trisodium (2S)-2,6-bis(3-carboxylatopropanamido)hexanoate

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance does not meet the criteria for classification as acute toxicity or STOT SE by the oral route and no systemic effects have been observed in in vivo studies with dermal exposure (e.g. skin irritation, skin sensitisation)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

skin irritation: in vitro / ex vivo

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

GLP compliance:

yes (incl. QA statement)

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

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Justification for test system used:

Method in compliance with current regulatory requirements

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM from MatTek Corporation (3D system of reconstructed epidermis of normal human keratinocytes)
- Tissue batch number(s): keratinocyte strain: 00267
- Production date: 16-01-2019
- Shipping date: n/a
- Delivery date: n/a
- Date of initiation of testing: 16-01-2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37±1°C
- Temperature of post-treatment incubation (if applicable): 37±1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure time the product and the controls were removed and the tissues rinsed for several times with DPBS (up to 25 times if necessary).
- Observable damage in the tissue due to washing: n/a
- Modifications to validated SOP: n/a

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT thiazolyl blue tetrazolium 5 mg/ml (MTT-100-CON) has been diluted with MTT diluent (MTT-100-DIL) up to 1 mg/ml.
- Incubation time: 3h
- Spectrophotometer: The absorbance is measured at 570 nm by microplate reader using a GEN5 software (Biotek).

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3

PREDICTION MODEL / DECISION CRITERIA
-The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%.
In case the test chemical is found to be non-corrosive (e.g., based on TG 430, 431 or 435), and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%, the test chemical is considered to be irritant to skin in accordance with UN GHS Category 2.
Depending on the regulatory framework in member countries, the test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50%.
According to EU classification, the irritancy potential of test substances is predicted to distinguish between H315 skin irritating (category 2) and not classified test substances (EU CLP).
In this study the irritancy potential of test substances is predicted by mean tissue viability of tissues exposed to the test substance.
The test substance is considered to be irritant to skin (H315), if the mean relative viability after 60 minutes exposure and 42 hours post incubation is less or equal to 50% of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µl
- Concentration (if solution): n/a applied neat

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): (H2O) 30 µl
- Concentration (if solution): n/a

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution):SDS 5% in DPBS

Duration of treatment / exposure:

25 min at room temperature
residual incubation time to 60 minutes has been done at 37±1°C, 5% CO2.

Duration of post-treatment incubation (if applicable):

At the end of the exposure time the product and the controls were removed and the tissues rinsed for several times with DPBS (up to 25 times if necessary).
Each tissue was then transferred in 6-well plate with 1 ml of Assay Medium and incubated for 24±2 hours at 37±1°C, 5% CO2, then the tissues will be transfer in a renewed Assay Medium for 18±2 h at 37±1°C, 5% CO2 before the MTT test.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean value

Value:

73.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTABILITY CRITERIA
Negative control: the mean OD570nm value of the negative control tissues should be ≥0.8 and ≤2.8.
Positive control: the mean viability value of positive control tissues, expressed as % against negative control tissues should be ≤20%.
Standard deviation (SD): the SD calculated from individual % tissue viabilities of the 3 treated replicates should be ≤18%.

RESULTS

Optical density (OD Value) at 570 nm.

	Tissue 1			Tissue 2			Tissue 3
Replicates	1	2	3	1	2	3	1	2	3
Negative control	2,283	2,307	2,283	2,386	2,339	2,346	2,480	2,460	2,471
Positive control	0,111	0,110	0,111	0,127	0,127	0,128	0,119	0,116	0,117
Sample	1,588	1,599	1,572	1,867	1,835	1,845	1,840	1,803	1,803

 

ASSAY VALIDITY CRITERIA		Value	Acceptability	Result
Negative control	Mean OD value	2.33	≥0.8 and≤2.8	Complies
Positive control	Mean Viability %	3.28	≤ 20	Complies
	SD	0.347	≤ 18	Complies
Sample	SD	6.139		Complies

 

SAMPLE	% VIABILITY
TRISODIUM (2S)-2,6-BIS(3- CARBOXYLATOPROPANAMIDO)HEXANOATE	73.30

Interpretation of results:

GHS criteria not met

Conclusions:

On the basis of the results, interpreted according to OECD 439 the test item “TRISODIUM (2S)-2,6-BIS(3-CARBOXYLATOPROPANAMIDO)HEXANOATE” must be considered NOT IRRITANT for the skin.

Executive summary:

On the test item “TRISODIUM (2S)-2,6-BIS(3-CARBOXYLATOPROPANAMIDO)HEXANOATE” an in

vitro toxicological study aimed to evaluate any potential cutaneous irritation was carried out.

The following test was performed:

-In vitro skin irritation test on Reconstructed Human Epidermis according to OECD N. 439:2015.

To perform the in vitro skin irritation test, three-dimensional Reconstructed Human Epidermis (RHE) tissues, consisting of normal human keratinocytes cultured for 17-days on an inert 0.63 cm²polycarbonate filter at the air-liquid interface, were used.

The test item was topically applied on three tissues replicates for 60 minutes. Exposure was followed by rinsing with phosphate buffer saline (DPBS) and dried, then tissues were transferred to fresh medium and incubated for 42 additional hours, then tissues were transferred to MTT for 3 hours. The aim of this assay was to assess quantitatively the effects of the tested product on cell survival through the MTT assay.

Cell viability determination is based on cellular dehydrogenase activity, measured by MTT reduction and conversion into blue formazan salt that is quantified after extraction from tissues.

\The percentage reduction in viability is used to predict the irritation potential.

On the basis of the results, interpreted according to OECD 439:2015 the test item “TRISODIUM (2S)-2,6-BIS(3-CARBOXYLATOPROPANAMIDO)HEXANOATE” must be considered NOT IRRITANT for the skin.

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

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

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

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.

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

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.

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.

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.

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