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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
22 Apr - 28 Nov 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study. The reliability was set to 2 due to read-across.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 22. March 1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650, July 2000
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium N-(2-carboxyethyl)-N-dodecyl-β-alaninate
EC Number:
222-899-0
EC Name:
Disodium N-(2-carboxyethyl)-N-dodecyl-β-alaninate
Cas Number:
3655-00-3
Molecular formula:
C18H35NO4.2Na
IUPAC Name:
disodium N-(2-carboxyethyl)-N-dodecyl-beta-alaninate
Details on test material:
- Name of test material (as cited in study report): Sodium coco β-iminodipropionate
- Physical state: liquid
- Analytical purity: 87%
- Active content: 30.89%
- Lot/batch No.: 184
- Expiration date of the lot/batch: 30. June 2008
- Storage condition of test material: room temperature (20 ± 5°C)

Test animals

Species:
rat
Strain:
other: HanRcc: WIST(SPF)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd., Switzerland
- Age at study initiation: 11 weeks
- Weight at study initiation: mean: males 294 - 344 g; females 176 - 221 g
- Housing: individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding
- Diet: Pelleted standard Kliba Nafag 3433 rat/mouse maintenance diet, ad libitum
- Water: community tap-water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
highly purified
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The dosage formulations were prepared weekly prior to administration using the test item as supplied by the Sponsor and using a factor of 3.72 taking in account the purity of 87% and the content of the active ingredient of 30.89% (CAS 3655-00-3). Sodium coco β-iminodipropionate (CAS 3655-00-3) was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Several application formulations were prepared and representative analytical samples were collected and dispatched to the analytical laboratories internally. The test item concentrations were determined by HPLC coupled to an ELSD detector and quantified with the area under the peak.
Duration of treatment / exposure:
Males: minimum of 4 weeks (2 weeks prior to mating and 2 weeks of mating)
Females: approximately 7 weeks (2 weeks prior to mating up to day 4 post partum)
Frequency of treatment:
daily, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
43 (group 2), 160 (group 3), 600 (group 4) mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosage levels were selected based on a previous dose range finding toxicity study in Han Wistar Rats.

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported

BODY WEIGHT: Yes
- Time schedule for examinations: daily from treatment start to day of necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE:
- was recorded for male animals weekly during pre-pairing, for females weekly in during the pre-pairing period, gestation days 0-7, 7-14 and 14-21 post coitum, and days 1-4 post partum; food consumption was not recorded during the pairing period

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day before or day of necropsy for males, for females on day 5 post partum
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 males, 5 females
- Parameters examined: Complete Blood Cell Count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Leukocyte count (total), Differential leukocyte count, Platelet count, Coagulation, Prothrombin time (= Thromboplastin time), Activated partial Thromoplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day before or day of necropsy for males, for females on day 5 post partum
- Animals fasted: Yes
- How many animals: 5 males, 5 females
- Parameters examined: Glucose, Urea, Creatinine, Bilirubin (total), Cholesterol (total), Triglycerides, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Gamma-glutamyl-transferase, Bile acids, Creatine kinase, Sodium, Potassium, Chloride, Calcium, Phosphorus, Protein (total), Albumin, Globulin, Albumin/Globulin ratio

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males shortly before the scheduled sacrifice and females on day 3 or 4 post partum
- Dose groups that were examined: each group
- Battery of functions tested: sensory activity / grip strength / motor activity / other: cage-side and hand-held observations
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

ORGAN WEIGHTS
- testes and epididymides of all parental males were weighed as pairs, from 5 males and females selected randomly from each group, the following organs
were trimmed from any adherent tissue, as appropriate, and their wet weight taken: Adrenal glands (weighed as pairs), Brain, Heart, Kidneys (weighed as pairs), Liver, Thymus, Spleen

HISTOPATHOLOGY: Yes
- the following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution:
Prostate, Seminal vesicles with coagulating gland, Testes (in Bouin’s fixative), Epididymides (in Bouin’s fixative)
- the following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:
Ovaries
- In addition, from the five males and females per group selected for organ weights, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
Gross lesions, Brain (cerebrum, cerebellum, pons), Spinal chord, Small and large intestines (incl. Peyer’s patches), Stomach, Liver, Kidneys, Adrenals, Spleen, Heart, Thymus, Thyroid and Parathyroid, Trachea and lungs (preserved by inflation with fixative and then immersion), Uterus (with vagina), Urinary bladder, Lymph nodes (mandibular and mesenteric), Peripheral nerve (sciatic), Bone marrow
Statistics:
The following statistical methods may be used to food consumption, body weight, macroscopic findings, organ weights and reproduction data:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied to the macroscopic findings.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
One female in group 2 died accidentally after blood sampling. This death was not considered to be test item-related.
In group 4, all animals were noted to push the head through the bedding material starting on day 9 of the pre-pairing period onwards. One female had salivation after administration of the test item on day 9 of the gestation period. These signs are considered to be signs of discomfort and not to be adverse. In groups 2 and 3, no clinical signs or observations were noted.

BODY WEIGHT AND BODY WEIGHT GAIN
- Males - pre-pairing period: In group 4, mean body weight gain was statistically significantly decreased during the prepairing period (+6.6% versus +13.7% in the control group). Although this decrease had no statistically significant impact on the mean body weight, it was considered to be a test item related effect. During the pairing period, mean body weight and mean body weight gain were not affected by treatment with the test item. In groups 2 and 3, mean body weight and mean body weight gain were not affected by the treatment with the test item for the entire duration of the study. In group 3, mean body weight gain was statistically significant lower on day 6 of the pre-pairing period however, statistical significance occurred on a single day only and therefore it was not considered to be an adverse effect. During the pairing period, mean body weight gain was lower (+8.2% versus +12.0% in the control group) attaining statistically significance on days 9, 13 and 14. However, this was considered to be incidental as there was no-dose dependency.
- Females - pre-pairing, gestation and lactation periods: In group 4, mean body weight gain was statistically significant decreased during the prepairing period (+3.5% versus +8.2% in the control group). This was considered to be a test item related effect, although it did not affect the mean body weight. During the gestation and lactation periods, mean body weight and mean body weight gain were not affected by treatment with the test item. In group 3, mean body weight gain was lower during the pre-pairing period (+5.0% versus +8.2% in the control group) attaining statistical significance between day 6 and 8 and on days 11
and 14. This was considered an effect of the test item although mean body weight was not affected. During the gestation and lactation periods, mean body weight and mean body weight gain were not affected by treatment with the test item. In group 2, mean body weight and mean body weight gain were not affected for the entire duration of the treatment with the test item.

FOOD CONSUMPTION
- Males - pre-pairing period: In group 4, mean food consumption was statistically significant reduced between days 1-8 of the pre-pairing period (-19.2% compared to the control group) and it remained lower between days 8-14 (-10.6% compared to the control group) without attaining statistical significance. In group 3, mean food consumption was not considered to be affected by the treatment with the test item. As mean food consumption was slightly lower between days 1 and 8 of the pre-pairing period (-4.9% compared to the control group) and slightly higher between days 8 and 14 of the pre-pairing period (+2.1% compared to the control group), these variations were considered to be incidental. In group 2, no test item-related effects were noted.
- Females - pre-pairing, gestation and lactation periods: In group 4, mean food consumption was statistically significant decreased between days 1-8 of the pre-pairing period (-15.6% compared to the control group). This was considered to be a test item-related effect. During the gestation and lactation periods mean food consumption was not considered to be affected by treatment with the test item. The statistically significant higher food consumption during the lactation period (+23.0% compared to the control group) was considered to be incidental. In group 3, mean food consumption was decreased between days 1-8 of the pre-pairing period (-10.8% compared to the control group). This was considered to be a transient test item-related effect even though it was not statistically significant. During the gestation and lactation periods, no test item-related effects were noted. In group 2, mean food consumption was not affected by treatment with the test item. During the lactation period, the statistically significant higher mean food consumption was considered to be incidental.

HAEMATOLOGY
- Males: In group 4, the absolute count of neutrophils was statistically significant higher (+40.6% compared to the control group). Since the relative count was not statistically significant increased, this was not considered to be adverse. Mean corpuscular hemoglobin concentration was slightly but statistically significant decreased (-2.8% compared to the control group). Since the mean corpuscular hemoglobin was not affected, it was not considered to be an adverse
effect. In group 3, the statistically significant higher relative red cell volume distribution width (+19.0% compared to the control group) was within the range of historical reference value. In group 2 no changes were noted.
- Females: The assessment of the hematology data did not reveal any test item-related effects in females.

CLINICAL CHEMISTRY
- Males: In group 4, urea and potassium concentrations were statistically significant increased (+25.0% and +15.2%, respectively, compared to the control group). These alterations correlate with the histopathological findings noted in the kidney, thus they were considered to be test item-related. In group 3, the statistically significant lower phosphorus concentration (-13.6% compared to the control group) was within the range of historical reference values. The statistically significant lower concentration of total bilirubin in groups 2 and 3 (-27.6% and -26.8% compared to the control group, respectively) was not considered to be a test item-related effect because there was no dose-dependency.
- Females: The assessment of the clinical biochemistry parameters did not reveal any test item-related effect.

NEUROBEHAVIOUR
- Functional Observational Battery: There was no indication of any test item-related effect during the open field phase. Mean values of grip strength (fore- and hind paws) and landing foot splay gave no indication of any test item-related effects. In groups 3 and 4, mean body temperature was statistically significant reduced in males compared to the control group (37.5°C and 37.4°C, respectively, compared to 38.6 °C in the control group).
- Locomotor Activity: Locomotor activity was assessed quantitatively in terms of low beam counts in an activity monitor. In all groups there was no indication of a test item-related effect. The statistically significant lower locomotor activity noted at 30 min in group 4 males, and the statistically significant lower total locomotor activity noted in group 4 females were within the range of the historical control data.

ORGAN WEIGHTS
- Males: In group 4, mean relative liver and kidney weights were statistically significant increased.
- Females: In group 4, mean absolute and relative liver weights were statistically significant increased.
These findings correlated with the histopathological findings and were therefore considered to be test item-related.

GROSS PATHOLOGY
The findings noted during the macroscopic examinations did not give any indication of test item related effects.
- Males: In group 4, one male was noted to have reddish foci on the thymus, a second male was noted to have a dark red discoloration of the mandibular lymph node and a third male to have enlarged liver. Dark red or reddish foci were noted on the thymus of one male in group 3, in three males in group 2 and in one male in group 1. One male in group 1 was noted to have a subcutaneous yellowish-soft nodule on the right side of thoracic dorsal region.
- Females: In group 4, an isolated reddish focus in the lung of one female and dark red foci in the stomach of a second female were noted. In group 3, one female was noted to have a dark brown content in the stomach, jejunum and ileum. Dark red foci were also noted on the mucosa of fundus. The other findings noted were dark red discoloration of both ovaries in a second female and enlargement of the renal lymph nodes in a third female. In group 2, dark red foci were noted on the thymus in two females (one of these females died after the blood sampling).

HISTOPATHOLOGY:
- Liver: Minimal centrilobular hepatocellular hypertrophy was observed in four males in group 3 and slight centrilobular hepatocellular hypertrophy in one male in group 4; minimal to slight diffuse hepatocellular hypertrophy was noted in the remaining four males and all five females in group 4. Both types of liver cell hypertrophy were considered to represent an adaptive response to the increased need for metabolizing the test item.
- Thyroid gland: In group 4, minimally increased incidence and severity of diffuse follicular cell hypertrophy was noted in males and females. This change was considered to be secondary to the enhanced liver cell metabolism due to hepatocellular hypertrophy.
- Stomach: Minimal acanthosis of nonglandular stomach was present in all males and in three females in group 4 and was associated with minimal to slight focal/multifocal chronic inflammation in two of these males. Isolated, slight and multifocal chronic inflammation occurred in two females in group 3 and one male in group 4. Minimal multifocal chronic inflammation in the glandular stomach was noted in one male in group 3, and slight focal chronic inflammation in one female in group 3. The findings in the female were associated with minimal and multifocal congestion. Slight multifocal congestion occurred in two females in group 4, in one female associated with an acute thrombus and in the other with minimal and multifocal chronic inflammation. All of the stomach findings were considered to represent a local irritating effect of the test item.
- Kidneys: Minimally increased incidence of hyaline droplets/granules was observed in males in groups 3 and 4 (five males in each group, compared to three in the control group). These eosinophilic hyaline droplets/granules, which reflected an increased content of α-2μ-globulin, occur within the cytoplasm of proximal convoluted tubules of sexually mature male rats. Because a slightly increased severity was also noted (mean severity of 2.2 compared to 1.0 in the control males), this was considered to be an adverse effect of the test item in group 4 males.
- Spleen: Extramedullary hematopoiesis was minimally increased in severity in females in group 4, possibly secondary to or an adaptation to stress and/or impaired health conditions. In group 4, the minimally increased incidence of thymus atrophy in females was also considered to be secondary to stress rather than to represent a direct effect of the test item.
All other microscopic findings noted in various organs and in all groups examined were considered to be incidental in nature because their morphology, severity, and incidence did not distinguish treated rats from controls.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
43 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: statistically significant reductions in food consumption and body weight gain, histopathological findings and clinical biochemistry changes, reduced body temperature
Dose descriptor:
NOAEL
Effect level:
160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: effects on kidneys (histopathology + clinical biochemistry), food consumption and body weight gain

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: differences in mean Food consumption (Males in g/animal/day), pre-pairing period

Group

Days

 

1 – 8

8 - 14

1 - 14

mg/kg

g

(%)*

g

(%)*

g

(%)*

1 (0)

24.5

 

23.5

 

24

 

2 (43)

24.3

-0.8

25.6

+8.9

25

+4.2

3 (160)

23.3

-4.9

24

+2.1

23.7

-1.3

4 (600)

19.8

-19.2

21

-10.6

20.4

-15.0

*percentages refer to the values of group1

 

Table 2: Differences in mean Body weight gain males, pre-pairing period

Group

Days

 

1 – 8

8 - 14

1 - 14

mg/kg

g

(%)*

g

(%)*

g

(%)*

1 (0)

22

+7.0

21

+6.3

43

+13.7

2 (43)

20

+6.3

23

+6.8

43

+13.5

3 (160)

16

+5.0

17

+5.1

33

+10.4

4 (600)

10

+3.1

11

+3.3

21

+6.6

*increase within the respective time interval

 

Table 3: differences in mean Food consumption (Females in g/animal/day), pre-pairing period

Group

Days

 

1 – 8

8 - 14

1 - 14

mg/kg

g

(%)*

g

(%)*

g

(%)*

1 (0)

16.7

 

15.3

 

16

 

2 (43)

16.6

-0.6

16.7

+9.2

16.6

+3.8

3 (160)

14.9

-10.8

16.3

+6.5

15.6

-2.5

4 (600)

14.1

-15.6

15.7

+2.6

14.9

-6.9

*percentages refer to the values of group1

 

Table 4: Differences in mean Body weight gain females, pre-pairing period

Group

Days

 

1 – 8

8 - 14

1 - 14

mg/kg

g

(%)*

g

(%)*

g

(%)*

1 (0)

5

+2.6

11

+5.5

16

+8.2

2 (43)

6

+3.0

7

+3.4

13

+6.4

3 (160)

0

+0.0

10

+5.0

10

+5.0

4 (600)

1

+0.5

6

+3.0

7

+3.5

*increase within the respective time interval

 

Table 5: Clinical Biochemistry

 

 

Group 1

Group 2

Group 3

Group 4

Days (pre-pairing period)

 

0

43 mg/kg bw

160 mg/kg bw

600 mg/kg bw

Male (before necropsy)

Urea

MEAN

6.16

5.93

6.79

7.70*

mmol/L

% of control

100

96

110

125

 

SD

0.63

0.93

0.72

1.16

 

N

5

5

5

5

Potassium

MEAN

4.09

4.27

4.34

4.71*

mmol/L

% of control

100

104

106

115

 

SD

0.39

0.24

0.18

0.49

 

N

5

5

5

5

Female (day 5 post partum)

Urea

MEAN             

9.34

8.16

8.03

8.79

mmol/L

% of control

100

87

86

94

 

SD

1.82

1.28

0.63

2.12

 

N

5

5

5

5

Potassium

MEAN

4.10

4.22

3.92

3.77

mmol/L

% of control

100

103

96

92

 

SD

0.59

0.80

0.53

0.50

 

N

5

5

5

5

Significant different compared to control value, *p<0.05

 

Table 6: Organ/body weight ratios

 

 

Group 1

Group 2

Group 3

Group 4

Days (pre-pairing period)

 

0

43 mg/kg bw

160 mg/kg bw

600 mg/kg bw

Male

Liver (%)

MEAN

2.57

2.53

2.63

3.02**

 

SD

0.1

0.07

0.16

0.17

 

N

5

5

5

5

Kidneys (%)

MEAN

0.61

0.61

0.61

0.68**

 

SD

0.02

0.02

0.03

0.02

 

N

5

5

5

5

Female

Liver (%)

MEAN

3.18

3.35

3.19

3.93**

 

SD

0.21

0.07

0.25

0.23

 

N

5

5

5

5

Kidneys (%)

MEAN

0.63

0.65

0.61

0.67

 

SD

0.06

0.02

0.03

0.05

 

N

5

5

5

5

Significant different compared to control value, **p<0.01

Applicant's summary and conclusion

Conclusions:
Based on the statistically significant reductions in food consumption and body weight gain observed in males and females at 600 mg/kg/day and the transient effects in females at 160 mg/kg/day, on histopathological findings and clinical biochemistry changes observed in males at 600 mg/kg, and on reduced body temperature recorded in males at 160 and 600 mg/kg/day, the systemic NOEL for Sodium coco β-iminodipropionate (CAS 3655-00-3) was established at 43
mg/kg bw/day. The NOAEL was set to be 160 mg/kg bw.

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