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Toxicological information

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation

Data source

Reference
Reference Type:
publication
Title:
A 90-day toxicity study of L-asparagine, a food additive, in F344 rats
Author:
M Yokohira, K Hosokawa, K Yamakawa, N Hashimoto, S Suzuki, Y Matsuda, K Saoo, T Kuno, K lmaida
Year:
2008
Bibliographic source:
Food and Chemical Toxicology 46; 2568-2572

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Molecular weight: 132.12 g/mol
C4H8N2O3
isoelectric point: 5.41

Test animals

Species:
rat
Strain:
Fischer 344/DuCrj
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan (Atsugi, Japan)
- Age at study initiation: 5 weeks
- Housing: Five per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 2°C
- Humidity (%): 60 ± 10%
- Photoperiod (hrs dark / hrs light): 12h light/12h dark

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): 1.25%, 25.%, 5% of asparagine were mixed with synthetic diet (AIN-93G, Oriental yeast Co., Ltd, Tokyo, Japan) containing minerals and vitamins and constarch substituted for L-asparagine.


Analytical verification of doses or concentrations:
yes
Remarks:
performed by the Institute of Life Sciences, Ajinomoto Co. Inc.
Details on analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
continuously with diet
Doses / concentrationsopen allclose all
Dose / conc.:
1.25 other: % in diet
Remarks:
753 mg/kg bw/d in males, 857 mg/ kg bw/d in females
Dose / conc.:
2.5 other: % in diet
Remarks:
1537 mg/kg bw/d in males, 1708 mg/kg bw/d in females
Dose / conc.:
5 other: % in diet
Remarks:
3242 mg/kg bw/d in males, 3466 mg/kg bw/d in females
No. of animals per sex per dose:
10
Control animals:
yes, plain diet

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: once a week

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the 90 days study after 16 h starvation collection of blood samples from the abdominal aorta under deep anesthesia and subsequently the animals were exsanguinated
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: 20


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the 90 days study after 16 h starvation the animals were exsanguinated
- Animals fasted: Yes
- How many animals: 20
- Parameters examined: TP, A/G, ALB, BIL, TC, GLU, PL, TG, BUN, CRN, Ca, P, Na, Cl, K, AST, ALT, ALP, γ-GT

CAGE SIDE OBSERVATIONS: Not specified

URINALYSIS: No
- Metabolism cages used for collection of urine: No
- Animals fasted: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. A complete autopsy was then performed. Macroscopic pathological findings were noted during autopsy and weights of the brain,lungs, heart, spleen, liver, adrenals, kidneys, testes, salivary glands and ovaries were determined and values relative to the body weights were calculated.

HISTOPATHOLOGY: Yes. In addition to the above-mentioned organs, organs and tissues including the esophagus, stomach, duodenum, jejunum, ileum (including Peyer’s patches), colon, cecum, rectum, pancreas, urinary bladder, trachea, thyroid gland, tongue, skeletal muscle, ischiatic nerve, spinal cord, aorta, nasal cavity, eyeballs, Harderian glands, Zymbal’s glands, skin, mammary gland, mesenteric and mandibular lymph nodes, bone marrow in femur and sternum, epididymis, prostate, seminal vesicles, uterus and vagina were fixed in 10% buffered formalin solution. Testes were fixed in 2% glutaraldehyde, 15% formalin and 3% acetic acid (GFA) solution overnight before being transferred to 10% buffered formalin solution. The pituitary, prostate, seminal vesicle and uterus were weighed at the time of processing. Paraffin sections of all tissues from the 5% treated group and control groups were stained with hematoxylin and eosin for histopathological assessment.
Statistics:
Analysis of the data for body and organ weights, hematology and serum biochemistry was performed using the following general procedures as appropriate. Dunnett's multiple comparison (P < 0.05) was applied. If significant heterogeneity of variance was detected, the Student's t-test for comparing treatment and control groups was employed. The Spearman’s rank correlation test was applied to examine dose - response relationships. The Fisher's exact probability test was used for histopathological data to analyze increases in incidence between treated and control rats. incidences of differing severities of histopathological findings were also compared using the one-sided Mann - Whitney U-test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Slight decrease of body weights was observed in L-asparagine treated groups of males at autopsy, but there were no significant differences in final body weights in either male or female groups.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
ALP was significantly decreased by 9.4% in 2.5% treated males but there was no dose - response relationship. In 5% treated females, GLU, PL, TG, K and ALT were significantly increased to 123%, 125%, 316%, 117% and 121%, respectively. In 2.5% treated females, TG was significantly increased to 300%. In 1.25% treated females, GLU and TG were also significantly increased to 113% and 246%, respectively, and CRN was significantly decreased to 11%. The other serum biochemical parameters were not affected by the treatment.
Urinalysis findings:
not 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):
Final bodyweights of 1.25% and 5% treated males, 273 ± 15 and 271 ± 14 g, respectively, and these values were significantly decreased compared to the control value, 290 ± 8 g. Significant increases of relative organ weights of the brain, kidney and testes were observed in 5% treated males,
0.72 ± 0.04%, 0.56 ± 0.03% and 1.07 ± 0.05%, compared with controls, 0.66 ± 0.03%, 0.52 ± 0.03% and 1.00 ± 0.02%, respectively. Other organs in males did not demonstrate significant variation and final body - and organ weights of females did not significantly vary across groups.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Organs of 5% and 0% treated males and females were analyzed in line with the OECD Test Guideline. In some rats, inflammatory findings or leukemia in the lung, simple cysts of the ovaries and sebaceous adenomas in the Zymbal’s glands were observed. However, there were no significant intergroup differences by the Fischer's exact test and the Mann-Whitney U-test. Additional examinations of 2.5% and 1.25% treated rats were therefore not performed, in line with the OECD Test Guideline.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The monocytic lymphomas in the lungs and sebaceous adenomas observed in the present study as proliferative lesions in male or female 5% groups are known to occur spontaneously in this strain of rats
Other effects:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 650 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
body weight and weight gain
clinical biochemistry
Key result
Dose descriptor:
NOAEL
Effect level:
1 730 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
female
Basis for effect level:
body weight and weight gain
clinical biochemistry

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Table 1: Selected clinical chemistry and pathology findings

Doses (unit)

0

1.25%

2.5%

5%

0

1.25%

2.5%

5%

male

female

Number of animals/group

10

10

10

9

10

10

10

9

Blood chemistry(day 90)

 

 

 

 

 

 

 

- TP (g/dL)

7.0±0.1

 7.1±0.2

 7.2±0.2

7.0±0.2 

7.2±0.2 

7.1±0.4 

7.3±0.3 

 7.1±0.3

- A/G

1.7±0.1

 1.7±0.2

 1.5±0.2

 1.6±0.2

 2.0±0.1

 2.0±0.3

 2.0±0.1

 2.0±0.2

- ALB (g/dL)

4.4±0.1

 4.4±0.2

 4.3±0.2

 4.3±0.1

 4.8±0.1

 4.7±0.2

 4.9±0.2

 4.7±0.1

- BIL (mg/dL)

0.05±0.01

 0.050.01

 0.04±0.00

 0.04±0.01

 0.05±0.00

 0.05±0.01

 0.05±0.01

 0.05±0.01

- TC (mg/dL)

70.1±3.8

 66.8±12.9

 61.2±9.2

 72.6±14.0

 63.2±9.2

 69.9±10.2

 68.6±13.5

 78.4±18.4

- GLU (mg/dL)

168±29

 165±18

 149±14

 165±17

 107±7

 121±8

 116±8

 132±13

- PL (mg/dL)

116±7

114±20

103±14

117±19

115±13

128±14

130±19

114±27

- TG (mg/dL)

112±24

122±49

112±32

143±38

22±8

54±23

66±8

69±21

- BUN (mg/dL)

15.2±0.9

16.9±1.6

15.5±1.6

17.3±2.3

16.1±1.4

16.3±1.8

16.2±0.9

17.1±1.6

- CRN (mg/dL)

0.25±0.01

0.25±0.02

0.26±0.01

0.26±0.02

0.27±0.02

0.24±0.03

0.27±0.01

0.25±0.02

- Ca (mg/dL)

10.6±0.3

10.6±0.5

10.8±0.2

10.5±0.2

10.3±0.3

10.4±0.2

10.7±0.1

10.9±1.5

- P (mg/dL)

5.3±0.7

5.4±1.0

5.2±0.6

4.6±0.7

5.9±0.7

5.8±1.4

4.8±0.4

5±0.9

- Na (mEq/L)

140±1

139±2

140±1

139±1

141±2

141±2

143±1

140±2

- Cl (mEq/L)

102±1

101±1

101±1

101±1

104±2

104±1

103±1

101±5

- K (mEq/L)

3.9±0.6

4.1±0.5

3.8±0.3

4.0±0.3

4.1±0.3

4.4±1

4.5±0.4

4.8±0.6

- AST (IU/L)

61.2±7

65.7±6.3

65.4±4.7

68.3±9.2

68.6±4.6

69.3±5.5

69.3±5.5

77.2±12.2

- ALT (IU/L)

33.4±5.4

3.3±6.4

38.2±5.1

39.6±7.9

26.8±2.4

27.7±2.6

28.5±2.0

32.4±3.4

- ALP (IU/L)

437±23

432±39

397±27

424±34

307±44

340±50

339±58

349±49

- γ-GTP (IU/L)

<2

<2

<2

<2

<2

<2

<2

2.4±1.3

Histopathology

 

 

 

 

 

 

 

Lung bronchial

 

 

 

 

 

 

 

- Accumulation, foam cell/slight lesion

2

 

 

 5

 

 

 6

- Accumulation, foam cell/moderate lesion

0

 

 

 0

 2

 

 

 1

- Acute inflammation/slight lesions

2

 

 

 1

 0

 

 

 1

-Acute inflammation/moderate lesions

0

 

 

 0

 1

 

 

 0

- Acute inflammation/severe lesions

0

 

 

 0

 0

 

 

 0

- Granulation

0

 

 

 0

 0

 

 

 1

- Monocytic cell leukemia

0

 

 

 2

 0

 

 

 2

Ovary

 

 

 

 

 

 

 

- simple cyst

0

 

 

 0

 1

 

 

 0

Zymbal´s gland

 

 

 

 

 

 

 

- Sebaceuos adenoma

0

 

 

 1

 0

 

 

 0

Statistics: For clinical chemistry ANOVA + Dunnetts tests (two sided): * P < 0.05   ** P < 0.01 and for histopathology Fischer´s exact test.

Applicant's summary and conclusion

Conclusions:
In the present 90-days repeated dose study in rats the animals received either 0, 1.25, 2.5 or 5% L-asparagine via daily diet. There were no adverse effects observed except an increase in relative organ weight and some clinical biochemistry biomarkers at the 5% dose level. Therefore, significant effects of the 5% treatment were concluded and the NOAEL was determined to be 2.5%, i.e. 1650 mg/kg bw in males and 1730 mg/kg bw in females.