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

Endpoint summary

Description of key information

Based on a combined chronic toxicity / carcinogenicity study in Wistar rats (GLP & OECD Guideline 453) the NOAEL was determined to be 262.2 mg/kg bw/day (male) and 333.9 mg/kg bw/day (female) (BASF, 82S0059/95121, 2006).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: charge 4

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 37 ±1 days (males) and 32 ±1 (females) days
- Housing: singly in type DK III stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm2). Underneath the cages, waste trays were fixed containing absorbent material (type 3/4 dust free embedding, supplied by SSNIFF, Soest, Germany).
- Animal identification: ear tattoo
- Diet: ground Kliba maintenance diet mouse/rat meal (GLP), supplied by Provimi Kliba SA, Kaiseraugst, Switzerland. ad libitum
- Water: ad libitum from water bottles
- Acclimation period: 5 days for males and 10 days for females

ENVIRONMENTAL CONDITIONS
- Temperature: 20 – 24° C
- Humidity: 30 – 70 %
- Air changes (per hr): fully air-conditioned rooms.
- Photoperiod (hrs dark / hrs light): 12 hours (12 hours light from 06.00 a.m. - 06.00 p.m., 12 hours dark from 06.00 p.m. - 06.00 a.m.)
- Cleaning/ desinfection of animal room: The room was completely disinfected prior to the study using a disinfector ("AUTEX", 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 % lncidin (supplied by Henkel, Düsseldorf, FRG).

GENERAL ANALYSIS:
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.

Bedding analyses
The bedding is regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals).

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Remarks:

dietary administration

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For each concentration, the test substance was weighed out and mixed with a small amount of food. Then corresponding amounts of food, depending on dose group, were added to this premix in order to obtain the desired concentrations. Mixing was carried out for about 10 minutes in a laboratory mixer. Details of the mixers used are retained with the raw data. The test substance preparations were mixed in such intervals that the stability was guaranteed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in the diet over a period of 49 days at room temperature was proven before the start of the study. At the beginning of the study, the homogeneity was demonstrated using each 3 samples of the highest and lowest concentration, and concentration control analyses were performed with each one sample. During the study, analyses of the test substance preparations with respect to concentration control and/or homogeneity were conducted every three months. Homogeneity was demonstrated using 3 samples of one concentration in uprising row, and concentration control analyses were performed with 1 sample. The homogeneity analyses also served as concentration controls for the respective concentration. The analyses from 3 months onwards were usually performed with samples taken at the end of the time period for which the respective test substance preparations were used. The samples were taken out of randomly selected reserve food boxes being stored in the animal room. Thus, the stability of the test substance in the diet was also proven under test conditions.

Duration of treatment / exposure:

24 months (main group); 12 month (satellite group)

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg diet

Dose / conc.:

1 000 ppm

Dose / conc.:

5 000 ppm

Dose / conc.:

19 200 ppm

No. of animals per sex per dose:

Groups of 60 male and 60 female Wistar rats, 50 animals as main group (administration for 24 months) and 10 animals as satellite groups (administration for 12 months)

Control animals:

yes, plain diet

Details on study design:

CHANGES IN DOSE LEVELS
From the second week on, test item dosage was reduced from 20000 ppm to 19200 ppm. For details see field "positive control".

ANALYSES
Stability analyses
The stability of the test substances in the diet over a period of up to 49 days at room temperature was verified. As the mixtures were stored no longer then this time period, the stability was guaranteed.
 
Homogeneity and Concentration control analyses
Considering the low standard deviation in the homogeneity analysis, it can be concluded that the test item and the positive substance was distributed homogeneously in the diet.
 
Food anddrinking water analyses
On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants the food was found to be suitable.
 
Bedding analyses
On the basis of the analytical findings the bedding was found to be suitable.
 

Positive control:

- CAS: 5064-31-3
- Purity 94 %

After the first week of administration of 20000 ppm positive substance (CAS# 5064-31-3) body weight loss was observed in male animals. Due to this clear sign of exceeding the maximum of tolerated dose (MTD), the concentration in the diet was reduced down to 15000 ppm. In order to administer an equimolar concentration of the test substance, dosage was reduced from 20000 ppm to 19200 ppm.

Observations and examinations performed and frequency:

CLINICAL EXAMINATIONS
Clinical observations
The animals were examined for overt signs of toxicity or mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays. Additionally, further clinical examinations were carried out daily in all satellite animals.
Detailed clinical observations were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The following parameters were examined:
abnormal behavior during handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmos, feces (appearance/consistency), urine, pupil size

Food consumption
Food consumption was determined once a week over a period of 7 days during the first 13 weeks of the administration period, thereafter at 4-week intervals, and prior to start of necropsy.

Water consumption
Water consumption was determined at 4-weeks intervals over a period of 4 days.

Body weight data
Body weight was determined before the start of the administration period in order to randomize the animals. During the conduct of the study, body weight was determined on day 0 (start of the administration period), at weekly intervals during the first 13 weeks of the study, thereafter at 4-week intervals, and prior to start of necropsy.

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

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

Ophthalmoscopy
Prior to the start of the administration period the eyes of all animals were examined for any changes using an ophthalmoscope after administration of a mydriatic. Around study day 363, only animals of the dose groups 0 ppm, 19200 and Trilon A (pos. ctrl.) of the satellites were examined. Additionally, on day 724-725 the eyes of the corresponding main group animals of dose groups 0 ppm, 19200 ppm and the Trilon A (pos. ctrl.) were examined.

CLINICAL PATHOLOGY
Blood was taken from the retroorbital venous plexus in the morning from fasted animals. The animals were anesthetized using isoflurane (Essex GmbH. Munich, Germany). 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 list of randomization instructions was compiled with a computer using a random number generator. The assays of blood and serum parameters were performed under internal Iaboratory quality control conditions with commercial reference controls to assure reliable test results.
The following examinations were carried out in 10 animals per test group and sex. On day 362, however, urine specimens of 9 females of test groups 1000, 5000 and 19200 ppm were analyzed.

Hematology
The following parameters were determined in blood with EDTA-K 3 as anticoagulant using a particle counter:
leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count, reticulocytes

The clotting analyses were carried out using a ball coagulometer (KC 10 A model; Amelung, Lemgo, Germany). The following parameter was determined: prothrombin time (Hepato Quick's test)

Clinical chemistry
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters. The following parameters were determined:
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 semi-quantitatively using test strips (Combur-9-test M, Roche, Mannheim, Germany) and a reflection photometer (Miditron M; Roche, Mannheim, Germany). The specific gravity was determined using a urine refractometer. The sediment was evaluated microscopically. The following examinations were carried out:
volume, color, turbidity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment

Sacrifice and pathology:

PATHOLOGY
Necropsy
At study termination, all animais were sacrificed by decapitation under CO2-anesthesia. The exsanguinated animais were necropsied and assessed by gross pathology. The weights of the following organs were determined:
Anesthetised animals, Liver, Kidneys, Adrenal glands, Testes, Epididymides, Ovaries, Uterus, Spleen, Brain, Heart
The following organs / tissues were preserved in neutral buffered 4% formaldehyde:
All gross lesions, salivary glands (mandibular gland, sublingual gland), esophagus, stomach (fore- and glandular stomach), duodenum, jejunum, ileum, cecum, colon, rectum, liver, pancreas, brain, pituitary gland, sciatic nerve, spinal cord (cervical, thoracic and lumbar cord), eyes with optic nerve, thyroid glands, parathyroid glands, trachea, lungs, pharynx, larynx, nasal cavity, aorta, heart, bone marrow (femur), lymph nodes (mesenteric and mandibular Iymph nodes), spIeen, thymus, femur with knee joint, kidneys, urinary bladder, testes, ovaries, uterus, oviducts, vagina, epididymides, prostate, seminal vesicle, female mammary gland, skin, skeletal muscle, sternum with marrow, extraorbital Iacrimal glands, harderian glands

All the fixed organs derived from animals of dose group 0 ppm, 19200 ppm and the positive ctr. were embedded in paraffin, stained with hematoxylin-eosin and examined light-microscopically. From selected animals of the dose groups 1000 and 5000 ppm, testes, kidneys, pancreas and liver were as well analyzed histologically. A correlation between gross lesions and histopathological findings was performed. Of few organs of single animais, the following special stains were performed: Periodic acid Schiff reaction (PAS) for mucopolysaccharides, iran staining (Peris, Turnbull) for hemosiderin, Mallory-Heidenhain for protein, Ziehl Neelsen for ceroid, and Schmort for lipofuscin. Of few organs of single animals, the following immunhistochemical examinations were performed: Calcitonin, ACTH, Cytokeratin for epithelial cells, S-100 for Schwann cells, Vimentin for soft tissue, and Factor VIII far endothelial cells.
Following the initial examination, peer review of the findings in target organs was performed by internal peer reviewer (Dr. Wolfgang Kaufmann, BASF AG, Ludwigshafen, Germany).

Statistics:

PATHOLOGY:
Weight parameters: two-sided Kruskal-Wallis test and Wilcoxon test
CLINICAL EXAMINATIONS:
For dose groups 1000 ppm, 5000 ppm, 19200 ppm:
Food consumption, food efficiency, water consumption, body weight and body weight change: two-sided Dunnett's test
Feces, rearing, grip strength length forelimbs, grip strength length hindlimbs, footsplay test, motor activity: two-sided Kruskal-Wallis test and Wilcoxon test
For negative and positive ctrls:
Food consumption, body weight, body weight change, food efficiency: two-sided Welch t-test

CLINICAL PATHOLOGY:
Urinalysis, except volume, color, turbidity and specific gravity: Fisheer's exact test
Dose groups 0 ppm, 1000 ppm, 5000 ppm and 19200 ppm:
Clinical pathology, parameters, except reticulocytes and differential blood count: two-sided Kruskal-Wallis test and two-sided Wilcoxon-test
Positive and negative ctrls.:
Clinical pathology, parameters, except reticulocytes and differential blood count: two-sided Wilcoxon-test

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item: Dose group 19200 ppm: piloerection, urine discolored red, anogenital and abdominal region smeared with urine and/or feces as well as feces discolored light brown

Positive substance: abdomen palpable mass, paleness and reduced general condition (only males), piloerection, alopecia (females), urine discolored red, anogenital and abdominal region smeared with urine and/or feces as well as feces discolored light brown

These findings were assessed as related to treatment with the test substances. All other clinical findings were equally distributed between control and treated animals or occurred in single animals, only. Therefore, these findings were spontaneous in nature and do reflect the range of biological variation.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Test item: No substance-related increase in mortality was determined.
Positive substance: The mortality was substance-related increased only in male animals.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test item: Body weight was statistically significantly decreased in males of dose group 19200 ppm throughout the whole treatment period. This finding was assessed to be related to the test substance. In females no influence on body weight was observed.
Body weight change was statistically significantly decreased in males of dose group 19200 ppm on all days of the study. This finding correlates well with decreased body weight as above-mentioned and is therefore assessed as related to the test compound.

Positive substance: Body weight was statistically significantly and severe decreased in animals of both sexes during the whole study. Caused by this impaired body weight, body weight change was also distinctly as well as statistically significantly decreased in both sexes during the whole study. This finding was clearly a treatment-related sign of general systemic toxicity and therefore assessed as adverse.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean daily test substance intake in mg/kg body weight/day over the entire administration period is shown in the following:

Substance intake (mg/kg bw/day):
Test group 1 (1000 ppm): 53.9 (males); 65.9 (females)
Test group 2 (5000 ppm): 262.2 (males); 333.9 (females)
Test group 3 (19200 ppm): 1132.3 (males); 1316.9 (females)
Positive substance (15000 ppm): 1166.7 (males); 1039.1(females)

Test item: food consumption was not affected by substance treatment.

Positive substance: Food consumption was statistically significantly decreased in males throughout the whole administration period and in females on most days of the study. These continuous reductions were assessed as substance-related.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Test item: Water consumption was statistically significantly increased in all animals of dose group 19200 ppm throughout the whole study. This finding was clearly substance-related.

Positive substance: Water consumption was severe increased in animals treated with Trilon A 92 R. This finding was statistically significant in males throughout the entire study with a maximum of +90.9 %. In females water consumption was increased on most days of the study, statistically significantly until day 448.

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

Test item: No test substance-related changes were observed in the results of the hematological examinations and in the differential blood count of the treated male and female animals in the course of the study. Clotting analyses revealed prolonged prothrombin times in the blood of males of group 19200 ppm at each time interval.

Positive substance: In the peripheral blood of males statistically significantly decreased hemoglobin concentrations and hematocrit values were found at each time interval. In addition, decreased red blood cell counts but increased reticulocytes and platelet counts were noted in animals of both sexes on single occasions. Slightly increased polychromasia was also seen in the erythrocytes of males at each time interval. No treatment-related effects were observed in the other hematology parameters. White blood cell counts were significantly increased in the circulation of males on single occasions. In the differential blood count the increases in leukocytes were associated with increases in polymorphonuclear neutrophils. In the females no test substance related changes in white blood cell parameters were seen.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Test item: Compound-related differences in serum enzyme activities were not evident at any dose level.
19200 ppm: Increased total protein, globulins, prothrombin times and urinary erythrocytes in males
No treatment-related changes were observed in the other blood chemistry parameters.

Positive substance: Compound-related differences in serum enzyme activities were not evident in either males or females. Blood chemistry examinations showed increased urea levels in males and reduce creatinine and elevated cholesterol concentrations in females at each time interval. The fall in creatinine in females, however, was not considered to be test article related because decreases in serum creatinine are, in general, pathologically meaningless (except there was marked decrease in muscle mass) and due to the inconsistency of changes between both sexes. In the serum of males lower magnesium levels were found on single days. The test compound did not affect the other blood chemistry parameters.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Test item: In urine sediments of males of dose group 19200 ppm an increased number of red blood cells were seen on few time intervals. No other changes occurred in the urine parameters of treated males and females.

Positive substance: Males excreted higher amounts of turbid urine with lower specific gravity and increased blood (hematuria) during the course of the study. Due to the marked increase in red blood cells urine specimens of males were discolored from dark yellow to dark red brown on few time intervals. Urine sediment examinations of males revealed an increased number of erythrocytes at each time interval, elevated number of leukocytes and atypical cells on few time intervals. The test substance did not affect the other urine parameters of the males. Females produced slightly higher amounts of cloudy urine with decreased specific gravity. In addition, their urine contained an increased number of unidentified crystals and a higher number of erythrocytes in the urine sediments. No treatment-related chances occurred in the other urine parameters examined.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute weights

Satellite group: When compared to control group 0, the mean absolute weights of following organs were considered to be treatment related:
Test item: Male animals displayed increased kidney weights at 19200 ppm.
Positive substances: Increased organ weights: kidney (both animals); decreased organ weights: kidneys (all observations exclusive to males)

Main group: When compared to control group 0, the mean absolute weights of following organs were considered to be treatment related:
Test item: Increased organ weights: Kidneys in both sexes; decreased organ weights: Body weight (males of the highest dose group)
Positive substance: Increased organ weights: kidney in both sexes; decreased organ weights: body weight (both sexes)

Test item: The increase of absolute adrenal weight in females treated with 19200 ppm was not statistically significant because it is due to one female with a malignant pheochromocytoma of the adrenal medulla that resulted in very high adrenal weight
Positive substance: The increased absolute uterus weight of females is due to two females with very high uterus weights (endometrial stromal sarcoma and severe dilation of the uterus).


Relative Organ Weights

Satellitegroup: When compared to control group 0, the mean relative weights of following organs were considered to be treatment related:
Test item: Relative increase: kidneys (highest dose in both sexes)
Positive substances: Relative increase: kidney (both sexes)

Main group: When compared to control group 0, the mean relative weights of following organs were considered to be treatment related:
Test item: Relative increase: kidneys (both sexes),
Positive substance: Relative increase: kidneys (both sexes)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Satellite group:
Test item: No treatment trelated findings.
Positive control: Kidneys: cysts, a granular surface, pelvic dilation and retraction. Pancreas: ectatic vessels.Ureters: dilated. All other gross lesions occurred either singly or were biologically equally distributed over the control group and the treatment groups.

Main group
Test item: Kidneys: cysts, pelvic dilation, abnormal mass and enlarged kidneys were observed in males treated with 19200 ppm.

Positive control: Kidneys: Females showed pelvic dilation and cysts. Ureter: the ureters were dilated; Pancreas, mesentery: Ectatic vessels (males); Testes: calcification, abnormal mass and organ size (males); Glandular stomach: erosions or ulcers in the glandular stomach (males); Abdominal cavity: effusion (males). All other gross lesions occurred either singly or were biologically equally distributed over the control group and the treatment groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Satellite group:
Kidneys:
Test item 19200 ppm: multifocal tubular hyperplasia (both sexes), papillary transitional cell hyperplasia (partly with ectasia of ducts or vessels), chronic necropathy and dilated renal pelvis, cytoplasmic vacuolation which coincides with thes storage of a light brownish pigment (partly iron) (all findingsonly males)
Positive substance: multifocal hyperplasia, chronic necropathy, papillary transitional cel hyperplasia (partly with ectasia of ducts or vessels), chronic fibrotic, partly necrotising pyelitis and dilated renal pelvis (both sexes), cytoplasmic vacuolation which coincides of a light brownish pigment (partly iron) (all findings in both sexes)

Testes:
Test item: No treeatment related observations.
Positive substance: (multi)focal tubular degeneration in both testes coinciding with moderate chronic progressive arteritis in the testes.

Pancreas:
Test item: No treatment related findings
Positive substance: Slight or moderate chronic progressive arteritis occurred in the pancreas with comparable findings in the testes.
All other findings noted were either single observations or they were biologically equally distributed between control and treatment groups. All of them were considered to be incidental or spontaneous in origin and without any relation to treatment.

Main group:
Kidneys:
Test item: Chronic nephropathy occurred in most males and females with highest gradings in animals treated with 19200 ppm; other findings restricted to animals treated with 19200 ppm: cytoplasmatic vacuolization and pigment storage in tubular epithelial cells, (multi) focal tubular hyperplasia, (multi)focal ectasia of ducts or vessels in the papillar region
Positive susbtance: chronic nephropathy, cytoplasmatic vacuolization and pigment storage in tubular epithelial cells, (multi) focal tubular hyperplasia, (multi)focal ectasia of ducts or vessels in the papillar region, cyst(s) in the kidneys, pelvic dilation, chronic fibrotic, partly necrotising pyelitis, mineralization in the papilla, nodular or papillary hyperplasia of the transitional cell epithelium in the renal pelvis (mainly in males)

Ureter:
Test item: No treatment related observations.
Positives substance: Most of the macroscopically diagnosed dilated ureters could be confirmed histopathologically with a diffuse hyperplasia of transitional cells

Pancreas:
Test item: No treatment related observations.
Positive substance: The macroscopically seen ectatic vessels in the pancreas correlated with chronic progressive arteritis. These findings coincided frequently with chronic progressive arteritis in the mesentery.

Testes:
Test item: No treatment related findings.
Positives substance: Multifocular tubular degeneration and mineralization, chronic progressive arteritis
The occurrence of diffuse tubular degeneration was comparable between control and treatment groups.

Epididymides:
Test item: No treatment related findings.
Positive substance: chronic progressive arteritis, hypospermia

Glandular stomach:
Test item: No treatment related findings.
Positive substance: Most of the macroscopically diagnosed erosions/ ulcers in the glandular stomach could be confirmed histopathologically in males.

Bone marrow, sternum:
Test item: No treatment related findings.
Positive substance: Increase of hematopoiesis was observed in the bone marrow and sternum.

Femur with joint:
Test item: No treatment related findings.
Positive substance: Osteodystrophia fibrosa

Nasal cavity:
Test item: Inflammation in the area of Steno's glands was observed mainly in females treated with 19200 ppm
Positive substance No treatment related findings.

Parathyroid glands:
Test item: No treatment related findings.
Positive substance: The incidence of diffuse hyperplasia of parathyroid glands was increased in males

Spleen:
Test item: No treatment related findings.
Positive substance: Reduced hemosiderin storage in the spleen.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Satellite group:
There were single neoplastic findings which were biologically equally distributed over the control group and the treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Main group:

Test item: No treatment related observations.

Positive substance: Kidneys: only in males: benign mesenchymal tumor (not further classified), malignant mesenchymal tumors (not further classified) and hemangiosarcomas. The males with the hemangiosarcoma and with the malignant mesenchymal tumor died prematurely.Testes: Leydig ceII adenomas (males). All other neoplastic findings occurred either singly or were biologically equally distributed over the control group and the treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Tumor bearing animals
The numbers of animals with neoplasms, benign and malignant neoplasms and systemic neoplasms were comparable between control animals and animals treated with 19200 ppm test item or the pos. ctrl.. Similar, the total numbers of primary, benign, malignant, and systemic neoplasms was comparable between control animals and animals of the highest dose group and the pos. ctr.. They were biologically equally distributed over the control and treatment groups.

Other effects:

no effects observed

Description (incidence and severity):

Decedents:
Test item: Independent of the sex of the decedents, there were no treatment-related histopathological findings that caused an earlier death. Comparable to control animals, most of these animals showed different kinds of tumors in different organs or inflammatory lesions.

Positive substance: male descendents showed severe pathological findings in the kidneys: mesenchymal tumors in the renal pelvis, severe or extreme chronic progressive nephropathy, severe or extreme dilation of the renal pelvis, moderate to extreme chronic pyelitis, moderate to extreme chronic progressive arteritis in the pancreatic vessels. The severity of these findings might have caused the earlier death. In female decedents treated with the positive substance, there were no treatment-related histopathological findings that caused an earlier death.

Key result

Dose descriptor:

NOAEL

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Effect level:

262.2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical signs

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Effect level:

333.9 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical signs

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

262.2 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

GLP and guideline study

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

To assess toxicity of the test item upon chronic oral exposure, Wistar rats of both sexes were administered with 0, 1000, 5000 and 19200 ppm (54 -1317 mg/kg bw) test substance by dietary uptake for 24 month in a GLP and OECD guideline study. In parallel, a satellite group comprising all dose groups was sacrificed and examined after 12 month exposure (BASF, 82S0059/95121, 2006). Adverse effects were exclusively seen in the high dose group. Clinical findings include a red urine, increased water consumption and a decreased body weight. Parameters of clinical chemistry revealed a treatment related increase of total protein, globulins, prothrombin times and urinary erythrocytes in male rats. Increased kidney weights correlated with cysts and pelvic dilation in males. Histopathology revealed chronic nephropathy with highest gradings, focal tubular hyperplasia and cytoplasmic vacuolisation. Importantly, males seem to be more susceptible to test item induced renal toxicity, as most of the toxicological effects are more severe in male rats. Overall, the test item displayed renal toxicity exclusively in the highest dose group. Thus, under the chosen test conditions the NOAEL is 262.2 mg/kg bw/day.

The observations of the key study are supported by two further studies:

To assess toxicity of the test item upon sub-chronic oral exposure, Wistar rats of both sexes were administered with 4 dose levels between 170 -2097 mg/kg bw/d test substance by dietary uptake in a GLP and OECD guideline study (BASF, 50S0059/95106, 1998). Adverse effects included reduced body weight, increased haemoglobin in the urine specimen, decreased magnesium and creatinine concentration, focal tubular hyperplasia and vacuolization. Under the chosen test conditions, the NOAEL was 2400 ppm (170 mg/kg bw/d for males and 204 mg/kg bw/d for females).

In a GLP and OECD 407 guideline study, Wistar rats of both sex were administered with 3 dose levels between 76 - 1504 mg/kg bw/d test substance by drinking water (BASF AG, 39C0059/95008, 1995). Adverse effects included decreased food consumption, decreased body weight, decreased magnesium concentrations in the sera, focal tubular vacuolization and calcification. All toxic effects were completely reversible during a 14 days recovery period. Under the chosen test conditions, the NOAEL was 750 ppm (76 mg/kg bw/d for males and 88 mg/kg bw/d for females).

Toxicity of the test item seems to be independent of the duration of treatment and restricted to higher dose levels. The severity of nephrotoxic symptoms was similar in all studies ranging from 28 days to 24 month (BASF AG, 39C0059/95008, 1995; 50S0059/95106, 1998; 82S0059/95121, 2006). Moreover, their NOAELs are within a comparable range. Individual differences can be explained by the selection and spacing of the different dose levels in the particular studies. In conclusion, the overall NOAEL is 262.2 mg/kg bw/day.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The NOAEL for repeated dose toxicity was determined to be 262.2 mg/kg bw/d. As a result the substance is not considered to be classified for specific target organ toxicity under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/218.