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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: A non-GLP study performed to sound scientific principles with a sufficient level of detail to assess the quality of the submitted data.

Data source

Reference
Reference Type:
publication
Title:
Toxicity Study of Sodium L-2-Pyrrolidone-5-carboxylate (PCA-Na) Administered to Rats in Diet for 13 and 26 Weeks
Author:
Ishii H, Fujimoto T, Ninomiya K & Torii K
Year:
1992
Bibliographic source:
Ishii H et al (1992) Toxicity Study of Sodium L-2 Pyrrolidone-5-Carboxylate Administered to Rats in Diet for 13 and 26 Weeks. Iyakuhin Kenkyu, 23(6): 717-36

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
no
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
sodium (2S)-5-oxopyrrolidine-2-carboxylate
Cas Number:
28874-51-3
Molecular formula:
C5H6NO3. Na
IUPAC Name:
sodium (2S)-5-oxopyrrolidine-2-carboxylate
Test material form:
not specified

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 4 weeks old
- Weight at study initiation: Approximately 100 g
- Housing: In groups of two, in a wire bracket cage (26 x 26 x 45cm).
- Diet: Basic commercial diet
- Water: Tap water, ad libitum
- Acclimation period: Males were acclimatised for 7 days whereas females were acclimatised for 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1 °C
- Humidity (%): 60 ± 10%

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Preparation: Diet containing 2, 4 or 8 % test material was given to animals in test groups 2, 3 and 4, respectively. Additionally, a diet containing 6 % Prop-Na in which sodium contents is approximately equivalent to that of 8 % PCA-Na was given freely to the sodium control group (group 5). From week 4 after the start of administration, the contents of the test material in the diet were increased 50 % in order to keep the dosages per body weight to the prescribed levels in each group (1000, 2000 and 4000 mg/kg bw).
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
13 and 26 weeks.
Frequency of treatment:
Continuously (in diet)
Doses / concentrations
Remarks:
Doses / Concentrations:
2, 4 or 8 % test material
Basis:
other: in diet. From week 4 after the start of administration, the contents of the test material in the diet were increased 50 % in order to keep the dosages per body weight to the prescribed levels in each group.
No. of animals per sex per dose:
Eight per sex per dose.
Control animals:
yes, plain diet
Details on study design:
- Study design: Two separate studies were run in parallel, where animals were either treated with the test material (and controls) for 13 or 26 consecutive weeks.
Positive control:
- Sodium control: A sodium control was included in the study, where 8 animals per sex per were exposed to sodium propionic acid at 3000 mg/kg. Approximately equivalent to 4000 mg/kg of the test material.

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: Daily, except Sundays.
- Observations: General appearance, behaviour, and conditions of excretions.

BODY WEIGHT: Yes.
- Time schedule for examinations: Measurements were taken on the day of study initiation, weekly thereafter, and then again at autopsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Each 24 hour period (per cage)

HAEMATOLOGY: Yes.
- Time schedule for collection of blood: Blood was collected after urine collection on the day before autopsy.
- Anaesthetic used for blood collection: Yes, diethyl ether gas.
- Animals fasted: Yes.
- How many animals: All ten animals.
- Parameters checked: Specific gravity of whole blood (only in the 26-week study) was measured by cupric sulfate method immediately after the blood collection. Erythrocyte count and leukocyte count were measured using a counting chamber. Hemoglobin content was measured by cyanmethemoglobin method and red blood cell volume was measured by haematocrite method. Differential leukocyte count was measured by microscopic observation of stained blood smear slides.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected after urine collection on the day before autopsy.
- Animals fasted: Yes
- How many animals: All animals were sampled.
- Parameters checked: Serum glutamic-oxaloacetic transaminase, glutamic pyruvic transaminase (S-GOT, S-GPT, by Reitman-Frankel method), serum alkaline phosphatase (S-ALP, by Kind-King method), total cholesterol (by modified Zurkowski method), glucose (by oxidizing enzyme method), urea nitrogen (by Urease method), total protein (by refractometry), and sodium and calcium (only in the 26-week study, by atomic absorption analysis). Albumin/globulin ratio (A/G ratio) was calculated from the fractions of albumin and globulin obtained by electrophoresis of the serum melted after cryopreservation.

URINALYSIS: Yes
- Time schedule for collection of urine: On the day before necropsy.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes.
- Parameters checked: Parameters analyzed were protein, glucose, urobilinogen, bilirubin, occult blood, ketone bodies, specific gravity by refractometry, pH by electrode reaction, and urinary electrolyte (Na+ and Ca++) by atomic absorption analysis. Ketone bodies, occult blood, specific gravity, Ca and urine volume were, however, analyzed only in the 26-week study.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- The weights of the following organs were recorded: liver, heart, spleen, kidneys, adrenals, testes and ovaries.
In addition to the above the cerebrum, cerebellum, pituitary, lungs, thymus, thyroid, stomach, small intestine, pancreas, urinary bladder, prostate, and uterus were examined macroscopically.

HISTOPATHOLOGY: Yes
The liver, heart, spleen, kidneys, adrenals, testes, ovaries, cerebrum, cerebellum, pituitary, lungs, thymus, thyroid, stomach, small intestine, pancreas, urinary bladder, prostate, and uterus were examined histopathologically with a light microscope. organs were fixed in neutral buffered 10 % formalin (pH 7.4) after excision and embedded in paraffin, sectioned and stained with hematoxylin and eosin. the kidney sections were stained additionally with Kossa method for calcium. All sections were observed microscopically to record histological changes which had occurred.
Statistics:
Data obtained were calculated to give group means and standard deviations, and statistical analyses were conducted by Student’s t-test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Soft faeces were observed in all treated groups.
Mortality:
mortality observed, treatment-related
Description (incidence):
Soft faeces were observed in all treated groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Dose-dependent reduction in male body weight.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment related effects were observed.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Dose-dependent increase in males and females.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment related effects were observed.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment related effects were observed.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment related effects were observed.
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Significant inter-group differences in organ weights were noted in the liver, spleen, adrenals, and ovaries, however these changes were not dose-dependent.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related effects were observed.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
No treatment related effects were observed.
Histopathological findings: neoplastic:
not examined
Details on results:
- Two animals were excluded from the evaluation:
> At week 3 after the start of administration, one female animal in the 1000 mg/kg bw group was turned out to be a male.
> At week 4 of administration, one male animal of the control group damaged the teeth with the cage, which led to eating difficulty. Thereafter the body weight extremely decreased and the animal was necropsied. No abnormalities were observed in the visceral organs.

CLINICAL SIGNS AND MORTALITY
- No deaths were noted in animals treated for either 13 or 26 weeks.
- Soft faeces were observed in several animals dosed at 2000 and 4000 mg/kg bw, from one week after the start of administration. At week 4 when the mixing proportions of the test material in the diet were increased, soft faeces were observed in most of animals in the groups administered with 1000, 2000 and 4000 mg/kg bw. Thereafter, soft faeces were gradually decreased and disappeared at and after week 9. No other special abnormalities were observed.
- Soft faces observed at the early period of both studies did not developed to diarrhoea, but was recovered by week 9 of administration. Therefore the change is considered to be caused by the transient insufficient adaptation of the intestinal tract due to the intake of food which contains large amount of highly-moisturizing test material.

BODY WEIGHT AND WEIGHT GAIN
- In the males of the groups administered with the test material, the body weight gain tended to be suppressed dose-dependently. In the 4000 mg/kg bw dose group, the body weight was significantly lower than the control group at week 10, 11, 13 and thereafter until the end of the administration period.
- The male animals of the sodium control group showed delayed growth immediately after the start of administration and the body weights were significantly lower than the control group from week 2 until the end of administration.
- No differences in the body weights were noted in females between each administration group and the control group.

FOOD CONSUMPTION AND COMPOUND INTAKE
- No differences in food intakes were noted between the each administration group and the control group either in males or females.
- Mean daily dose intake per bodyweight can be seen in Table 1 for the 13 week study and in Table 2 for the 26 week study.
- Although there was a significant suppression of body weight gain in male of the sodium control group from week 2, and from week 10 in the high dose group (4000 mg/kg bw), almost no difference was noted in the food intake in these groups. Body weight gain suppression was therefore considered to be due to transient disturbance of digestion and absorption because the administration method was diet mixing and the mixing ratio were as high as 6-9 % in the sodium control group and 8-12 % in the high dose group.

WATER CONSUMPTION AND COMPOUND INTAKE
- The water intake of the all groups dosed with the test material increased dose-dependently both in males and females, and the water intake of the sodium control group showed high levels, following the high dose group administered with the test material (4000 mg/kg bw).
- The reason for the increase in water intake is considered to be induced by the physiological demand in order to maintain a constant osmolarity and mineral concentration of body fluid because the sodium ion contained in the diet is assumed to be absorbed from the intestine and excreted into urine.

HAEMATOLOGY
- Compared to the control group, statistically significant differences were noted in the leukocyte count and the ratio of segmented neutrophil count in some animals of all groups administered with the test material in the 13-week study, and haemoglobin concentration in the 26-week study. However, each value of these parameters was within the normal variations, and no relation with the dose level was noted in the changes.

CLINICAL CHEMISTRY
- Total cholesterol value was significantly low compared to the control group in the male sodium control group and all female dose groups in the 13-week study and in the male sodium control group in the 26-week study. Urea nitrogen was significantly high compared to the control group in the males of high dose group administered with test material at 4000 mg/kg bw and in the females of the sodium control group in the 13-week study, and in the 26-week study in the males of 2000 and 4000 mg/kg bw dose groups as well as in males and females of the sodium control group. However, the values of these two parameters in each animal were within the normal variation. Significant difference between the control group and the dose groups were noted sporadically in other parameters. However, no relation with dose level was noted and the measured values of each animal were within the normal variations.

URINALYSIS
- The 26-week study, urinary pH of each group showed a slightly higher value compared to the results in the 13-week study. However, the results were due to time lapse of urine collection for 24 hours in the 26-week study. No other reportable changes were noted.

ORGAN WEIGHTS
- In the 13-week study significantly high values in the absolute kidney weight was noted in the left kidney of males in the high dose group (4000 mg/kg bw), and in the both kidneys of females in the middle and high dose groups (2000 and 4000 mg/kg bw) and sodium control group. Significant increase in relative kidney weights of both sides was noted in the middle and high dose groups (2000 and 4000 mg/kg bw) and sodium control group both in males and females.
In the 26-week study, absolute kidney weight significantly increased in the male high dose group (4000 mg/kg bw) and all female dose groups. Significantly high values in the relative kidney weight compared to the control group were noted in the middle dose group and high dose group (2000 and 4000 mg/kg bw) and the sodium control group in male, and all female dose groups.
Significantly high values compared to the control group was noted in the absolute heart weight of the females in the middle and high dose groups (2000 and 4000 mg/kg bw), and in the relative heart weight of the high dose group (4000 mg/kg bw) and the sodium control group both in males and females in the 13-week study.
In the 26-week study, significantly high values compared to the control group were noted in the absolute heart weights of females in the middle and high dose groups (2000 and 4000 mg/kg bw) and sodium control group, and in the relative heart weights of all of male dose groups and the female high dose group (4000 mg/kg bw), and the sodium control group.
Significantly high values compared to the control group was noted in absolute testis weight of the sodium control group and the relative testis weight of the high dose group (4000 mg/kg bw) and of the sodium control group in the 13-week study. In the 26-week study, the relative weights of bilateral testes showed significantly high values in the middle dose and the high dose groups (2000 and 4000 mg/kg bw) and in the sodium control group compared to the control group. In the 26-week study, slight dose-dependent tendency was noted in the increase of the relative testes weight.
Significant inter-group differences between the control group and the dose groups were sporadically noted in the weights of the liver, spleen, adrenals, and ovaries. However, these changes were not dose-dependent.
- Regarding the kidney, the correlation especially of the relative kidney weight with the dose level is considered to be due to compensatory hypertrophy of kidney caused by the load of sodium ion. In addition, the correlation of heart weight (relative heart weight) with dose level is considered to be caused by dose-dependent decrease of the body weight accompanied by no change of heart weight, which led to the increase of the relative heart weight.
The number of animals with the submucosal mineralization of renal pelvis and the severity of the change showed correlation with the dose level of the sodium salt. The mineralization in the medulla of kidney is noted at high frequency in adult rats. In the rats used in this study, the change was noted in all groups including the control group. However, the mineralization showed no correlation with the dose level of the test material in its incidence or development of the change.

GROSS PATHOLOGY
- Several findings were noted sporadically in macroscopic pathological observation both in the 13-week study and in the 26-week study. However, relation with test material administration was not noted.

HISTOPATHOLOGY: NON-NEOPLASTIC
- Very slight chronic focal myocarditis consisting of the degeneration and necrosis of heart muscles associating with large mononuclear cell infiltration were noted in a male rat of the low dose group (1000 mg/kg bw) and a female of the middle dose group (2000 mg/kg bw) in the 13-week study. In the 26-week study, this change was noted in a female rat of the low dose group (1000 mg/kg bw) and sporadically in several males of each group including the control group.
- In the lung, chronic interstitial pneumonia consisting of perivascular cell infiltration, enlargement of alveolar epithelial cells, histiocyte infiltration into the interstitium of alveolar wall, and macrophage infiltration into alveolar space were noted sporadically in each group including the control group. This change was very slight and the incidence was low in the 13-week study. However, in the 26-week study, the incidence of the change was high and developed change was also observed. However, no relation with administration was noted in the incidence and the severity of this change.
- In the kidney, chronic nephritis consisting of acidophilic casts with flattened tubular epithelium, regenerated tubules, thickening of tubular and glomerular basement membrane, perivascular infiltration of lymphocytes, and submucosal mineralization in renal pelvis and closely relevant acute or chronic pyelonephritis, and mineralization in the inner medulla were noted. Chronic nephritis was noted in a female of the low dose group (1000 mg/kg bw) in the 13-week study, and several males sporadically in each group including the control group in the 26-week study.
- Submucosal mineralization in renal pelvis was noted in 2 males of the high dose group (4000 mg/kg bw), 3 males of the sodium control group, 2 females of the middle dose group (2000 mg/kg bw), and 1 female of the sodium control group in the 13-week study. In the 26-week study, the mineralization was noted in about half the number of animals in all groups except female control group and low dose group (1000 mg/kg bw). The incidence and the severity of the change were highly dose-dependent according to the administration of the test material, and were similar in degree between the sodium control group and the high dose group (4000 mg/kg bw). Mineralization at the inner medulla was noted in each group including the control group in the 26-week study.
- In the testes, very slight atrophy of seminiferous tubules was observed sporadically in each group including the control group in the 13-week as well as in the 26-week study.
- Other histopathological changes were sporadically observed in the liver, stomach, urinary bladder, adrenals, pituitary gland and thyroid. However, no administration-related changes were noted.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
8 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Effect level based on the mean daily intake of the test material over 26 weeks.
Dose descriptor:
NOAEL
Effect level:
7 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Effect level based on the mean daily intake of the test material over 26 weeks.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Mean (± SD) Daily Dose Intake per Body Weight

Nominal Concentration mg/kg bw

Male (mg/kg bw) (range)

Female (mg/kg bw)

Test Material, 1000

1800 ± 400

2000 ± 300

Test Material, 2000

3800 ± 700

4000 ± 600

Test Material, 4000

7200 ± 1400

8200 ± 1100

Sodium Control, 3000

5500 ± 1000

5900 ± 600

 

Table 2: Mean Daily Dose Intake per Body Weight, 26 week Study

Nominal Concentration mg/kg bw

Daily Male Intake (mg/kg bw) (range)

Daily Female Intake (mg/kg bw) (range)

Test Material, 1000

1800 (1300 - 2800)

2000 (1600 – 2800)

Test Material, 2000

3800 (2800 – 5700)

4000 (3100 – 5600)

Test Material, 4000

7200 (5400 – 11300)

8200 (6500 – 12100)

Sodium Control, 3000

5600 (4400 – 9000)

5900 (4700 – 7000)

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, repeated oral exposure to the test material over a 26 consecutive week period induced no toxic effects in male or female rats. Therefore the NOAEL can be said to be the maximum daily intake, which was determined to be 7200 mg/kg bw/day and 8200 mg/kg bw/day for males and females, respectively.
Executive summary:

The repeat dose toxicity of the test material was determined in an oral feeding study which was performed according to a methodology similar to OECD 408. Male and female rats were exposed to the test material, prepared in their diet, in two parallel tests conducted over either a 13 or 26 consecutive week period. In both tests the animals were exposed to nominal concentrations of 1000, 2000 and 4000 mg/kg bw. Blank and sodium (3000 mg/kg bw) controls were run concurrently for comparison.

Under the conditions of the study several effects were observed, soft faeces, a dose-dependent reduction in male body weight, and a dose-dependent increase in male and female water intake. However, these were considered to be incidental. Significant inter-group differences in organ weights were noted in the liver, spleen, adrenals, and ovaries, however these changes were not dose-dependent and again not considered to be of toxicological significance.

Significant inter-group differences in organ weights and/or histopathology changes were noted in the kidneys, heart, liver, spleen, adrenals, lung, renal pelvis, ovaries and testes, however these changes were considered to be incidental . As the kidney is the responsible organ for excretion of sodium ions. It was suggested that the observed hypertrophy of the kidney (compensatory hypertrophy) was induced to enhance renal function in the rat during growing phase in order to adapt the load.

Since repeated exposure to the test material over a 26 consecutive week period induced no toxic effects in male or female rats the NOAEL can be estimated to be the maximum daily intake, which was determined to be 7200 mg/kg bw/day and 8200 mg/kg bw/day for males and females, respectively.