L-tryptophan

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-11-21 to 2008-03-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to OECD Guideline 408

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

L-tryptophan

EC Number:

200-795-6

EC Name:

L-tryptophan

Cas Number:

73-22-3

Molecular formula:

C11H12N2O2

IUPAC Name:

L-tryptophan

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Harlan Italy S.r.l.
Weight (g): 75 – 99
Age (days): 27 - 29
Pre-dose acclimatisation period: ca. 2 weeks
Housing: groups of 5 animals/sex in polycarbonate cages equipped with a stainless steel mesh lid and floor
Diet: laboratory powdered rodent diet (4 RF 21) ad libitum
Water: ad libitum

Environmental conditions:
Temperature (°C): 22 ± 2
Relative humidity (%): 55 ± 15
Photoperiod (hrs dark / hrs light): 12 / 12 by artificial light
Air changes (per hr): 15-20

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Rate of diet preparation: weekly
The test item was formulated, using powdered rodent diet (4RF21), by initial preparation of a pre-mix followed by dilution with further quantities of diet and mixing at fixed concentrations of 5000, 15000 and 50000 ppm. Formulations were prepared separately for each group.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before start of treatment, formulation analysis was performed in order to confirm the acceptability of the analytical method and if the stability of the formulation was satisfactory. The stability of the formulated test item was assessed over a period of 20 days. In addition, samples of formulations prepared in weeks 1 and 13 of the study were analysed to check the homogeneity and concentration. Results were satisfactory being inside the limits of acceptance (90-110%).

Duration of treatment / exposure:

90 days
Recovery groups (control and high dose group) were sacrificed after 4 weeks of recovery

Frequency of treatment:

continuously via diet

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 5000, 15000 or 50000 ppm (m: ca. 0, 366, 1069 or 3582 mg/kg bw/d; f: ca. 0, 401, 1214 or 3946 mg/kg bw/d)
Basis:
nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

Dose selection rationale: Based on a 4 week range-finding study
Rationale for animal assignment: The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and there is ample experience and background data on this species and strain

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: yes (twice daily)
DETAILED CLINICAL OBSERVATIONS: yes (once before the administration (day -1), as well as once daily during treatment)
BODY WEIGHT: yes (on the day of allocation to treatment groups, on the day that treatment commenced, twice weekly thereafter and just prior to necropsy)
FOOD CONSUMPTION AND COMPOUND INTAKE: yes (at weekly intervals)
WATER CONSUMPTION: no
OPHTHALMOSCOPIC EXAMINATION: yes (just prior to the commencement of treatment by means of an ophthalmoscope, and by a slit-lamp microscope, after the instillation of 0.5% Tropicamide. The eyes of all animals from high dose and control groups were re-examined during week 12 of treatment. The examination was also extended to the other treated groups. Since no treatment-related changes were observed the examination was not repeated during week 4 of recovery)
HAEMATOLOGY: yes (according to guideline)
CLINICAL CHEMISTRY: yes (according to guideline)
URINALYSIS: yes (according to guideline)
NEUROBEHAVIOURAL EXAMINATION: yes (observations included changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system). Motor activity of all animals was measured once during week 12 of treatment and once during week 4 of recovery by an automated activity recording.

Sacrifice and pathology:

GROSS PATHOLOGY: yes (determination of weights of adrenal glands, brain, epididymides, heart, liver, kidneys, ovaries, pituitary gland, testes, spleen, thymus, uterus – cervix)
HISTOPATHOLOGY: yes (all major/relevant organs according to guideline)

Statistics:

For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett's test using a pooled error variance. The homogeneity of the data was verified by Bartlett's test before Dunnett's test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied. The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off. Statistical analysis of histopathology findings was carried out by means of the non-parametric Kolmogorov-Smirnov test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Mortality:
No mortality occurred during the study.

Clinical signs, observations of cage tray, motor activity and neurotoxicity assessment:
Hair loss was the only relevant sign at clinical examination and was observed mainly in animals dosed with 3582 mg/kg bw/d. Complete reversibility was observed during the recovery period. No other toxicologically relevant signs were seen during the study including neurotoxicity evaluation, motor activity and sensory reactivity to stimuli assessment. Moderate yellow staining on the cage tray was reported at >= 3582 mg/kg bw/d indicating that the substance was excreted via urine.

Body weight:
Statistically significant reduction (up to –12%) in mean body weight was observed in animals dosed with >= 1069 mg/kg bw/d of both sexes during the study. Partially (females) or complete (males) recovery was noted during the recovery period. Compared with controls these differences were insufficient in magnitude to be considered of toxicological significance.

Food consumption:
Food consumption in both sexes was reduced at >= 1069 mg/kg bw/d, while no relevant differences were noted between controls and dosed animals during the recovery period.

Terminal body weight and organ weights:
Terminal body weight reflected the decreases seen in body weight in animals given >= 1069 mg/kg bw/d. Slight increases in absolute and/or relative liver weights in >= 3582 mg/kg bw/d animals of both sexes and in spleen weight only in 3946 mg/kg bw/d females were seen. All these changes, although statistically significant, were not considered treatment-related or with biological significance since no effects were found at the histopathological evaluation.

At the end of the recovery period, kidney and spleen weights recovered. Liver weight completely recovered in males and partially in females. All the above mentioned changes were not considered to be treatment-related or of biological significance since no effects were found at the histopathological evaluation.

Effect levels

Dose descriptor:

NOAEL

Effect level:

3 764 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

L-tryptophan was tested in a 90 d oral study (0, 5000, 15000 or 50000 ppm in diet) with GLP compliance according to OECD guideline 408. Recovery groups had a 4 week treatment-free period. The calculated mean achieved dosages for 13 weeks of treatment were 0, 366, 1069 or 3581 mg/kg bw/d for males and 0, 401, 1214 or 3946 mg/kg bw/d for females. No relevant toxicological findings were noted during the study including neurotoxicity assessment, motor activity and sensory reactivity to stimuli, and there were also no ophthalmic lesions. The slight decreases in body weight at >= 1069 mg/kg bw/d and the decreases in food consumption at >= 3582 mg/kg bw/d were of insufficient magnitude to be considered of toxicological significance. Changes observed in some biochemical parameters at the end of the treatment period showed a complete/partial reversibility at the end of the recovery period. Therefore, these findings were considered of no toxicological significance. No toxicological significance was attributed to some changes recorded at the macroscopic observations (dark coloration and/or swelling shape of the spleen seen in treated animals mainly at >= 3582 mg/kg bw/d), as the histopathological evaluation did not confirm these changes. The histopathological examination did not reveal differences in the incidence of the findings observed in treated animals when compared with controls.
Therefore, from this study a NOAEL of ca. 3764 mg/kg bw/d (mean value for males and females) corresponding to 50000 ppm can be derived.