly contribute to the observed effects in this study.
Seen effects may be caused only by this extremely artificial situation of protein-restriction in combination with amino acid excess. Such situations are very artificial and unlikely for humans to occur.

Justification for classification or non-classification

Additional information