Reaction mass of calcium 2,6-bis(3-carboxylatopropanamido)hexanoate and isomers of calcium amino-(3-carboxylatopropanamido)hexanoate

Endpoint summary

Description of key information

The test item is considered as non-irritant in the EPISKIN in vitro skin irritation test. Furthermore, the test item, applied to the rabbits’ eye mucosa, caused slight conjunctival irritant effects which were fully reversible within 24 hours.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 15, 2016 - October 6, 2016

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)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

06 July 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiSkin™ SOP, Version 1.8

Version / remarks:

February 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult human donors

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. It showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin(TM)SM, EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model
- Tissue batch number(s): 16-EKIN-034
- Expiry date: August, 29, 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: approximately 25 mL PBS 1 x solution; rest of the PBS was removed from epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging to the epidermis)
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 3 mg/mL stock solution; 2 mL of 0.3 mg/mL per well
- Incubation time: 3 hours
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- N. of replicates : 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42 hours post incubation is less than or equal to 50 % of the negative control.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 hours post incubation is greater than 50 % of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: The test item is a highly viscous material therefore exact weighting of treatment volume could not be performed. The test item was applied with spatula, in such a way that the entire surface of a plastic film was covered with test substance.

NEGATIVE CONTROL
- Amount(s) applied: 10 µL
- Lot/batch no.: SLBH9016

POSITIVE CONTROL
- Amount(s) applied: 10 µL
- Concentration: 5 % aq. solution

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1. Experiment

Value:

103

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

15%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be excluded.
- Colour interference with MTT: As the test item has an intrinsic colour, two additional test item-treated tissues were used for the non-specific OD evaluation. The mean OD of these tissues was determined as 0.075. The Non Specific Colour % (NSC %) was calculated as 12.2 %. Therefore additional data calculation was necessary

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392.554.2938) using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD value of the three negative control tissues was 0.618.
- Acceptance criteria met for positive control: The mean OD value obtained for the positive control was 0.094 and this result corresponds to 15 % viability when compared to the results obtained from the negative controls.
- Acceptance criteria met for variability between replicate measurements: Each calculated standard deviation value (SD) for the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is considered as non-irritant in the EPISKIN in vitro skin irritation test.

Executive summary:

The skin irritation potential of the test item on reconstructed human epidermis in the EPISKIN model was determined according OECD TG 439 and EU method A.46.

Reconstructed human epidermis was treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically.

As the test item has an intrinsic colour (brown), two additional test item treated tissues were used for the non-specific OD evaluation.

SDS 5 % aq. and 1 x PBS treated (three units / positive and negative control) epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control.

The test chemical is identified as irritant, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % when compared to the viability values obtained from the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 6, 2016 - September 20, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2 October 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

31 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: S & K LAP Kft. 2173 Kartal, Császár út 135, HUNGARY
- Age at study initiation: Young adult rabbits
- Weight at study initiation: 2884-3118 g
- Housing: housed individually in metal cages
- Diet: ad libitum, C.HYF rabbit mixed diet produced by Cargill Takarmány Zrt., 5300 Karcag, Madarasi út 0399., Hungary
- Water: ad libitum, tap water
- Acclimation period: 5 days in first animal and 6 days in second and third animal

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): above 10 changes
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g

Duration of treatment / exposure:

The eyes of the test animals were not washed out 24 hours after the application of test item, because the test substance was removed from the eye of the test animal by physiological mechanisms and the test item did not cause immediately severe irritation or corrosion after test item application.

Observation period (in vivo):

eyes examined at 1, 24, 48 and 72 hours after the application

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: none
SCORING SYSTEM: The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (2nd October 2012)
A. Opacity-degree of density (Area most dense taken for reading)
No uncleration or opacity: 0
Scattered or diffuse areas of opacity (other than slight
dulling of normal lustre): details of iris clearly visible: 1
Easily discernible translucent area: details of iris slightly obscured: 2
Nacreous area: no details of iris visible: size of pupil barely discernible: 3
Opaque cornea: iris not discernible through the opacity: 4

Area of cornea involved
One quarter (or less), but not zero: 1
Greater than one quarter, but less than half: 2
Greater than half, but less than three quarters: 3
Greater than three quarters, up to whole area: 4

B. Iris
Values
Normal: 0
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia: or injection: iris reactive to light
(a sluggish reaction is considered to be an effect): 1
Hemorrhage, gross destruction, or no reaction to light: 2

C. Conjunctivae
Redness (Palpebral and bulbar)
Normal: 0
Some blood vessels hyperaemic (injected): 1
Diffuse, crimson colour, individual vessels not easily discernible: 2
Diffuse beefy red: 3
Chemosis
Normal: 0
Some swelling above normal (includes nictitating membrane): 1
Obvious swelling with partial eversion of lids: 2
Swelling with lids about half closed: 3
Swelling with lids more than half closed: 4
D. Discharge
No discharge: 0
Any amount different from normal (does not include small amounts observed in inner canthus of normal animals): 1
Discharge with moistening of the lids and hairs just adjacent to lids: 2
Discharge with moistening of the lids and hairs, on considerable area around the eye: 3

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

3

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

other: Discharge

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

3

Remarks on result:

no indication of irritation

Other effects:

No systemic toxicity was observed on the day of the treatment and during the 72-hour observation period. The body weight of animals corresponded to their species and age. Sign of pain and distress as discharge was observed in one animal on the treatment day at first observation.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item, applied to the rabbits’ eye mucosa, caused slight conjunctival irritant effects which were fully reversible within 24 hours.

Executive summary:

A study according OECD TG 405, EU method B5 and EPA OPPTS 870.2400 was conducted to determine the eye irritation potential of the test item. The test item was placed into the conjunctival sac of the left eye of three New Zealand White rabbits. The untreated right eye served as control. A weight of 0.1 g of the test item was used, as a single dose. The eyes of the test animals were not washed out after the application of test item. The irritation effect of the test item was evaluated according to the Draize method (OECD No.: 405, 2012).

The eyes were examined at 1, 24, 48 and 72 hours after the application.

One hour after the treatment, slight conjunctival redness, chemosis and discharge were observed in animals. 24 hours after the treatment, all animals became free of symptoms. 72 hours after the treatment all animals were free of symptoms and the study was terminated.

No systemic toxicity was observed on the day of the treatment and during the 72-hour observation period. The body weight of animals corresponded to their species and age. Sign of pain and distress as discharge was observed in one animal on the treatment day at first observation.

In conclusion, the test item applied to the rabbits’ eye mucosa, caused slight conjunctival irritant effects which were fully reversible within 24 hours. According to Regulation (EC) No 1272/2008, the test item has not been classified into any category.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro eye irritation study does not need to be conducted because adequate data from an in vivo eye irritation study are available

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation in vitro

The skin irritation potential of the test item on reconstructed human epidermis in the EPISKIN model was determined according OECD TG 439 and EU method A.46.

Reconstructed human epidermis was treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically.

As the test item has an intrinsic colour (brown), two additional test item treated tissues were used for the non-specific OD evaluation.

SDS 5 % aq. and 1 x PBS treated (three units / positive and negative control) epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control.

The test chemical is identified as irritant, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % when compared to the viability values obtained from the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

 

Eye irritation in vivo

A study according OECD TG 405, EU method B5 and EPA OPPTS 870.2400 was conducted to determine the eye irritation potential of the test item. The test item was placed into the conjunctival sac of the left eye of three New Zealand White rabbits. The untreated right eye served as control. A weight of 0.1 g of the test item was used, as a single dose. The eyes of the test animals were not washed out after the application of test item. The irritation effect of the test item was evaluated according to the Draize method (OECD No.: 405, 2012).

The eyes were examined at 1, 24, 48 and 72 hours after the application.

One hour after the treatment, slight conjunctival redness, chemosis and discharge were observed in animals. 24 hours after the treatment, all animals became free of symptoms. 72 hours after the treatment all animals were free of symptoms and the study was terminated.

No systemic toxicity was observed on the day of the treatment and during the 72-hour observation period. The body weight of animals corresponded to their species and age. Sign of pain and distress as discharge was observed in one animal on the treatment day at first observation.

In conclusion, the test item applied to the rabbits’ eye mucosa, caused slight conjunctival irritant effects which were fully reversible within 24 hours. According to Regulation (EC) No 1272/2008, the test item has not been classified into any category.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin and eye irritation, the test item is not classified as skin irritant according to Regulation (EC) No 1272/2008 (CLP), as amended for the seventeenth time in Regulation (EU) 2021/849. In addition, based on in vivo data available on eye irritation, the substance is not classified as eye irritant according to Regulation (EC) No1272/2008 (CLP), as amended for the seventeenth time in Regulation (EU) 2021/849.