Alanine, N,N-bis(carboxymethyl)-, sodium salt (1:3)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Reference 2

Reference Type:

other: amendment

Title:

Unnamed

Year:

2001

Report date:

2001

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)

Test material

Test material information

Constituent 1

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 34/9

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species and strain: Wistar rats (Chbb:THOM (SPF)) were supplied by Dr. Karl Thomae GmbH, Biberach/Riss, FRG
- Age of animals: 42 days at the beginning of the study
- Acclimatization period: 10 days
- Bodyweight at begin of the study: 173.7 - 198.4 g for males; 127.3 - 154.5 g for females
- Animal identification: The rats were unambiguously identified by ear tattoo.
- Reason for species selection: Rats are recommended in the respective test guideline. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.
- Housing: The rats were housed singly in type DK III stainless steel wire mesh cages supplied by Becker & Co. Underneath the cages, waste trays were fixed containing absorbent material (type 3/4 dustfree embedding, supplied by SSNIFF, Soest, FRG).
- Diet: ad libitum; Kliba maintenance diet rat/mouse/hamster, meal, supplied by KLIBA-MUHLEN AG, Kaiseraugst, Switzerland.
- Water: water from bottles ad libitum

ENVIRONMENTAL CONDITIONS:
Humidity: 30-70 %
air changes: fully air-conditioned
temperature: 20 – 24°C
Photoperiod (hrs dark / hrs light): 12 h/12 h

OTHER:
The animal room was completely disinfected using a disinfector (“AUTEXI", fully automatic, formalin-ammonia-based terminal disinfector). The floor and the walls were cleaned once a week. The cleansing liquid used was water containing about 0.1% Incidin perfect (supplied by Henkel, Düsseldorf, FRG).

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: in a Gebr. Loedige laboratory mixer

DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: daily diet

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses of the test substance preparations were carried out at the Bioanalytical Laboratory of Toxicology of BASF AG. The stability of the test substance in the diet for 49 days at room temperature was tested prior to the study. The homogeneity of the test substance preparations was proven at the start of the administration period (highest and lowest concentration). These analyses served also as concentration controls. Further concentration control analyses were carried out with samples of all concentrations drawn at the start as well after 10 weeks of the administration period.

Food analyses
The food used in the study was assayed for chemical as well as for microbiological contaminants.

Drinking water analyses
The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and the Technical Services of BASF AG as well as for the presence of microorganisms by a contract laboratory.

Duration of treatment / exposure:

90 days

Frequency of treatment:

continuously

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 400 ppm

Doses / concentrations 2

Dose / conc.:

12 000 ppm

Doses / concentrations 3

Dose / conc.:

18 000 ppm

Doses / concentrations 4

Dose / conc.:

24 000 ppm

No. of animals per sex per dose:

10 males and 10 females per dose and control group

Control animals:

yes, plain diet

Details on study design:

INTRODUCTION AND DOSE SELECTION

SELECTION OF DOSES
As highest concentration, 24,000 ppm was selected. This is aequimolar to 20,000 ppm NaNTA, a concentration which caused tumors in a long-term study (NCI 1977). As further concentrations, 18,000 ppm, 12,000 ppm and 2,400 ppm were selected.

RATIONAL FOR ANIMAL ASSIGNMENT:
Animals were assigned randomely. The list of randomization instructions was compiled by a computer (laboratory data processing, Department of Toxicology, BASF AG).

Examinations

Observations and examinations performed and frequency:

CLINICAL EXAMINATIONS
Clinical observations
The animals were examined for evident signs of toxicity or mortality twice a day from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays. Once a week an additional comprehensive clinical examination was carried out.

Food consumption
Food consumption was determined weekly over a period of 7 days.

Water consumption
Water consumption was determined weekly over a period of 3 days.

Body weight data
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period, the body weight was determined on day 0 (start of administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

Intake of test substance
The mean daily intake of the test substance (group means) was calculated based upon individual values for body weight and water consumption

Food efficiency
Food efficiency (group means) was calculated based upon individual values for body weight and food consumption.

Ophthalmoscopy
One day prior to the start of the administration period the eyes of all animals were examined for any changes using an ophthalmoscope. On day 94 the animals of high dose and control group were examined.

CLINICAL PATHOLOGY
Blood was taken from the retroorbital venous plexus in the morning from non-fasted, unanesthetized animals. The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. The urine samples were evaluated in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with commercial reference controls to assure reliable test results.

Hematology
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Technicon H 1 E model; Bayer, Munich, FRG): leukocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count. Furthermore, differential blood smears were prepared and stained according to Wright without being evaluated.
The clotting analyses were carried out using a ball coagulometer (KC 10 A model; Amelung, Lemgo, FRG) and the prothrombin time (Hepato Quick's test) was determined.

Clinical chemistry
An automatic analyzer (Hitachi 917; Boehringer, Mannheim, FRG) was used to examine the clinicochemical parameters:
alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-y-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium

Urinalysis
With the exception of volume, color, turbidity, sediment examination and the specific gravity, all the urine constituents were determined semiquantitatively using test strips (Combur-9-test RL; Boehringer, Mannheim, FRG) and a reflection photometer (Urotron RL9 model; Boehringer, Mannheim, FRG). The specific gravity was determined using an urine refractometer. The sediment was evaluated microscopically. The following examinations were carried out:
volume, color, turbidity, nitrite, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment.
Calcium concentrations were determined by atomic emission spectrometry and zinc levels by atomic absorption spectrometry.

Sacrifice and pathology:

PATHOLOGY
Necropsy
The animals were decapitated under CO2 anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

Organ weights
The weight of the anesthetized animals as well as the weights of liver, kidneys, adrenal glands, testes, ovaries, spleen, and brain from all animals sacrificed at scheduled dates were determined.

Histopathology
The following organs or tissues were fixed in 4 % formaldehyde solution:
all gross lesions, brain, pituitary gland, thyroid glands, parathyroid glands, thymus, trachea, lungs, heart, aorta, salivary glands (mandibular and sublingual glands), liver, spleen, kidneys, adrenal glands, pancreas, testes/ovaries, uterus/vagina/oviducts, epididymides, prostate, seminal vesicle, skin, esophagus, stomach (glandular and non-glandular), duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, lymph nodes (mandibular and mesenteric lymph nodes), female mammary gland, skeletal musculature, sciatic nerve, sternum with sternal bone marrow, bone marrow (femur), eyes, femur with knee joint, spinal cord (cervical, thoracic and lumbar cord), extraorbital lacrimal glands, calotte, ureter
Haematoxylin-Eosin staining was performed with gross lesions, thyroid glands, parathyroid glands, liver, kidneys, urinary bladder and calotte

Statistics:

Means and standard deviations of each test group were calculated for several parameters:
Food consumption, water consumption, food efficiency, body weight and body weight change: two-sided F-test (Anova) and two sided Dunnet's test.
Clinical pathology parameters except differential blood count: two-sided Kruskal-Wallis test and two-sided Mann-Whitney-U-test.
Urinalysis, except volume, color and turbidity: Fisher's exact test
Weight parameters: (two-sided) Dunnett's test

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

24000 ppm: Discoloration of urine, dark discoloration of feces. Both findings were assessed as being related to treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

24000 ppm: reduced body weight and body weight changes in males
18000 ppm: body weight changes were reduced in males
This was assessed as being treatment-related.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

24000 ppm: Food consumption was significantly decreased (males). A substance-relationship cannot be ruled out with certainty.

The mean daily test substance intake in mg/kg body weight over the entire study period is shown in the following (mg/ kg bw per day):

Test group 1 (2400 ppm): 170 (males) and 204 (females)
Test group 2 (12000 ppm): 874 (males) and 1056 (females)
Test group 3 (18000 ppm): 1325 (males) and 1588 (females)
Test group 4 (24000 ppm): 1774 (males) and 2097 (females)

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food efficiency was significantly impaired in males of dose groups 12000-24000 ppm. A substance-relationship cannot be ruled out with certainty.

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption was dose-dependently increased when treated with 12000, 18000 and 24000 ppm with males were slightly more affected than females. This was assessed as being treatment-related.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No substance-related effects were observed. All findings were spontaneous in nature and equally distributed between treated animals and controls.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

males: test group treated with 24000 ppm displayed reduced mean corpuscular volume and haemoglobin
females: females of all treatment groups displayed reduced mean corpuscular volume

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There are no treatment-related changes in the serum enzyme activities.
Blood chemistry examinations: decreased creatinine concentration (24000 ppm) and serum magnesium levels (18000-24000 ppm).
The other blood chemistry parameters were unaffected.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

24000 ppm:
males: increased hemoglobin levels and numbers of erythrocytes in the urine specimens, macrohematuria, discoloration from yellow to orange/brown and cloudy appearance

females: trend toward increased amounts of hemoglobin and erythrocytes in the urine specimens.

The other urinalyses revealed no treatment-related changes.
Increased Zinc levels were observed in the urine specimens which were upt to 100 times higher than the control.

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Weight parameters
Absolute weights
24000 ppm increased kidney weight, decreased body weight (males)
18000 ppm increased kidney weight (females)
12000 ppm increased kidney weight (males)
The other mean absolute weight parameters did not show significant differences when compared with the control group.

Relative organ weights
24000 ppm: increased kidney weight, increased liver weight (females)
18000 ppm: increased kidney weight (females), increased liver weight (females)
12000 ppm: increased kidney weight (males)

The other mean relative weight parameters did not show significant differences when compared with the control group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The few gross lesions recorded occurred singly. They are of spontaneous origin and are hence not regarded to be related to treatment.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Kidneys: focal tubular vacuolization (unilateral) (24000 ppm), focal hyperplasia of the urothelium in the renal pelvis/ureter (12000-24000 ppm)
Liver: zonal fatty infiltration reduced (12000-24000 ppm)

Histopathology did not reveal any further findings that may be treatment related in any of the other organs investigated. All findings noted were either single observations or they were biologically equally distributed over the treatment groups and the control group, both when looking at their incidence as with respect to the graded severity.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

2 400 ppm

Sex:

male/female

Basis for effect level:

food efficiency

histopathology: non-neoplastic

water consumption and compound intake

2

Key result

Dose descriptor:

NOAEL

Effect level:

170 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

food efficiency

histopathology: non-neoplastic

water consumption and compound intake

3

Key result

Dose descriptor:

NOAEL

Effect level:

204 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

food efficiency

histopathology: non-neoplastic

water consumption and compound intake

Target system / organ toxicity

Key result

Critical effects observed:

no