L-methionine

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

125 d

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study. The study itself was regarded as reliable without restrictions (Kl.1). However, according to the "Prctical guide 6: How to report read-across and categories" the maximum score for read-across is 2.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

DL-methionine

EC Number:

200-432-1

EC Name:

DL-methionine

Cas Number:

59-51-8

Molecular formula:

C5H11NO2S

IUPAC Name:

methionine

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 31 days (at study initiation / at test item administration)
- Weight at study initiation: males 96.5 - 120.1g, females 94.8 - 118.0g
- Fasting period before study: overnight
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 55% ± 15%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: -

Administration / exposure

Route of administration:

oral: feed

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
DIET PREPARATION
- Rate of preparation of diet (frequency): The test item-food mixture was freshly prepared once a week.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the determination of the methionine concentration in the exposure diets (groups 1 - 4), samples of approximately 10 g were taken at the following times and stored at -20°C or colder until analysis at LPT.
Total number of samples: 28
The samples were labelled with study number, test species, type of sample, concentration, test day, sampling time and date.
The analytical method for the determination of methionine is commonly available and was re-validated by LPT.

Duration of treatment / exposure:

90 d + 28 additional days for the animals scheduled for the recovery period

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.5%
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1%
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
2%
Basis:
nominal in diet

No. of animals per sex per dose:

Main study:
3 dose groups + 1 control group; 6 animals per sex per group
Recovery animals:
3 dose groups + 1 control group; 6 animals per sex per group

Control animals:

yes

Details on study design:

- Dose selection rationale: The dose levels for this study were selected by the Sponsor based on available toxicological data.

Examinations

Observations and examinations performed and frequency:

- Cage side observations: once daily
- Weighing: day 0 (prior to dosing), weekly thereafter

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection, all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart. The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

HISTOPATHOLOGY: Yes

The following organs or parts of organs with the exception of the eyes, epididymides and testicles of all animals were fixed in 7% buffered formalin. The eyes were preserved in Davidson’s solution for optimum fixation. The epididymides and testicles were preserved in Bouin’s fixative.
adrenal gland (2)
aorta abdominalis
bone marrow (os femoris)
brain (3 levels: cerebrum, cerebellum,medulla/pons)
coagulating gland with seminal vesicle
epididymis (2)
eye with optic nerve (2)
gross lesions observed
heart (3 levels: right and left ventricle, septum)
intestine, large (colon, rectum)
intestine, small (duodenum, jejunum, ileum, incl. Peyer´s patches), Swiss roll method
kidney and ureter (2)
liver
lungs (with mainstem bronchi and bronchioles (preserved by inflation with fixative and then immersion))
lymph node (1, cervical)
lymph node (1, mesenteric)
mammary gland (male and female)
nerve (sciatic)
oesophagus
ovary (2)
pancreas
pituitary
prostate
salivary glands (mandibular, sublingual and parotid gland)
skin (left flank)
spinal cord (3 levels: cervical, mid-thoracic, lumbar)
spleen
sternum (bone and bone marrow)
stomach
testicle (2)
thymus
thyroid (2) (incl. parathyroids)
tissue masses or tumours (including regional lymph nodes)
trachea (incl. larynx)
urinary bladder
uterus (incl. cervix and oviducts)
vagina

The afore-listed organs of all animals of groups 1 and 4 were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining. In addition, frozen sections of the heart, liver and one kidney were made, stained with Oil Red O and examined microscopically.
Parathyroids cannot always be identified macroscopically. They were examined microscopically if in the plane of section and in all cases where they were noted as grossly enlarged.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see details on results

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Details on results:

Body weight and body weight gain
The body weight of the male and female animals treated with 2.0% Mepron® via the diet was slightly reduced as of test week 1 by up to 20% for the males and by up to 14% for the females compared to the body weight of the control group. If compared to the natural control group in test weeks 1 to 13 (males) and test weeks 1 to 10 (females) the males displayed a reduced body weight up to 21% whereas for the females a reduction in body weight up to 14 % was documented. Body weight gain was reduced accordingly. The reduced body weight observed at the beginning of the study is linked to a decreased food consumption caused by a decreased palatability of the Mepron®-containing feed and, therefore, not considered to be of toxicological significance.
Body weight at necropsy was still decreased by 12% for the male animals dosed with 2.0% Mepron® compared to the control group. No changes in body weight of body weight gain occurred for male and female animals dosed with 0.5 or 1% Mepron.
Recovery period:
At the end of the recovery period the body weight and body weight gain of the male and female animals were again within the normal range.

Food and drinking water consumption
The male animals treated with 1.0% Mepron® via the diet revealed a decreased relative food intake in test week 1 by 13% compared to the
control group. The male and female animals treated with 2.0% Mepron® via the diet revealed a decreased relative food intake in test week 1 by 20% and 13%, respectively, compared to the control group. The reduction of food consumption is most likely due to a decreased palatability of the Mepron®-containing feed. The relative food intake of the male animals treated with 2.0% Mepron® via the diet was also decreased in test week 1 by 12% compared to the natural control group. The visual appraisal of the drinking water consumption did not reveal any test item-related influence at any of the tested dose levels.
Recovery period:
No test item-related influence was noted. All values were within the normal range of variation.

Haematology and coagulation
Starting at the intermediate dose group treated with 1.0% Mepron® via the diet the following non-specific changes were noted on test days 41 and/or 91 compared to the control group or the natural control group: slight decreases in the number of leucocytes, neutrophilic granulocytes, lymphocytes, monocytes, eosinophilic granulocytes, large unstained cells and basophilic granulocytes most likely caused by an excessive intake of essential amino acids. At the high dose of 2.0% Mepron® via the diet, additionally, a slight decrease in the haemoglobin content, a slight decrease in the number of erythrocytes, a slight increase in the number of reticulocytes, a slight increase in MCV and a slight decrease in MCHC were noted. The changes observed were not considered to be of any noteworthy toxicological relevance since no effects were found at the histopathological evaluation.
Recovery period:
The animals previously treated with 1.0% or 2.0% Mepron® via the diet still revealed slight decreases in the number of leucocytes, neutrophilic granulocytes, lymphocytes, monocytes, eosinophilic granulocytes, large unstained cells and basophilic granulocytes, and a slight increase in MCV in the previously high dosed animals compared to control group or natural control group at the end of the recovery period. However, these parameters revealed a tendency towards normalisation. The changes observed were not considered to be of any noteworthy toxicological relevance since no effects were found at the histopathological evaluation

Clinical biochemistry
The male and female animals treated with 2.0% Mepron® via the diet revealed an increased LDH activity on test day 41 by up to 59% compared to the control group or the natural control group. The change observed was not considered to be of any noteworthy toxicological relevance since no effects were found at the histopathological evaluation.
Recovery period:
The above mentioned change had completely normalised at the end of the 4-week recovery period.

Organ weights
The male and female animals treated with 1.0% or 2.0% Mepron® via the diet revealed an increase in the relative and absolute kidney and/or spleen weights compared to the control group or natural control group, most probably due to an enhanced metabolism.
Recovery period:
The previously intermediate and high dosed males still revealed an increase in the relative and absolute kidney weights compared to control group or natural control group at the end of the 4-week recovery period. The changes observed were not regarded to be of any biological or any toxicological significance since no effects were found at the histopathological evaluation

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 474 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 647 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The no-observed-adverse-effect level (NOAEL) was considered to be above 2% Mepron® in the diet, equivalent to 1474 and 1647 mg/kg b.w./day for
the male and female animals, respectively.