Registration Dossier

Administrative data

Description of key information

There is an In-vitro skin irritation: human skin model test according to OECD guideline 439 using EpiSkin tissues. There is an old non-standard CAM test for eye irritation and modern GLP studies the Bovine Corneal Opacity (BCOP) test and a rabbit eye irritation test.

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:
Experimental start date: 27 September 2017 Experimental completion date: 13 October 2017
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: Sodium cocopropylenediamine propionate, CAS 97659-50-2
Batch: 48724
CAS No.: 97659-50-2
Purity: 29.7% (w/w), the substance is a UVCB, purity is equal to total solids
Appearance: Liquid, yellowish to brown
Expiry Date: 17 May 2020
Storage Conditions: At room temperatue
Stability in Solvent: Stable in water (not quantified)
Purpose of Use: Industrial chemical
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
Cell Culture
EpiSkin™ kits are purchased from SkinEthic Laboratories (69007 Lyon, France). The EpiSkin™ tissue consists of NHEK, which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.
EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate and reached Envigo CRS GmbH on 10 October 2017. On the day of experiment EpiSkin™ tissues were transferred to 12-well plates with maintenance medium and the pre-incubation phase of the EpiSkin™ tissues started.

EpiSkin™ Kit Components Needed for the Assay
EpiSkin™ Kit Lot No.: 17-EKIN-041
1, Sealed 12-well plate, Contains 12 inserts with EpiSkin™ tissues on agarose
1, 12-well plate, For MTT viability assay
1 bottle, Assay Medium, Basic medium for use in MTT assays
1 bottle, EpiSkin™ Maintenance Medium, Basic medium for incubations
Vehicle:
unchanged (no vehicle)
Details on test system:
Test for Direct MTT Reduction and Colour Interference
Prior to the start of the test, the test item’s colour interference potential had to be evaluated. For this purpose the test item (10 μL) was mixed with 90 μL of deionised water in a pre-experiment. The test item/water mixture was gently shaken for 15 minutes at room temperature.
The colour of the test item/water mixture did not change during the incubation period compared with the colour of the pure test item.
For correct interpretation of results it is necessary to assess the ability of the test item to directly reduce MTT. To test for this ability 10 μL of the test item was added to 2 mL of MTT solution (0.3 mg/mL) and the mixture will be incubated in the dark at 37 ± 1.5 °C (5 ± 0.5% CO2) for 3 hours. MTT solution was used as the control. Since the MTT solution did not turn blue/purple, the test item was not considered to reduce MTT and an additional test with freeze-killed tissues did not have to be performed.

Pre-warming of EpiSkin™ Tissues
Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium. The EpiSkin™ tissues were incubated for about 22 hours.
Treatment
The negative control, positive control and the test item were added into the insert atop the concerning EpiSkin™ triplicate tissues for 15 minutes.
After the end of the treatment interval the inserts were immediately removed from the 12-well plate. The tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for about 42 at 37 ± 1.5 °C, 5 ± 0.5% CO2.
IL-1 α Immunoassay
Samples of all treatment groups were taken from the wells. The plates were shaken for approximately 15 minutes to homogenise the released mediators in the medium before sampling. At least 1.6 mL medium from each well was taken and was stored in the freezer at –20 °C. Since the results derived from the MTT assay (see chapter 4.4 and 8.1) were not unclear or borderline, the IL-1 α concentration in the medium was not determined and the taken samples will be discarded after report finalization.
MTT Assay
A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.
After the 42 ± 1 hour incubation period was completed for all tissues the cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was rinsed three times with PBS and the tissues were plotted. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol containing 0.04 N HCl) into each vial. The tissue samples were completely covered by isopropanol. The formazan salt was extracted for 2.5 hours at room temperature with gentle agitation.
Per tissue sample 2  200 μL aliquots of the formazan extract were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Softmax Pro Enterprise, version 4.7.1) with 570 nm filter. Mean values were calculated from the 2 wells per tissue sample.
Data Recording
The data generated were recorded in the laboratory protocol. The results were presented in tabular form, including experimental groups with the test item, negative and positive controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 μL (26 μL/cm2) of the undiluted test item were applied to each of the triplicate tissues.
10 μL of negative and positive were applied to each of triplicate tissues for 15 minutes.
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
triplicate
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
100.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not lead to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent did not show blue/purple colour.
The mean relative absorbance value of the test item, corresponding to the cell viability, was 100.6% (threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.
The acceptance criteria were met:
• the mean OD of the three negative control exposed tissues is ≥ 0.6 till ≤ 1.5 (range: 1.164 to 1.292).
• the standard deviations between tissues of the same treatment group was ≤ 18 (range: 3.0 to 5.5).
• the mean relative tissue viability of the positive control was ≤ 40% (22.1%).
• the acceptance limit of the IC50 of the respective EpiSkin™ lot was between 1.5 and 3.0 mg/mL after 18 hours treatment with SLS (1.9 mg/mL).
• the results for the positive and negative controls are within the historical data (means, rel. standard deviation, and ranges) of Envigo CRS GmbH

Treatment Group

Tissue No.

OD 570 nm
Well 1

OD 570 nm
Well 2

Mean OD of 2 Wells

Mean OD

of 2 Wells blank

corrected

Mean

OD

of 3 tissues

blank corrected

Rel. Viability [%] Tissue
1, 2 + 3*

Standard Deviation

of viability values

Mean Rel. Viability

[%]**

Blank

 

0.037

0.040

0.039

0.000

 

 

 

 

Negative Control

1

1.306

1.278

1.292

1.253

1.184

105.9

5.5

100.0

2

1.209

1.214

1.211

1.173

99.1

3

1.176

1.152

1.164

1.125

95.1

Positive Control

1

0.263

0.255

0.259

0.221

0.261

18.6

3.0

22.1

2

0.312

0.318

0.315

0.277

23.4

3

0.324

0.325

0.324

0.286

24.2

Test Item

1

1.180

1.227

1.203

1.165

1.191

98.4

3.2

100.6

2

1.280

1.265

1.273

1.234

104.2

3

1.208

1.219

1.213

1.175

99.2

* Relative viability [rounded values]: [100 x (absorbance test item/ postive control/ negative control)]/ mean absorbance negative control

** Mean relative viability [rounded values]: [100 x (mean absorbance test item/ postive control/ negative control)]/ mean absorbance negative control

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, Sodium cocopropylenediamine propionate, CAS 97659-50-2 is not irritant to skin according to UN GHS and EU CLP regulation.
Executive summary:

This in vitro study was performed to assess the irritation potential of Sodium cocopropylenediamine propionate, CAS 97659-50-2 by means of the Human Skin Model Test according to OECD TG 439.

The test item did not reduce MTT (pre-test for direct MTT reduction), and it did not dye water, when mixed with it (pre-test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative control (PBS) or the positive control (5% sodium lauryl sulfate) for 15 minutes.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD  0.6 till ≤ 1.5 thus showing the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control thus ensuring the validity of the test system.

After treatment with the test item the mean relative absorbance value was 100.6%. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, Sodium cocopropylenediamine propionate, CAS 97659-50-2 is not irritant to skin according to UN GHS and EU CLP regulation.

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:
1993
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Batch no is given; limited details on composition. Limited reported study (max reliability score can be 2).
Reason / purpose:
reference to same study
Qualifier:
no guideline available
Principles of method if other than guideline:
Method according to Luepke N.P., Hen's Egg Chorioallantoic Membrane Test for Irritation Potential. Fd. Chem. Toxicol. 23, 287-291
(1985).
GLP compliance:
no
Remarks:
The study was not subjected to formal QA inspection or report audit. However, all data were recorded in accordance with the principles of GLP and all procedures carried out as detailed in SOPs. The report and data were rigorously checked.
Species:
other: day 10 fertilised chicken eggs
Strain:
other: Ross variety
Details on test animals or tissues and environmental conditions:
TEST EGGS
- Source: Suffolk Sovereign Hatcheries, Eye, Suffolk, UK
- Age at study initiation:10 day fertilised

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 39

IN-LIFE DATES: not indicated (1993)
Vehicle:
unchanged (no vehicle)
Controls:
other: sterile water (negative control) and glacial acetic acid (positive control)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 200 µl
Duration of treatment / exposure:
A line was etched on the air sac end of each egg using a diamond marker pen. The tip of a pointed scalpel blade was used to make a small incision through the line. The shell was carefully chipped away, using small dissection scissors, to reveal the inner shell
membrane. This was wetted with warm sterile water and gently peeled away from the delicate chorioallantoic membrane (CAM)
beneath, using fine forceps. Excess water was poured off. Only undamaged, well formed CAMs were used in the test. The test
compound was tested in triplicate, as supplied, and applied directly to the CAM surface: solvent and positive control materials, and
the test material, were applied as 200 ul aliquots. The test material was left in contact with the membrane for 20 sec, after which
time it was washed away by irrigation with warm sterile water. Observations of reactions in the CAM were made during a 5-min
period immediately after irrigation. The intensity of vascular injection, the extent of haemorrhage and the occurrence of coagulation of cytosolic proteins were assessed.
Observation period (in vivo):
See above
Number of animals or in vitro replicates:
3 eggs
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, warm sterile water
- Time after start of exposure: 20 sec

SCORING SYSTEM:
1 - Light injection remaining to 5 mins
2 - Light injection increasing to heavy injection by 5 mins
3 - Light injection increasing to light haem &/or TCL by 5 mins
4 - Light injection increasing to heavy haem &/or TCL by 5 mins
5 - Heavy injection remaining to 5 mins
6 - Heavy injection increasing to 1 ight haem &/or TCL by 5 mins
7 - Heavy injection increasing to heavy haem &/or TCL by 5 mins
8 - Light haem increasing to heavy haem by 5 mins
9 - Heavy haem
10 - Coagul at i on
Heam - haemorrhage; TCL - terminal capillary leakage

INTERPRETATION
0 non-irri tant
0.1 - 0.9 no significant effect
1.0 - 1.9 mildly irritant
2.0 - 4.9 moderately irritant
> 5 severely irritant
Irritation parameter:
in vitro irritation score
Remarks:
CAM
Run / experiment:
Mean of three eggs
Value:
2.3
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
positive indication of irritation
Irritant / corrosive response data:
Based on a mean score of 2.3 (see below), the test compound is considered a moderate eye irritant

egg no 22: light injection; light TCL score 3

egg no 23: light injection score 1

egg no 24: light injection; light TCL score 3

mean score: 2.3

Interpretation of results:
study cannot be used for classification
Remarks:
Criteria used for interpretation of results: other: Luepke (1985)
Conclusions:
Based on the results of this CAM test, Ampholak YCE would be a moderate eye irritant in vivo.
Executive summary:

Twenty-two materials were assessed in the CAM test, an in vitro prescreen for predicting in vivo ocular irritancy. The test also included negative (sterile water) and positive (glacial acetic acid) controls. Day 10 fertilised chicken eggs were used to obtain chorioallantoic membranes (CAMs).

Only undamaged, well formed CAMs were used in the test. The test material was tested in triplicate, as supplied, and applied directly to the CAM surface: solvent and positive control materials, and the test material, were applied as 200 µl aliquots. The test material was left in contact with the membrane for 20 sec, after which time it was washed away by irrigation with warm sterile water. Observations of reactions in the CAM were made during a five minute period immediately after irrigation. The intensity of vascular injection, the extent of haemorrhage and the occurrence of coagulation of cytosolic proteins were assessed. Based on the results of this study, Ampholak YCE would be a moderate irritant in vivo. The sensitivity of the test system to a known irritant was shown by the response to the positive control material, glacial acetic acid.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 21 November 2017 Experimental completion date: 21 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: Sodium cocopropylenediamine propionate, CAS 97659-50-2
Batch: 48724
CAS No.: 97659-50-2
Purity: 29.7% (w/w), the substance is a UVCB, purity is equal to total solids.
Partition coefficient (n-octanol/water): log Pow: not indicated by the Sponsor
Water solubility: Soluble (not quantified)
Appearance (Envigo): Clear amber colored liquid
Appearance (Sponsor): Liquid, yellowish to brown
Expiry Date: 17 May 2020
Storage Conditions: At room temperature in the dark
Stability in Solvent: Stable in water (not quantified)
Purpose of Use: Industrial chemical
Safety Precautions: Routine hygienic procedures were sufficient to ensure personnel health and safety. For further information see Material Safety Data Sheet.
Species:
other: Eyes from adult cattle
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
Source of Bovine Eyes
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
Vehicle:
other: 0.9% w/v sodium chloride
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL of the test item or control items were applied to the appropriate corneas.
For the purpose of this study the test item was formulated at a concentration of 10% v/v in 0.9% w/v sodium chloride.
The negative control item, sodium chloride 0.9% w/v, was used as supplied.
The positive control item, ethanol, was used as supplied.
Duration of treatment / exposure:
120 minutes
Duration of post- treatment incubation (in vitro):
90 minutes
Number of animals or in vitro replicates:
3
Details on study design:
Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.
Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Treatment of Corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed.
The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.
After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein
Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 µL of media representing each cornea was dispensed into the appropriate wells of a pre labeled 96 well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.
No histopathology was required for this study.
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
6.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No prediction of eye irritation can be made
Other effects / acceptance of results:
Corneal Epithelium Condition
The corneas treated with the test item were clear post treatment and clear post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

Criteria for an Acceptable Test
The positive control In Vitro Irritancy Score was within the range of 31.6 to 58.7. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity of ≤3.0 and permeability ≤0.077. The negative control acceptance criteria were therefore satisfied.

Individual and Mean Corneal Opacity and Permeability Measurements

Treatment

Cornea Number

Opacity

Permeability (OD)

In Vitro Irritancy Score

Pre-Treatment

Post-Treatment

Post Incubation

Post-Incubation - Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control

10

3

2

4

1

 

0.020

 

 

11

3

3

4

1

 

0.007

 

 

12

4

2

4

0

 

0.008

 

 

 

 

 

 

0.7*

 

0.012¨

 

0.8

Positive Control

13

3

51

49

46

45.3

0.979

0.967

 

14

3

32

38

35

34.3

1.198

1.186

 

15

3

33

35

32

31.3

1.995

1.983

 

 

 

 

 

 

37.0·

 

1.379·

57.7

Test Item

16

2

1

4

2

1.3

0.388

0.376

 

17

3

1

2

-1

0.0

0.417

0.405

 

18

5

5

9

4

3.3

0.217

0.205

 

 

 

 

 

 

1.6·

 

0.329·

6.5

OD= Optical density           * = Mean of the post-incubation -pre‑treatment values           ¨= Mean permeability                     ·= Mean corrected value

Corneal Epithelium Condition Post Treatment and Post Incubation

Treatment

Cornea Number

Observation

Post Treatment

Post Incubation

Negative Control

10

Clear

Clear

11

Clear

Clear

12

Clear

Clear

Positive Control

13

Cloudy

Cloudy

14

Cloudy

Cloudy

15

Cloudy

Cloudy

Test Item

16

Clear

Clear

17

Clear

Clear

18

Clear

Clear

Interpretation of results:
other: No prediction of eye irritation can be made
Conclusions:
No prediction of eye irritation can be made.
Executive summary:

Introduction

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short‑term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

Method

The test item was applied at a concentration of 10% v/v in 0.9% w/v sodium chloride for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). 

Data Interpretation

The test item is classified according to the prediction model as follows:

IVIS

UN GHS

EU CLP
(Regulation (EC) No 1272/2008)

≤ 3

No Category

Not classified for irritation

>3; ≤ 55

No prediction can be made

No prediction can be made

> 55

Category 1

Category 1
H318: Causes serious eye damage

 

Results

The In Vitro irritancy scores are summarized as follows:

Treatment

In Vitro Irritancy Score

Test Item

6.5

Negative Control

0.8

Positive Control

57.7

 

Conclusion

No prediction of eye irritation can be made.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date 15 January 2018 Experimental completion date 13 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: Sodium cocopropylenediamine propionate, CAS 97659-50-2
Batch: 48724
CAS No.: 97659-50-2
Purity: 29.7% (w/w), the substance is a UVCB, purity is equal to total solids.
Partition coefficient (n-octanol/water): log Pow: not indicated by the Sponsor
Water solubility: Soluble (not quantified)
Appearance: Liquid, yellowish to brown
Expiry Date: 17 May 2020
Storage Conditions: At room temperature
Stability in Solvent: Stable in water (not quantified)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Animal Information
Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Envigo RMS (UK) Limited, Leicestershire, UK. At the start of the study the animals weighed 3.34 or 4.13 kg and were 12 to 52 weeks old. After an acclimatization period of at least 5 days each animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface of the ear and on the cage label.

Animal Care and Husbandry
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
0.1 mL of the test item
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
2
Details on study design:
Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a
standard ophthalmoscope. Only animals free of ocular damage were used.
A subcutaneous injection of buprenorphine 0.01 mg/kg was administered 60 minutes prior to test item application to provide a therapeutic level of systemic analgesia. Five minutes prior to test item application, a pre-dose anesthesia of ocular anesthetic (two drops of 0.5% proxymetacaine hydrochloride) was applied to each eye.
A volume of 0.1 mL of the test item was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to a six point scale.
Eight hours after test item application, a subcutaneous injection of post-dose analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg, was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 hours later. No further analgesia was required. After consideration of the ocular responses produced in the first treated animal, a second animal was similarly treated.
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment.
Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
Any clinical signs of toxicity, if present, were also recorded.
Additional observations were made on Days 7 and 14 to assess the reversibility of the ocular effects.
Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 14 days
Remarks on result:
positive indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 14 days
Remarks on result:
positive indication of irritation
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 7 days
Remarks on result:
positive indication of irritation
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 7 days
Remarks on result:
positive indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 14 days
Remarks on result:
positive indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 14 days
Remarks on result:
positive indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 14 days
Remarks on result:
positive indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.7
Max. score:
4
Reversibility:
fully reversible within: 14 days
Remarks on result:
positive indication of irritation
Irritant / corrosive response data:
Ocular Reactions
Scattered or diffuse corneal opacity was noted in both treated eyes 1 hour after treatment and at the 24, 48 and 72-Hour observations and persisted in one treated eye at the 7-Day observation.
Iridial inflammation was noted in both treated eyes at the 24, 48 and 72-Hour observations.
Severe conjunctival irritation was noted in one treated eye with moderate conjunctival irritation in the other treated eye at the 1-Hour observation. Moderate conjunctival irritation was noted in both treated eyes at the 24 and 48-Hour observations. Moderate conjunctival irritation persisted in one treated eye with minimal conjunctival irritation in the other treated eye at the 72-Hour observation. Minimal conjunctival irritation was noted in both treated eyes at the 7-Day observation.
Both eyes appeared normal at the 14–Day observation.
Other effects:
Body Weight
Both animals showed expected gain in body weight during the study.

Individual and Mean Scores for Cornea, Iris and Conjunctivae

Rabbit Number and Sex Time After Treatment Corneal Opacity Iridial Inflammation Conjunctival Redness Conjunctival Chemosis
75885 Female 24 Hours 1 1 2 2
48 Hours 1 1 2 2
72 Hours 1 1 2 2
Total 3 3 6 6
Mean 1 1 2 2
75947 Female 24 Hours 1 1 2 2
48 Hours 1 1 2 2
72 Hours 1 1 2 1
Total 3 3 6 5
Mean 1 1 2 1.7
Interpretation of results:
Category 2A (irritating to eyes) based on GHS criteria
Conclusions:
The test item meets the criteria for classification as ‘Irritating to eyes (Category 2)’ according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.
The test item meets the criteria for classification as ‘Irritating to eyes (Category 2/2A)’ according to the Globally Harmonized System of Classification and Labelling of Chemicals.
Executive summary:

Introduction

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit.

Results

A single application of the test item to the non-irrigated eye of two rabbits produced scattered or diffuse corneal opacity, iridial inflammation and moderate to severe conjunctival irritation.

Both eyes appeared normal at the 14–Day observation.

Conclusion

The test item meets the criteria for classification as ‘Irritating to eyes (Category 2)’ according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

The test item meets the criteria for classification as ‘Irritating to eyes (Category 2/2A)’ according to the Globally Harmonized System of Classification and Labelling of Chemicals.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salt CAS No 2136366-30-6, is sold as a ca. 30% solution in water. Inhalation is not an expected route of exposure so no inhalation tests are available.

Justification for classification or non-classification

In-vitro skin irritation: human skin model test according to OECD guideline 439 using EpiSkin tissues was assessed. The viability of the tissues was 100.6%, as this was greater than 50% the test substance was not classified as a skin irritant. . Therefore it is not classified as a skin irritant.

The Bovine Corneal Opacity Permeability Assay (BCOP) was carried out following OECD guideline 437. The irritancy score was 6.5 which is higher than the cut off for none classification of 3.0. It was also less than the limit for classification as Category 1 for eye damage which is >55. It was there concluded that no prediction can be made. A rabbit study was required to establish if Category 2 for eye irritation was required.

In the rabbit eye irritation study OECD 405. The test item produced a maximum group mean score of27.0and was classified as amoderateirritant (Class4on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

The test item meets the criteria for classification as ‘Irritating to eyes (Category 2)’ according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. The mean score 24/48/72 hours was 1 for corneal opacity, 1.0 for iridial inflammation, 2.0 for conjunctival redness and 1.7 for chemosis, the corneal opacity and iridial inflammation which recovered within the 14 days of the study resulted in this classification.