Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Additional information

The database concerning toxicity to reproduction for L-tryptophan is very limited, as both available studies cannot be finally evaluated (insufficient information for assessment or study focussed on non-typical endpoints).

L-tryptophan was tested in several acute and longer-term studies which gave no indication for adverse effects on reproductive organs leading to C&L. Therefore, there is no need for further testing on reproductive toxicity (e.g. 1- or 2-generation toxicity). In addition further testing on vertebrates is not considered to be required for animal welfare reasons.

Effects on developmental toxicity

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented publication/study report which meets basic scientific principles
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Source: Tyler Laboratories, Inc., Bellevue
Age at study initiation: no data
Housing: housed individually in screen bottom cages in a university-approved vivarium
Diet: ad libitum
Water: ad libitum
Acclimation period: no data

ENVIRONMENTAL CONDITIONS
Temperature (°C): 20
Humidity (%): no data
Air changes (per hr): no data
Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Rats of the control group or the pair-fed control group were fed with modified American Institute of Nutrition AIN-76 semi-purified diet. To minimize variations, the diet was mixed in large batches and sifted several times. After addition of oik, a part of the diet was separated as basis diet (control diet). The other part was mixed with L-tryptophan. The diets were stored in plastic bags, labeled and frozen until use.
Analytical verification of doses or concentrations:
no
Details on mating procedure:
animals were time-mated by the breeder
Duration of treatment / exposure:
20 days
Frequency of treatment:
continuously via diet
Duration of test:
termination on day 20 of gestation
No. of animals per sex per dose:
7
Details on study design:
Pilot projects
Two pilot studies were performed to determined if diets with excess of tryptophan support growth and health in non-pregnan female rats. 5 non-pregnant animals each received experimental diet with different tryptophan concentrations (500, 1000, 5000, 10000%). The body weight, behaviour and general apperance were determined for 4 wekks, before the animals were sacrificed and analyzed for alteration in organs or fat deposits. The group receiving 10000% of tryptophan had to be sacrificed after 10 days because of weight loss and changes in general apperance.

A second pilot project was designed to determine if the chosen supplementation level of tryptophan would support pregnant rats so that delivery of viable offspring would be possible. Virgin Sprague Dawley rats were time mated by the supplier.
With the appearance of a seminal plug, the following morning was designated as the beginning of day 0. The animals were delivered to the university vivarium on day 0, weighed and randomly assigned to groups. Six animals were fed the standard control diet and groups of two rats each were fed excess tryptophan (500 or 1000%). Animals were fed ad libitum and food intake and spillage were monitored five times weekly from day 0 through day 20 of gestation. Body weight was measured three times weekly. General appearance of the animals was carefully noted throughout the course of pregnancy. On day 20, the animals were sacrificed, the uteri and ovaries were removed and the numbers of corpora lutea and foetuses plus resorption sites were compared to verify the number of resorptions. Placentas were weighed on a top loading balance after removal of membranes and a general examination for external malformations was completed. The foetuses were measured and sexed.

Main study
Virgin Sprague-Dawley rats were time mated at night by the supplier. Because it was determined in earlier phases of the study that in some cases the experimental diet was associated with reduced food intake, pair-fed control groups were necessary for this final phase of the investigation. Controls (n=11) were fed the control diet ad libitum, while 7 rats each received excess tryptophan (500, 1000 or 2500%). In addition, 7 seven pair-fed control animals were fed the control diet, but at the level recorded for the experimental animals that they were paired with. Food intake was monitored daily from day 0 until day 20 of gestation.
On day 20 of pregnancy, the necropsy procedure referred to above was completed. Approximately three-fourths of the foetuses from each litter were placed in Bouin’s solution for subsequent examination of any structural variations. The remaining one-fourth of the foetuses from each litter was placed in 95% ethanol for later processing with alizarin stain which facilitated skeletal examination. After two weeks’ fixation in Bouin’s solution, the foetuses were razor sectioned. The head, neck and lower trunk were serially sectioned with a razor blade and after opening the thoracic cavity; sagittal sections were made of the heart. The foetuses stained for approximately 24 hr with alizarin red stain and subsequently placed in increasing concentrations of glycerine were examined generally for skeletal defects and variations in ossification. The number of ribs and phalanges were counted.
Maternal examinations:
CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: No data
BODY WEIGHT: Yes
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
POST-MORTEM EXAMINATIONS: No data
Ovaries and uterine content:
Ovaries and uterine content: Yes
Examinations included number of corpora lutea, number of implantations and number of early / late resorptions
Fetal examinations:
External examinations: No data
Soft tissue and skeletal examinations: Yes (3/4 and 1/4 per litter, respectively)
Statistics:
One-way analysis of variance (ANOVA) and Duncan’s multiple comparison procedure
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: significantly lower mean maternal weight gain

Details on maternal toxic effects:
MORTALITY
There were no substance-related or spontaneous mortalities in any of the groups.

Pilot projects
The first pilot project provided data related to the effects of amino acid supplementation on growth and behaviour of adult female rats. Excess tryptophan (10000%) promoted rapid weight loss and was therefore deemed unacceptable for further study. The animals fed the higher amino acid supplemented diets were more active when compared to the control animals or those on the lower supplemented diets. Otherwise, all of these animals appeared reasonably healthy with the exception of those provided excess tryptophan (5000%), which developed scaly tails and thinning hair. Based on these observations, 500 and 1000% excess tryptophan were chosen for further investigation.
The second pilot project examined the impact of the selected diets on the course of pregnancy. All animals appeared healthy and gained weight steadily throughout the course of pregnancy. Necropsy revealed viable foetuses and in total 13 visible resorptions were present among all experimental animals and 1 visible resorption among the 6 control animals. The placentas of all groups appeared normal. These results supported the use of the chosen diets for the next step in the study with an additional level of tryptophan supplementation (2500%).

Main study
The tryptophan groups and their pair-fed controls consumed decreasing amounts of diet with increasing levels of tryptophan. Rats fed the highest tryptophan-supplemented diet (2500%) had a significantly lower mean maternal weight gain than any of the other groups and also a significantly smaller food efficiency ratio.
Dose descriptor:
NOAEL
Effect level:
ca. 2 220 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
There were no statistically significant differences within tryptophan groups in the mean number of viable foetuses. There were no significant differences in the mean number of early resorptions. Rats dosed with 1000% had significantly more late resorptions than any of the other tryptophan dosed rats. Total foetal weights per litter differed significantly between the highest and lowest tryptophan-supplemented groups (2500%: 32.4 +/- 4.3 g; 500%: 47.3 +/- 2.1 g). Within the tryptophan groups, two groups differed significantly from each other in average foetal weight (2500%: 3.7 +/- 0.1 g vs. 4.1 +/- 0.1 g in 1000% pair-fed controls). Considerable variation was found among the tryptophan groups in foetal length (not further specified). Among the tryptophan groups, no groups of rats differed significantly from each other in total placental weight. No significant differences were found in the ratio of males to females. No obvious congenital malformations or abnormalities were observed in any of the foetuses. No skeletal variations were found consistently within any group although some variation in ossification of phalanges existed, particularly in the smaller foetuses. Sectioning of foetuses preserved in Bouin’s solution showed that the foetuses were structurally normal, only one foetus in the 1000% group showed a missing left kidney.
Dose descriptor:
NOAEL
Effect level:
ca. 2 220 mg/kg bw/day
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
ca. 5 540 mg/kg bw/day
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
The results of this study are indicating that variable levels of tryptophan supplementation do not cause grossly recognizable foetal malformations in rats. Highly supplemented tryptophan diets (2500% excess) caused significant decreases in both maternal weight gain and foetal size.
From this study NOAEL values for maternal toxicity and embryotoxicity of ca. 2220 mg/kg bw/d can be derived.
The NOAEL value for teratogenicity is ca. 5540 mg/kg bw/d.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
5 540 mg/kg bw/day
Species:
rat
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

The results of this study are indicating that variable levels of tryptophan supplementation do not cause grossly recognizable foetal malformations in rats. Highly supplemented tryptophan diets (2500% excess) caused significant decreases in both maternal weight gain and foetal size.

From this study NOAEL values for maternal toxicity and embryotoxicity of ca. 2220 mg/kg bw/d can be derived.

The NOAEL value for teratogenicity is ca. 5540 mg/kg bw/d.

Justification for classification or non-classification