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Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Qualifier:
according to guideline
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
GLP compliance:
yes (incl. QA statement)
Test type:
fixed dose procedure
Limit test:
yes

Test material

1
Chemical structure
Reference substance name:
trisodium (2S)-2,6-bis(3-carboxylatopropanamido)hexanoate
EC Number:
948-778-9
Molecular formula:
C14O8H19N2Na3
IUPAC Name:
trisodium (2S)-2,6-bis(3-carboxylatopropanamido)hexanoate
Test material form:
liquid
Details on test material:
Active ingredient (%): 48.9% dry substance
Purity (%): 95.50 %
Stability: 5 years
Sterilization: None
Solubility: Soluble in water
Storage: Room Temperature
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 23ADB/50
- Expiration date of the lot/batch: 21/11/2023

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
In order to get the test item in a solution or suspension, which is applicable to the animals, several vehicles (sterile water, corn oil) were evaluated and preparation and solubility tests were performed. The test material formed an emulsion in corn oil which could not be applied. The preparation with sterile water yielded an applicable solution and considered to be adequate.



Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 – 9 weeks
- Weight at study initiation: animal no. 1: 173 g; animal no. 2: 184 g; animal no. 3: 153 g; animal no. 4: 176 g; animal no. 5: 170 g
- Fasting period before study: n/a
- Housing: Full barrier in an air-conditioned room
- Diet: ad libitum
- Water : ad libitum
- Acclimation period: Adequate acclimatisation period (at least five days) under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22  3 °C
- Humidity (%): 55  10%
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Aqua ad injectionem (sterile water, DELTAMEDICA, lot no. 807634, expiry date: 06/2021). This vehicle was chosen due to its non-toxic characteristics.
Details on oral exposure:
Animal No. /
Sex Dose(mg/kg bw) Body Weight on Day of Administration (g) Volume of Test Item Preparation in Vehicle Administered per Animal (mL)
1 / Female 2000 173 1.7
2 / Female 2000 184 1.8
3 / Female 2000 153 1.5
4 / Female 2000 176 1.8
5 / Female 2000 170 1.7
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
5 animals, Fixed dose
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were weighed on day 1 (prior to the administration) and on days 8 and 15. A careful clinical examination was made several times on the day of dosing (at least once during the first 30 minutes and with special attention given during the first 4 hours post-dose). Thereafter, the animals were observed for clinical signs once daily until the end of the observation period.
- Necropsy of survivors performed: yes
- Other examinations performed: Cageside observations included changes in the skin and fur, eyes and mucous membranes. Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern were examined. Particular attention was directed to observations of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
All animals were subjected to gross necropsy and examined macroscopically for gross pathological changes. In the absence of gross pathological changes no tissues were preserved for a possible histopathological evaluation.

Results and discussion

Preliminary study:
Sighting Study
The starting dose for the sighting study was 2000 mg/kg body weight.
No compound-related mortality was recorded for the first animal dosed.
Effect levels
Sex:
female
Dose descriptor:
LD50
Effect level:
> 5 000 mg/kg bw
Based on:
test mat.
Mortality:
none
Clinical signs:
other: none
Gross pathology:
No specific gross pathological changes were recorded for any animal.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, a single oral application of the test item Trisodium (2S)-2,6-bis(3-carboxylatopropanamido) hexanoate_Disuccinyl lysine sodium salt to rats at a dose of 2000 mg/kg body weight was not associated with signs of toxicity or mortality.
The median lethal dose of Trisodium (2S)-2,6-bis(3-carboxylatopropanamido) hexanoate_Disuccinyl lysine sodium salt after a single oral administration to female rats, observed over a period of 14 days is:
LD50 cut-off (rat): ˃ 5000 mg/kg bw
Executive summary:

Five females WISTAR Crl: WI(Han) rats, were treated with the test item by oral gavage administration at a dosage of 2000 mg/kg body weight. The test item was dissolved in the vehicle aqua ad injectionem (sterile water) at a concentration of 0.2 g/mL and administered at a dose volume of 10 mL/kg.

All animals used in the study after their entrance at BSL were allowed to acclimatise to the laboratory conditions for at least 5 days. The animals were observed on delivery, on inclusion in the study and before administration for mortality/morbidity and other clinical signs. All animals were examined for clinical signs several times on the day of dosing and once daily until the end of the observation period. Their body weights were recorded on day 1 (prior to the administration) and on days 8 and 15. All animals were necropsied and examined macroscopically.

Table 1: Results

Animal No. / Sex

Dose

(mg/kg bw)

Number of Animals

Number of
Intercurrent Deaths

Sighting Study

1 / Female

2000

1

0

Main Study

2 / Female

2000

1

0

3 / Female

1

0

4 / Female

1

0

5 / Female

1

0

 

All animals survived until the end of the study without showing any signs of toxicity.Throughout the 14-day observation period, the body weight gain of the test animals was within the normal range of variation for this strain. At necropsy, no treatment-related macroscopic findings were observed in any animal of any step.

LD50cut-off (rat):                     ˃5000 mg/kg bw