Registration Dossier

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Administrative data

Description of key information

Skin and eye irrritation results

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 17 May 2022 to 20 May 2022
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:
14 June 2021
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch no.: 2044 supplied by the sponsor
Aspect: clear to light turbid liquid
Purity: 33.2%
pH as it is 7.1
Water solution
Storage condition of test material: room temperature (15 °C - 25 °C), stable for at least 12 months
Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium): stable for 12 months minimum
Solubility: soluble and stable in water
Reactivity of the test material with the incubation material used (e.g. plastic ware): not reactive
More information can be found on the attached report
Test system:
artificial membrane barrier model
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Vehicle:
water
Remarks:
Substance in already in water solution
Details on test system:
The test item was preliminary tested to evaluate its interference with MTT endopoint. 16µL of the test item were put in contact with 0.3 ml of MTT and incubated for 180 ± 5 minutes at 37°C and 5% CO2. A negative control (16 µ1 of sterile deionized water) was run concurrently. MTT solution color did not change its color into blue/purple, indicating that the test item was not reducing the MTT.

Furthermore the test item was preliminary tested to evaluate its colorant properties.

10µ,L of the test item were put in contact with 0.09m1 of water and incubated for 30 minutes at room temperature after shaking. A negative control (10 µl of sterile deionized water) was run concurrently. The absorbance was measured with a plate reader equipped with the MTT measurement wavelength filter. After subtraction of the OD of negative control, the OD of the test item solution was < 0.1 (-0.0018, See Annex 05 — Raw data test item colorant properties), indicating that the test item does not interact with the MTT measurement.

In the definitive test, after over-night incubation in the growth medium, the epidermis units were treated with the test item. 16µL of test item, and 16µ1 of positive (SDS 5%) and negative (PBS without Ca and Mg) controls were applied on epidermis units in three replicates. The exposure was carried out for 42 minutes at room temperature. At the end of the exposure period sample and controls were removed with multiple washings with PBS and the tissues were further incubated in 2m1 of growth medium at 37°C, 5% CO2 for 42 h. At the end of the incubation, the viability assay was performed to evaluate the cell survival in the epidermis units. Epidermis units were treated with 0,3mL 1 mg/ml MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) for 3 h at 37°C.
The solution was then removed and replaced with isopropanol, with further 2 h incubation at room temperature under medium speed shaking.

2 aliquots of isopropanol were sampled for each epidermis unit and transferred to a 96 well plate for the reading (6 readings total for the test item, positive and negative control).

The absorbance was read with a microplate spectrophotometer equipped with the 570 nm filter. The absorbance values were corrected by subtracting the reading of the blanks (diluent only).

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
16 microliter of test item was applied in three replicates
Duration of treatment / exposure:
42 minutes at room temperature
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
116.15
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
103.36
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
104.55
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Acceptance criteria

For the blank (isopropanol): OD has to be < 0.1 for each replicates
For negative control (NC): the mean OD value of the 3 tissues has to be ≥ 0.8 and ≤ 3.0

the standard deviation of the viability mean percentage has to be ≤ 18%



For positive control (CP): the viability mean 8expresses as % versus the NC) has to be < 40%

the standard deviation has to be ≤ 18%



For the test item: the standard deviation of the viability mean percentage has to be ≤ 18%
Interpretation of results:
GHS criteria not met
Conclusions:
The test item Reaction mass of L-Glutamic acid, N-(1-oxooctyl)-, sodium salt and N-L-glutamyl-L-glutamic acid, N'-(1-oxoocty1)-, sodium salt Batch 2044, ABICH code S012­22 tested on Reconstructed Human Epidermis according to OECD 439 and in compliance to GLP, results to be not irritant to the skin (NO CATEGORY), because its mean cell viability is higher than 50%. All acceptance criteria were passed.

In conclusion, it can be stated that, in the experimental conditions here described and according to this result, the test item Reaction mass of L-Glutamic acid, N-(1-oxooctyl)-, sodium salt and N-L-glutamyl-L-glutamic acid, N'-(1-oxoocty1)-, sodium salt Batch 2044, ABICH code S012-22 can be classified as NOT IRRITANT (NO CATEGORY according to UN GHS CLASSIFICATION).
Executive summary:

Purpose of the study is to evaluate the skin irritation potential on Reconstructed Human Epidermis (RHE SkinEthic) of the test item "Reaction mass of L-Glutamic acid, N-(1-oxoocty1)-, sodium salt and N-L-glutamyl-L-glutamic acid, N'-(1-oxoocty1)-, sodium salt", ABICH code S012-22 according to the method described in OECD 439 and in compliance with Good Laboratory Practice (GLP).


This method uses human artificial skin models to assess the skin irritation of chemical substances and mixtures and to properly label them to this respect, if applicable.


The test is based on the evaluation of cell survival after the exposure to the test item through MTT assay and by comparison with epidermis treated with phosphate buffer only (negative control). The MTT method is a colorimetric assay that allows to determinate the percentage of cells alive within an in vitro cultured tissue. This assay is based on the ability of the mitochondrial succinate dehydrogenase enzyme to metabolise the nitro-blue tetrazolium salt, giving a coloured compound that can be measured by spectrophotometer reading.


The test item "Reaction mass of L-Glutamic acid, N-(1-oxooctyl)-, sodium salt and N-L-glutamyl-L-glutamic acid, N'-(1-oxoocty1)-, sodium salt", ABICH code S012-22, in the experimental conditions here described, showed a mean cell survival of 108.02% (±7.07), so it resulted a NOT SKIN IRRITANT (UN-GHS — no Category), because its cell viability is higher than 50% .


The positive control Sodium Doceyl Sulphate (SDS) showed a mean cell viability of 1.58% (±0.03), so it gave the expected results. All the acceptance criteria were passed.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From November 10, 2000 to November 17, 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to guideline
Guideline:
other: Method T11C Skin Irritation through patch test
Version / remarks:
See Principles of method if other than guideline
Deviations:
no
Principles of method if other than guideline:
Method T11C: "Skin irritation through patch test"
The method consists in an occlusive application of the test substance by means of Finn Chambers (alluminium cells of 20 microlitres volume) on 20 selected subjects’ back or forearm. The irritating activity is clinically evaluated:
- 30 minutes (T1) after application (immediate irritative power);
- 48 hours later (T2) (irritative power);
by observing the erythema induced by the test substance.
Test substances are then ranked on a statement scale ranging from “non irritating” to “strongly irritating” on the basis of the severity of the irritating reactions observed as well as their frequency in the different subjects.
GLP compliance:
yes
Remarks:
and GCP
Specific details on test material used for the study:
Appearance: liquid and transaprent
Species:
other: Human
Type of coverage:
occlusive
Preparation of test site:
other: Cleaned from sebum
Vehicle:
other: Vaseline oil or distilled water
Controls:
yes, concurrent vehicle
Amount / concentration applied:
Tested at 7% in S.A.L (active matter)
20 microliter
Duration of treatment / exposure:
30 minutes and 48 hours
Observation period:
- 30 min and 48 hours after application. The evident redness after 30 minutes is considered as a sign of irritation caused by the test substance.
- A second check is made 24 hours after the removal of the plaster strip. A persistent redness is considered as subjective allergic reaction to some of the components of the test substance or as a prolonged irritation.
Number of animals:
20 (M/F)
Details on study design:
The test substance was put in Finn Chambers, aluminium cells with a diameter of 8 mm, 50 mm2 area, volume of 20 microlitres. It was put directly into the cells, filled slightly more than half their volume. The two cells containing each productwere then applied on the back or on the forearm of the subject, on healthy skin previously cleaned from sebum.
Criteria for inclusion
Race: caucasian. Age: adults aged from 18 to 65 (the number of subjects aged between 55 and 65 must not be higher than 10% of the total). Sex: male and female. Health condition: no pathologies either for the period immediately before or during the test. Knowledge of the italian language. Easily contactable.
Criteria for exclusion
Persons who cannot be included according to the criteria listed in Criteria for inclusion; Subjects undergone epicutaneous tests in the last four weeks or to sensitization tests in the last six months; Subjects using topical or systemic pharmaceutical treatments that may influence the test (ex. antihistaminics, cortisone); Pregnant and nursing women; Person showing either temporary or chronic cutaneous diseases; Subjects who usually expose or have recently exposed themselves to UV radiation; Persons who proved to be intolerant of drugs, cosmetic products, nickel.
Drop-out and Restrictions
Irritation parameter:
erythema score
Remarks:
irritative power (%)
Basis:
mean
Time point:
other: 30 min, 48 hours and 24 hours after removal
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
- Immediate irritative power (IIP) (30 minutes after product application) = 0%.
- Irritating power (IP) (48 hours after product application) = 0%
- 24 hours after the test substance removal, no cases of delayed irritation/allergic reactions to the test substance were either observed or reported.
Other effects:
None of the subjects reported itching phenomena

Interpretation of data:


The immediate irritative power (IIP) and the irritative power (IP) are separately evaluated. The irritative power of the product was evaluated considering:


- the number of reactions that the product caused to the total number of subjects;


- the severity of the irritative reactions.


The reaction of type 2 (light faint homogeneous erythema or “silk paper” skin reactions) disappearing within 24 hours after the test substance has been removed, are not considered. The reactions of type 5 and 6 (erythema and bulla, follicular bulla and pustules or necrosis reactions) are counted twice. The erythema cases appeared within 24 hours after the removal are not included in the final calculation. However they will be reported since they can be regarded as allergic reactions to one or more components of the test substance. On the basis of the data reported in literature, the tested product can be considered: from No reaction to Maximum (%).

Interpretation of results:
other: CLP criteria are not met
Conclusions:
Under the study conditions, the substance was considered to be non-irritant to human skin.
Executive summary:

A supporting study was conducted to determine the in vivo skin irritant/corrosion potential of the substance according to Method: T11C: "Skin irritation through patch test", in compliance with GLP. The method consists in an occlusive application of the test substance by means of Finn Chambers (alluminium cells of 20 microlitres volume) on the back or forearm of 20 selected subjects. The irritating activity/power (%) was clinically evaluated after 30 minutes (T1) following application (immediate irritative power) and 48 hours later (T2) (irritative power); by observing the erythema induced by the test substance. Test substances were then ranked on a statement scale ranging from “non irritating” to “strongly irritating” on the basis of the severity of the irritating reactions observed as well as their frequency in the different subjects. A second check was made 24 hours after the removal of the plaster strip. The irritating power of the test substance was 0% and 24 hours after the test substance removal; no cases of delayed irritation/allergic were either observed or reported. None of the subjects reported itching phenomena. Under the study conditions, the substance was not considered to be irritating to human skin ( Rigano, 2000).

Endpoint:
skin corrosion: in vitro / ex vivo
Remarks:
MTT Test
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From December 09, 2002 to December 16, 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to guideline
Guideline:
other: Citotoxicity by MTT test (Mossman, 1993)
Version / remarks:
see Principles of method if other than guideline
Deviations:
no
Principles of method if other than guideline:
- Cytotoxicity by MTT test (Mossman, 1993): cell survival assay using cultured human keratinocytes in monostratum cultures and human 3D artificial skin cultures to assess skin irritation potential and biocompatibility. Description:The key component is (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) or MTT. This product is of yellowish colour in solution. Mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring, leading to the formation of purple crystals which are insoluble in aqueous solutions. The crystals are re-dissolved in acidified isopropanol and the resulting purple solution is measured spectrophotometrically. An increase or decrease in cell number results in a concomitant change in the amount of formazan formed, indicating the degree of cytotoxicity caused by the test substance.
GLP compliance:
yes
Specific details on test material used for the study:
Liquid- dry residues 38% (purity)
Test system:
human skin model
Remarks:
human skin cells grown in vitro
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: pediatric foreskin
Vehicle:
unchanged (no vehicle)
Details on test system:
1. Monolayer cultures of human primary keratinocytes:
The epidermis was separated from dermis by incubation with dispase (2.5 mg/mL) for three hours at 37°C, and trypsinized (0.20%) to generate single cell suspension. Keratinocytes were cultived in Dulbecco’s modified Eagle’s and Ham’s F12 media (3:1) enriched with 10% fetal calf serum (v/v) and specific enrichements.
2. Three-dimensional reconstructed artificial human skin model:
A reconstructed artificial human skin model comprising normal human epidermal keratinocytes, growing as an integrated three-dimensional cell culture model, perfectly mimicking the human skin in vitro. The model exhibits normal barrier functions (presence of a differentiated stratum corneum). It was purchased from Skinethic (Nice).
Control samples:
yes, concurrent no treatment
yes, concurrent vehicle
Amount/concentration applied:
1.1, 0.37, 0.12, 0.04, 0.014, 0.0047, 0.0016 mg/ml
Duration of treatment / exposure:
24 hours
Number of replicates:
3 replicates for each dilution
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Three dimensional reconstructed artificial human skin model -MTT - Survival (%)
Value:
100.88
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
cultures of human primary keratinocytes -MTT-IC50 (mg/ml)
Value:
> 1.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The test substance has not shown any cytotoxic effect on monolayer keratinocytes and on 3D epidermis and it is as well tolerated as a finished product. The product in not considered to be irritant for skin derived cells and this results is predictive of absence of irritant potential in vivo

The cytotoxicity data obtained with the MTT for test performed on keratinocytes assay were plotted against the concentrations, which generates dose-response curves that allows to determine:


- the theoretical regression curve


- the theoretical IC50 value (Inhibiting Concentration 50), i.e. the concentration of the test substance which induces a decrease of cell survival by 50% as compared to untreated cultures


The IC50 value (Inhibiting Concentration 50) is the concentration of test compound which induces a decrease of cell growth/survival by 50%. It makes it possible to evaluate the potential irritating effect as described:


- IC50 < 1 mg/mL means a cytotoxic/irritating effect


- IC50 > 1 mg/mL means the absence of cytotoxic/irritating effect

Interpretation of results:
other: CLP criteria not met
Conclusions:
Under the study conditions, the substance was considered to be non-irritant to human skin.
Executive summary:

A supporting study was conducted to determine the in vitro skin irritation/corrosion potential of the substance, using a cytotoxicity assay, in compliance with GLP. MTT was used on two different models:, i.e. monolayer cultures of human primary keratinocytes and three-dimensional reconstructed artificial human skin model. The skin was exposed to the test substance for 24 h at concentrations of 0.0016, 0.0047, 0.014, 0.04, 0.12, 0.37 and 1.1 mg/mL for the first model or 5% for the second model. The IC50 for the 1st model was > 1.1 mg/mL and the percentage of survival in the 2nd model was 100.88. Under the study conditions, the substance was not considered to be irritating to human skin (Bochietto, 2003).


 

Endpoint:
skin irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From October 03, 1989 to October 10, 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
RA study
Justification for type of information:
Refer to the section 13 of IUCLID dataset for details on the read across justification. The skin irritation / corrosion study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
Test animals
- Source: David Percival Ltd., Moston, Sandbach, Cheshire, U.K.
- Age at study initiation: 12-16 weeks
- Weight at study initiation: 2.32 - 2.68 kg
- Housing: individually in suspended metal cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

Environmental conditions
- Temperature (°C): 15-20
- Humidity (%): 47-60
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
semiocclusive
Preparation of test site:
other: clipped free of fur
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
0.5 ml
Duration of treatment / exposure:
4 hours
Observation period:
1h, 24h, 47 h, 72 h
Number of animals:
3
Details on study design:
Test site
- Area of exposure: dorsal/flank area
- Type of wrap if used: elasticated corset (TUBIGRIP)

Removal of the test substance
- Washing (if done): removing by gentle swabbing with cotton wool soaked in diethyl ether
- Time after start of exposure: 4h
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
24/48/72 h
Score:
0.5
Max. score:
8
Reversibility:
fully reversible
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.7
Max. score:
4
Reversibility:
fully reversible within: 7 d
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 d
Irritant / corrosive response data:
Scoring system: Draize J.H. (1959) Association of Food and Drug Officials of the United States, Austin, Texas, "The Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics":

Erythema and Eschar Formation
0 No erythema
1 Very slight erythema (barely perceptible)
2 Well-defined erythema
3 Moderate to severe erythema
4 Severe erythema (beet redness) to slight eschar formation (injuries in depth)

Oedema Formation
0 No oedema
1 Very slight oedema (barely perceptible)
2 Slight oedema (edges of area well-defined by definite raising)
3 Moderate oedema (raised approximately 1 millimetre)
4 Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure)
Primary Irritation Index
0 Non-irritant
> 0 - 2 Mild irritant
> 2 - 5 Moderate irritant
> 5 - 8 Severe irritant
Interpretation of results:
GHS criteria not met
Conclusions:
Under the study conditions, the substance was not irritating to rabbit skin.
Executive summary:

A study was conducted to determine the in vivo skin irritation / corrosion potential of the substance according to OECD Guideline 404, in compliance with GLP. New-Zealand White rabbits were exposed to 0.5 mL of undiluted test substance for 4 h. The experiment consisted of a semiocclusive coverage on clipped skin free of fur. Following application, animals were observed after 1, 24, 48 and 72 h. The mean scores for erythema and oedema were between 0.7 and 1.0 and all effects were reversible within 7 d. Under the study conditions, the substance was not irritating to rabbit skin (Safepharm Laboratories Limited, 1989).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
November 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to guideline
Guideline:
other: Hens Egg Test on the Chorio-Allantoic Membrane (HET-CAM), Luepke NP
Version / remarks:
see Principlaes of method if other than guideline
Deviations:
no
Principles of method if other than guideline:
Alternative ocular irritation tensting: Hens Egg Test on the Chorio-Allantoic membrane (HET-CAM)
GLP compliance:
not specified
Specific details on test material used for the study:
Batch n° 261000
Species:
other: Chicken eggs (chorio-allantoic membrane)
Details on test animals or tissues and environmental conditions:
- "White Leghorn" eggs (From Allevamento "Morini" S. Polo d'Enza (RE))
- Age of the embryo: 10 days
- 50 - 60g
- Eggs are placed in an incubator (37.0 +/- 0.5°C, 62.0 +/- 7.5°C relative umidity)
- 8 eggs (4 controls/4 treated)
Vehicle:
water
Remarks:
1:104 to get a 0.5% solution
Controls:
yes, concurrent no treatment
yes, concurrent vehicle
Amount / concentration applied:
0.2ml of a 0.5% solution
Duration of treatment / exposure:
30 sec, 2 and 5 min
Observation period (in vivo):
After 30 sec, 2 and 5 min
Number of animals or in vitro replicates:
4
Details on study design:
Application of the test substance on the chorio-allantoic membrane
Irritation parameter:
other: hyperemia, bleeding and clots
Run / experiment:
Hens Eggs Test on the Chrorio-Allantoic Membrane
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Interpretation of results:
other: CLP criteria not met
Conclusions:
Under the study conditions, the substance was considered to be non-irritant to chicken egg chorio-allantoic membrane.
Executive summary:

A supporting study was conducted to determine the eye irritation potential of the substance according to the Hens Egg Test on the Chorio-Allantoic Membrane (HET-CAM), in compliance with GLP. Chicken egg chorio-allantoic membranes were exposed to 2 mL of the test substance at a concentration of 0.5% for 0.5, 2, or 5 minutes. The presence of hyperemia, bleeding and clots was evaluated and subsequently scored. Under the study conditions, the substance was not considered to be irritating to chicken egg chorio-allantoic membrane (Consonni, 2000).

Endpoint:
eye irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From October 09, 1989 to October 26,1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
RA Study
Justification for type of information:
Refer to the section 13 of IUCLID dataset for details on the read across justification. The eye irritation study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Test system
- Source: David Percival Ltd., Moston, Sandbach, Cheshire, U.K.
- Age at study initiation: 12-16 weeks
- Weight at study initiation: 2.32-2.74 kg
- Housing: individually house in suspended metal cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

Environmental conditions
- Temperature (°C): 14-20
- Humidity (%): 52-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:
unchanged (no vehicle)
Controls:
yes
other: negative control: the other eye of the animal
Amount / concentration applied:
0.1ml
Observation period (in vivo):
1,24, 48 and 72 h
Number of animals or in vitro replicates:
3
Details on study design:
Scoring system:
The data relating to the conjunctivae were designated by the letters A (redness), B (chemosis) and C (discharge), those relating to the iris designated by the letter D and those relating to the cornea by the letters E (degree of opacity) and F (area of opacity). For each tissue the total score was calculated as follows:

Total score for conjunctivae = (A + B + C) x 2
Total score for iris = D x 5
Total score for cornea = (E x F) x 5

Tool used to assess score: standard ophthalmoscope
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
3
Max. score:
4
Reversibility:
fully reversible within: 14 d
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
2.3
Max. score:
4
Reversibility:
fully reversible within: 14 d
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 7 d
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
3
Max. score:
3
Reversibility:
fully reversible within: 14 d
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
3
Max. score:
3
Reversibility:
fully reversible within: 14 d
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2.7
Max. score:
3
Reversibility:
fully reversible within: 7 d
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 14 d
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 14 d
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 7 d
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1.7
Max. score:
4
Reversibility:
fully reversible within: 7 d
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 14 d
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1.7
Max. score:
4
Reversibility:
fully reversible within: 7 d
Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Under the study conditions, the substance was considered to be irritating to rabbit eye.
Executive summary:

A study was conducted to determine the in vivo eye irritation potential of the substance according to OECD Guideline 405, in compliance with GLP. One eye of three New-Zealand White rabbits was exposed to 0.1 mL of the undiluted test substance. The other eye served as control. Following application, animals were observed after 1, 24, 48 and 72 h. The mean scores for cornea, conjunctiva, iris effects and chemosis were determined to be 1.8, 2.9, 1.0 and 2.4. All effects were reversible within 14 d. Under the study conditions, the substance was considered to be irritating to rabbit eye (Safepharm Laboratories Limited, 1989).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information


  1. Skin irritation


 


In vivo:


A study was conducted to determine the in vivo skin irritation / corrosion potential of the read-across substance L-glutamic acid, N-coco acyl derivs., monosodium salt according to OECD Guideline 404, in compliance with GLP. New-Zealand White rabbits were exposed to 0.5 mL of undiluted test substance for 4 h. The experiment consisted of a semiocclusive coverage on clipped skin free of fur. Following application, animals were observed after 1, 24, 48 and 72 h. The mean scores for erythema and oedema were between 0.7 and 1.0 and all effects were reversible within 7 d. Under the study conditions, the substance was not irritating to rabbit skin (Safepharm Laboratories Limited, 1989).


 


A supporting study was conducted to determine the in vivo skin irritant/corrosion potential of the substance according to Method: T11C: "Skin irritation through patch test", in compliance with GLP. The method consists in an occlusive application of the test substance by means of Finn Chambers (alluminium cells of 20 microlitres volume) on the back or forearm of 20 selected subjects. The irritating activity/power (%) was clinically evaluated after 30 minutes (T1) following application (immediate irritative power) and 48 hours later (T2) (irritative power); by observing the erythema induced by the test substance. Test substances were then ranked on a statement scale ranging from “non irritating” to “strongly irritating” on the basis of the severity of the irritating reactions observed as well as their frequency in the different subjects. A second check was made 24 hours after the removal of the plaster strip. The irritating power of the test substance was 0% and 24 hours after the test substance removal; no cases of delayed irritation/allergic were either observed or reported. None of the subjects reported itching phenomena. Under the study conditions, the substance was not considered to be irritating to human skin (Sirigu, 2000).


 


In vitro:


A supporting study was conducted to determine the in vitro skin irritation/corrosion potential of the substance, using a cytotoxicity assay, in compliance with GLP. MTT was used on two different models:, i.e. monolayer cultures of human primary keratinocytes and three-dimensional reconstructed artificial human skin model. The skin was exposed to the test substance for 24 h at concentrations of 0.0016, 0.0047, 0.014, 0.04, 0.12, 0.37 and 1.1 mg/mL for the first model or 5% for the second model. The IC50 for the 1st model was > 1.1 mg/mL and the percentage of survival in the 2nd model was 100.88. Under the study conditions, the substance was not considered to be irritating to human skin (Bochietto, 2003).


The key test was a  study is to evaluate the skin irritation potential on Reconstructed Human Epidermis (RHE SkinEthic) of the test item "Reaction mass of L-Glutamic acid, N-(1-oxoocty1)-, sodium salt and N-L-glutamyl-L-glutamic acid, N'-(1-oxoocty1)-, sodium salt", ABICH code S012-22 according to the method described in OECD 439 and in compliance with Good Laboratory Practice (GLP).


This method uses human artificial skin models to assess the skin irritation of chemical substances and mixtures and to properly label them to this respect, if applicable.


The test is based on the evaluation of cell survival after the exposure to the test item through MTT assay and by comparison with epidermis treated with phosphate buffer only (negative control). The MTT method is a colorimetric assay that allows to determinate the percentage of cells alive within an in vitro cultured tissue. This assay is based on the ability of the mitochondrial succinate dehydrogenase enzyme to metabolise the nitro-blue tetrazolium salt, giving a coloured compound that can be measured by spectrophotometer reading.


The test item "Reaction mass of L-Glutamic acid, N-(1-oxooctyl)-, sodium salt and N-L-glutamyl-L-glutamic acid, N'-(1-oxoocty1)-, sodium salt", ABICH code S012-22, in the experimental conditions here described, showed a mean cell survival of 108.02% (±7.07), so it resulted a NOT SKIN IRRITANT (UN-GHS — no Category), because its cell viability is higher than 50% .


The positive control Sodium Doceyl Sulphate (SDS) showed a mean cell viability of 1.58% (±0.03), so it gave the expected results. All the acceptance criteria were passed.


 



  1. Eye irritation


 


In vivo:


A study was conducted to determine the in vivo eye irritation potential of the read-across substance L-glutamic acid, N-coco acyl derivs., monosodium salts according to OECD Guideline 405, in compliance with GLP. One eye of three New-Zealand White rabbits was exposed to 0.1 mL of the undiluted test substance. The other eye served as control. Following application, animals were observed after 1, 24, 48 and 72 h. The mean scores for cornea, conjunctiva, iris effects and chemosis were determined to be 1.8, 2.9, 1.0 and 2.4. All effects were reversible within 14 d. Under the study conditions, the substance was considered to be irritating to rabbit eye (Safepharm Laboratories Limited, 1989).


 


In vitro:


A supporting study was conducted to determine the eye irritation potential of the substance according to the Hens Egg Test on the Chorio-Allantoic Membrane (HET-CAM), in compliance with GLP. Chicken egg chorio-allantoic membranes were exposed to 2 mL of the test substance at a concentration of 0.5% for 0.5, 2, or 5 minutes. The presence of hyperemia, bleeding and clots was evaluated and subsequently scored. Under the study conditions, the substance was not considered to be irritating to chicken egg chorio-allantoic membrane (Consonni, 2000).


 

Justification for classification or non-classification

Based on in vivo and in vitro skin irritation studies on the read-across substance and the substance itself, no classification is warranted for skin irritation according to EU CLP (1272/2008) criteria.


 


The HET-CAM study with the substance does not fulfill the current requirements for eye irritation endpoint. Therefore, the in vivo eye irritation study with the read-across substance L-glutamic acid, N-coco acyl derivs., monosodium salts has been used in the dossier to fulfill the requirement. Based on in vivo eye irritation studies with the read-across substance L-glutamic acid, N-coco acyl derivs., monosodium salts, the substance warrants classification as Eye Irrit. 2: H319 (Causes serious eye irritation) according to EU CLP (1272/2008) criteria.