Registration Dossier

Administrative data

Description of key information

Skin irritation/Corrosion

Two skin irritation / corrosion studies are available on Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1). The key study is an in-vivo study performed in 2016 according to the OECD Guideline 404 and under GLP. It has reliability rating 1 and classifies the substance as not irritating. This is backed up by an in-vitro study from 2016, as well as the available in-vivo and in-vitro data on the other two substances within the group Sodium cocoamphopolycarboxyglycinate (Amines, N-(3-aminopropyl)-N’-C12-18-alkyltrimethylenedi-, N-(carboxymethyl)derivs., sodium salts with CAS no 2060541-51-5) and Sodium oleylamphopolycarboxyglycinate (1,3-Propanediamine, N1-(3-aminopropyl)-N3-[3-[(9Z)-9-octadecen-1-ylamino]propyl]-, N-(carboxymethyl) derivs., sodium salts with CAS no 2060541-49-1) all showing consistent negative results.

 

Eye irritation/corrosion

Only in-vitro eye irritation tests are available on Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1). Results from in-vitro tests are similar within the group. Two in-vivo eye irritation studies are available on two of the amphoteric, glycinate substances: Sodium cocoamphopolycarboxyglycinate (Amines, N-(3-aminopropyl)-N’-C12-18-alkyltrimethylenedi-, N-(carboxymethyl)derivs., sodium salts with CAS no 2060541-51-5) and Sodium tallowamphopolycarboxyglycinate (Amines, N-[3-[(3-aminopro yl)amino]propyl]-N’-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, N-(carboxymethyl) derivs., sodium salts with CAS no 2060541-47-9). The effects seen, such as redness in conjunctiva and cornea, were reversible and mild, therefore not generating any classification. The effects in the in-vitro studies are mild, and do not indicate Cat 1 classification for eye irritation. Currently in-vivo data is required in order to classify eye irritation Cat 2. The weight of evidence from the available eye irritation data on this group of substances indicates no classification for eye irritation.      

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-10-06 to 2016-12-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2500 (Acute Dermal Irritation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Specific details on test material used for the study:
Name: Coco iminodiglycinate
Batch No.: 1259678
CAS No.: Active ingredient: CAS RN 97659-51-3
Aggregate State at RT: liquid
Color: yellow
Active Components: 30.2% Coco iminodiglycinate, 59.2% Water, 10.6% NaCl
Purity: 40.8% active ingredient
Stability: stable
Storage Conditions: room temperature
Expiry Date: 16 December 2016
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Housing and Feeding Conditions:
- Semi barrier in an air-conditioned room
- Temperature: 18 +/- 3 °C (recommendations of TVT, GV-SOLAS)
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Free access to autoclaved hay and to Altromin 2123 maintenance diet for rabbits, rich in crude fibre
- Free access to tap water (drinking water, municipal residue control, microbiological controls at regular intervals)
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Housed in ABS-plastic or Noryl rabbit cages, floor 4200 cm2
- Adequate acclimatisation period (at least 5 days) under laboratory conditions


Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
Dose Level
A dose of 0.5 g of the test item was applied to each test site.
Duration of treatment / exposure:
Exposure Period
The test item was held in contact with the skin throughout a 4-hour period.
At the end of the exposure period, the residual test item was removed with sterile water.
Observation period:
Observation Period
The animals were observed for 72 hours after the patch removal.
Number of animals:
3
Details on study design:
Clinical Observation
The animals were examined for signs of erythema and oedema 1 hour after the patch removal. For the determination of classification-relevant values, the animals were examined for signs of erythema and oedema 24, 48 and 72 hours after the patch removal. Dermal irritation was scored and recorded according to the grades in the table below. Any other signs such as hyperplasia, scaling, discolouration, fissures and scabs or any systemic effects were also recorded.
For the initial test in one animal, the test site was also examined immediately after the patch had been removed.

Evaluation of Results
Individual reactions for each animal were recorded according to the scoring system described in the table below at each time of observation.
Nature, severity and duration of clinical observations were described.
The body weight changes were summarised in a tabular form.

Irritation parameter:
edema score
Basis:
animal #1
Time point:
other: 0 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #1
Time point:
other: 1 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #1
Time point:
other: 24 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 72 h
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #1
Time point:
48 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 72 h
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #1
Time point:
72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Neither irritant nor corrosive effects were observed on the intact skin of three male rabbits (strain NZW) after a contact time of 4 hours. Two of the three rabbits showed no effects at all for any of the dermal irritation endpoints evaluated; the third rabbit had very slight Oedema (score 1) after 24 and 48 hours, which was fully reversed after 72 hours.
Other effects:
Neither mortalities nor significant clinical signs of toxicity were observed.
There were no significant body weight changes during the observation period.

I

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
Under the conditions of the present study, the single dermal application of the test item Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1) to three rabbits at a dose of 0.5 mL showed slight irritant but no corrosive effects in one animal which were fully reversible within up to 72 hours. Neither mortalities nor significant clinical signs of toxicity were observed.
Executive summary:

Summary Results

Species/strain:                               New Zealand White Rabbits Crl: KBL (NZW)

Number of animals:                               3

Duration of exposure:                          4 hours

Amount of substance:                          0.5 g per test site

Type of dressing:                                   semi-occlusive

Vehicle (moistening):                            none

First time of effects:                               animal no. 1: 24 hours after patch removalerythemagrade 1, animals no. 2 and no. 3: no effects observed

Last time of effects:                             animal no. 1: 48 hours after patch removalerythemagrade 1, animal no. 2 and no. 3: no effects observed

Reversibility of the observed effects:  no effects observed

Method:                                                 OECD 404; EC 440/2008, Method B.4; OPPTS 870.2500; OPPTS 870.1000

Average Irritation Scores – (24, 48, 72-hour reading) – and Total Mean Value

Statistic Average
Animal No. 1 Animal No. 2 Animal No. 3 Total Mean Value
Oedema Grade 0 0 0 0
Erythema Grade 0.67 0 0 0.22

Conclusion

Under the conditions of the present study, the single dermal application of the test item Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1) to three rabbits at a dose of 0.5 mL showed slight irritant but no corrosive effects in one animal which were fully reversible within up to 72 hours.

Neither mortalities nor significant clinical signs of toxicity were observed.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-05-25 - 2016-05-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
other: EU Method B.46 (Skin Irritation)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Amount / concentration applied:
10 µL
Duration of treatment / exposure:
15 minutes
Observation period:
42 +/- 1 h
Details on study design:
The test was performed on EpiSkin, an organotypic reconstructed three-dimensional model of the human epidermis. 3 replicate tissues are dosed with the test item, the negative control (10µL DPBS) and the positive control (10µL 5% SDS), respectively. After 15 minutes treatment period at room temperature the test item and the controls are rinsed off with DPBS and the tissues are post-incubated for 42 +/- 1 h. Then the tissues are stained via MTT for 3 hours. Isopropanol extracts are measured photometrically at 570 nm.
Irritation / corrosion parameter:
% tissue viability
Remarks:
Mean Relative Tissue Viability [%] from three tissues
Run / experiment:
In-vitro Skin irritation: Human Skin Model Test (EPISKIN-SM)
Value:
73.6
Negative controls validity:
valid
Remarks:
DPBS (Gibco, Cat. No. 14040-091, Lot No.: 1737107)
Positive controls validity:
valid
Remarks:
5% sodium dodecyl sulphate in aqua dest.
Remarks on result:
no indication of irritation

The test item showed no direct MTT reducing capability and no colouring potential.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: OECD 439
Conclusions:
The test item is classified as "non-irritant" (No Category).
Executive summary:

Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1)I was tested in the in-vitro skin irritation test using the EpiSkin human epidermis model. 10 µL was dispensed directly atop for 15 minutes. After 42 h post-incubation cytotoxic effects were determined via MTT reduction assay.

The mean relative tissue viability (% negative control) was > 50%.

The test item is therefore classified as "non-irritant" (UN GHS: No Category).

This study is classified as acceptable:

OD570 of the blank is < 0.1.

Mean OD570 of the three negative control tissues is >= 0.6 and <= 1.5.

Mean relative tissue viability of the three positive contol tissues is <= 40%.

Standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is < 18%.

This study satisfies the requirement for Test Guideline OECD 439 for in vitro skin irritation.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
20 september 1993 - 4 october 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Read-across (Category)

The available in vivo data on this endpoint is consistent within the group. Two in vivo eye irritation studies are available on two of the substances. The effects seen, such as redness in conjunctiva and cornea, were reversible and mild, therefore not generating any classification. Using this data for read across within the group would be extrapolating data to the extremes of the group. In order to support this read across, in vitro eye irritation studies on all the four substances were performed, both in EpiOcular and Isolated Chicken Eye test. Also the in vitro tests results were consistent within the group, indicating a mild to irritating results. There is no possibility to evaluate the reversibility of the effects seen in in vitro studies. The in vitro tests EpiOcular and Isolated chicken eye tests are considered to over-predict the actual eye irritation potential of the substance shown in the rabbit tests. The effects in the in vitro studies are mild, and do not indicate Cat 1 classification for eye irritation. Currently in vivo data is required in order to classify eye irritation Cat 2. The weight of evidence from the available eye irritation data on this group of substances indicates no classification for eye irritation.
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Amount / concentration applied:
0.1 mL
Observation period (in vivo):
24, 48, 72 h
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Reversibility:
fully reversible within: 5 days
Irritation parameter:
iris score
Basis:
mean
Time point:
other: 24-48-72 hours
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
other: 24-48-72 hours
Score:
1.3
Max. score:
3
Reversibility:
fully reversible within: 5 days
Interpretation of results:
GHS criteria not met
Conclusions:
A single instillation of the technical product into the eye of the rabbit elicited corneal opacificaton, iridial inflammation and well-defined conjunctival irritation. All reactions had resolved 3 or 7 days after instillation. The composition of the technical product is 40% active ingredient and 60 % water. The observed results do not warrant classification according to GHS.
Executive summary:

A study was performed to assess the eye irritation potential of a technical product to the rabbit. The method followed was that described in EEC Methods for the determination of toxicity, Amex to Directive 92/69/EEC (OJ No. L383A, 29.12.92), Part 3, Method B.5. Acute toxicity (eye irritation). Three rabbits were each administered a single ocular dose of 0.1 ml of the test substance and observed for seven days after instillation. A single instillation of the technical product into the eye of the rabbit elicited corneal opacificaton, iridial inflammation and well-defined conjunctival irritation. All reactions had resolved 3 or 7 days after instillation. The observed results do not warrant classification according to GHS.

Endpoint:
eye irritation: in vivo
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Justification for type of information:
The available in vivo data on this endpoint is consistent within the group. Two in vivo eye irritation studies are available on two of the substances. The effects seen, such as redness in conjunctiva and cornea, were reversible and mild, therefore not generating any classification. Using this data for read across within the group would be extrapolating data to the extremes of the group. In order to support this read across, in vitro eye irritation studies on all the four substances were performed, both in EpiOcular and Isolated Chicken Eye test. Also the in vitro tests results were consistent within the group, indicating a mild to irritating results. There is no possibility to evaluate the reversibility of the effects seen in in vitro studies. The in vitro tests EpiOcular and Isolated chicken eye tests are considered to over-predict the actual eye irritation potential of the substance shown in the rabbit tests. The effects in the in vitro studies are mild, and do not indicate Cat 1 classification for eye irritation. Currently in vivo data is required in order to classify eye irritation Cat 2. The weight of evidence from the available eye irritation data on this group of substances indicates no classification for eye irritation.
Irritation parameter:
cornea opacity score
Remarks:
redness
Basis:
mean
Time point:
other: 24-48-72 hours
Score:
0.15
Max. score:
3
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other: effects were only observed in one out of 6 animals
Irritation parameter:
conjunctivae score
Remarks:
chemosis
Basis:
mean
Time point:
other: 24-48-72 hours
Score:
0.55
Max. score:
4
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other: effects were only observed in one out of 6 animals
Irritation parameter:
conjunctivae score
Remarks:
discharge
Basis:
mean
Time point:
other: 24-48-72 hours
Score:
0.33
Max. score:
3
Reversibility:
not fully reversible within: 72 hours
Remarks on result:
other: effects were only observed in one out of 6 animals
Irritation parameter:
iris score
Basis:
mean
Time point:
other: 24-48-72 hours
Score:
0.05
Max. score:
2
Reversibility:
fully reversible within: 48 hours
Remarks on result:
other: effects were only observed in one out of 6 animals
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
other: 24-48-72 hours
Score:
0
Max. score:
4
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
The results of this study indicate that the test material may have little irritant effect in the eye. The composition of the test material is estimated to be 40% active ingredient (incl NaCl) and 60 % water. The active ingredient is Amines, N-[3-[(3-aminopropyl)amino]propyl]-N’-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, N-(carboxymethyl) derivs., sodium salts (CAS no 2060541-47-9). The response observed appears to have been in an unusually sensitive animal. The animals were only observed for 72 hours and therefore it can not be judges if the effects are reversible within 21 days. However after 72 hours compared with the 24 reading the severity of the effects had decreased some in the affected animal. The active ingredient Amines, N-[3-[(3-aminopropyl)amino]propyl]-N’-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, N-(carboxymethyl) derivs., sodium salts (CAS no 2060541-47-9) may not be classified as an irritant to the eye according to GHS.
Executive summary:

According to methods similar to OECD and under GLP 0.1 ml aliquots of the test material were instilled into the right eye of each of six rabbits, the other eye remaining untreated as a control. The irritation produced in the treated eye of all rabbits was assessed 24, 48 and 72 hours after dosing. 24 hours after dosing iris inflammation and severe conjunctival irritation were apparent in one rabbit of the group, no response to treatment being observed in other , animals o f the group. Moderate conjunctival irritation was maintained by the one rabbit throughout the study.

The results of this study indicate that the test material may have little irritant effect in the eye. The response observed appears to have

been in an unusually sensitive animal. The animals were only observed for 72 hours and therefore it can not be judges if the effects are reversible within 21 days. However after 72 hours compared with the 24 reading the severity of the effects had decreased some in the affected animal. The active ingredient Amines, N-[3-[(3-aminopropyl)amino]propyl]-N’-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, N-(carboxymethyl) derivs., sodium salts (CAS no 2060541-47-9) may not be classified as an irritant to the eye according to GHS.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2016-05-17 to 2016-05-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guidline study
Qualifier:
according to
Guideline:
other: OECD Guideline for the Testing of Chemicals No. 492: (Reconstructed human Cornea-like Epithelium (RhCE) test method)
Qualifier:
according to
Guideline:
other: EURL ECVAM DB-ALM Method Summary No. 164: EpiOcular™ Eye Irritation Test - Summary
Qualifier:
according to
Guideline:
other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) For the prediction of acute ocular irritation of chemicals
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)
Amount / concentration applied:
50 µL (undiluted)
Duration of treatment / exposure:
incubation: 30 min.
Observation period (in vivo):
post soak: 12 +/- 2 min.
post treatment: 120 min.
Details on study design:
The test was performed on EpiOcular, a reconstituted three-dimensional human corneal epithelium model. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 30 min exposure period and 120 min post-treatment period and compared to those of the concurrent negative controls.
Irritation parameter:
other: Mean Relative Tissue Viability [%] from two tissues
Run / experiment:
In-vitro Eye Irritation: Ocular Irritation Assay using the EpiOcular Human Tissue Model
Value:
7.5
Negative controls validity:
valid
Remarks:
Aqua dest.
Positive controls validity:
valid
Remarks:
Methyl acetate
Remarks on result:
positive indication of irritation

The mixture of 50 µL test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture turned to dark red, but not blue/purple.

Although the absorption spectra of the mixture showed a peak at 595 nm with an OD of 3.1261, the NSMTT was not determined since the viability of the test item treated tissues (TM) was below 60%

The mixture of 50 µL test item per 1 mL A. dest. and per 2 mL isopropanol showed no colouring as compared to the solvent.Therefore, NSC equaled 0%.

Conclusions:
In this study under the given conditions the test item showed irritant effects. The test item is classified as “irritant“ in accordance with UN GHS “Category 1” or “Category 2”.
Executive summary:

Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with Aqua dest.

The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 60% (7.5%).

The controls confirmed the validity of the study. The mean absolute OD570of the two negative control tissues was> 0.8 and < 2.5 (2.082). The mean relative tissue viability (% negative control) of the positive control was < 50% (32.5%). The maximum inter tissue difference of replicate tissues of all dose groups was < 20% (0.1 – 9.4%).

This study is classified as acceptable. This study satisfies the requirement for OECD Guideline for the Testing of Chemicals No. 492: (Reconstructed human Cornea-like Epithelium (RhCE) test method).

Endpoint:
eye irritation: in vitro / ex vivo
Remarks:
Isolated Chicken Eye Test
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
4 November 2016 - 5 December 2016
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
GLP compliance:
yes
Details on test animals or tissues and environmental conditions:
Approximately 7 weeks old, male or female chickens (ROSS, spring chickens), body weight range approximately 1.5-2.5 kg, were used as eye donors. Heads of these animals were obtained from poultry slaughterhouse v.d. Bor, Nijkerkerveen, the Netherlands. Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
30µL of the neat test samples were tested. The neat samples were 40% aqueous solutions.
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
The eyes were examined at approximately 0, 30, 75, 120, 180 and 240 minutes after treatment.
Number of animals or in vitro replicates:
3
Details on study design:
Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye-lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium 2.0% w/v (Minims, Chauvin, England) was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the head with the fluorescein-treated cornea was examined with a slit-lamp microscope (Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland) to ensure that the cornea was not damaged. If undamaged (e.g., fluorescein retention and corneal opacity scores of ≤ 0.5), the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye-ball from the orbit without cutting off the optical nerve too short.
The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus (Triskelion, Zeist, the Netherlands; see Figure 1). The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a target rate of 0.10-0.15 mL/min (peristaltic pump set at speed 5.00, Watson-Marlow 205CA, Rotterdam, the Netherlands). The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32oC (water pump set at 36.4oC; Lauda 103, Germany).
After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they were not damaged. Corneal thickness was measured using the Depth Measuring Attachment No. I for the Haag-Streit slit-lamp microscope, set at 0.095 mm.

Corneal thickness was expressed in instrument units. An accurate measurement was taken at the corneal apex of each eye.
Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes showing opacity (score higher than 0.5), or were unacceptably stained with fluores-cein (score higher than 0.5) indicating the cornea to be permeable, or eyes that showed any other signs of damage, were rejected as test eyes and replaced.
Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. For that purpose, after an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured again to determine the zero reference value for corneal swelling calculations.
At time t = 0 (i.e. immediately after the zero reference measurement), the following procedure was applied for each test eye: The clamp holding the test eye was placed on paper tissues outside the chamber with the cornea facing upwards.

The isolated chicken eyes were exposed to a single application of 30 μL test substance for 10 seconds followed by a 20 mL saline rinse. After rinsing, each eye in the holder was returned to its chamber. The eyes were examined at approximately 0, 30, 75, 120, 180 and 240 minutes after treatment, using the criteria and scoring system given in Annex 1. Fluorescein retention was only scored at approximately 30 minutes after treatment. All examinations were carried out with the slit-lamp microscope. After the final examination, the test substance treated eyes, the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at ca 4 μm and stained with PAS (Periodic Acid-Schiff). The microscopic slides were subjected to histopathological examination.
Irritation parameter:
other: Irritation index
Remarks:
Irritation Index = maximum mean corneal swelling + maximum mean opacity (x 20) + mean fluorescein score (x 20)
Run / experiment:
Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1)
Value:
70
Positive controls validity:
valid
Remarks:
BAC 5% in water
Remarks on result:
positive indication of irritation
Irritation parameter:
other: Irritation index
Remarks:
Irritation Index = maximum mean corneal swelling + maximum mean opacity (x 20) + mean fluorescein score (x 20)
Run / experiment:
Sodium cocoamphopolycarboxyglycinate (Amines, N-(3-aminopropyl)-N’-C12-18-alkyltrimethylenedi-, N-(carboxymethyl)derivs., sodium salts CAS number:2060541-51-5)
Value:
78
Positive controls validity:
valid
Remarks:
BAC 5% in water
Remarks on result:
positive indication of irritation
Irritation parameter:
other: Irritation index
Remarks:
Irritation Index = maximum mean corneal swelling + maximum mean opacity (x 20) + mean fluorescein score (x 20)
Run / experiment:
Sodium oleylamphopolycarboxyglycinate (1,3-Propanediamine, N1-(3-aminopropyl)-N3-[3-[(9Z)-9-octadecen-1-ylamino]propyl]-, N-(carboxymethyl) derivs., sodium salts with CAS number 2060541 -49 -1)
Value:
78
Positive controls validity:
valid
Remarks:
BAC 5% in water
Remarks on result:
positive indication of irritation

Slit-lamp examination

Sodium cocoamphopolycarboxyglycinate, Coco iminodiglycinate and Sodium oleylamphopolycarboxyglycinate caused corneal effects leading to a Category 2/2B classification, consisting of very slight or slight corneal swelling (range of mean scores 8-12%),

slight-to-moderate or moderate opacity (range mean scores 1.5-2.0) and slight-to-moderate fluorescein retention (mean score 1.5). The opacity and fluorescein were observed as focal areas.

 

The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate.

The positive control Benzalkonium Chloride 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

 

Microscopic examination

Microscopic examination of the corneas treated with Sodium cocoamphopolycarboxyglycinate revealed very slight erosion with slight necrosis of the epithelium.

Microscopic examination of the corneas treated with Coco iminodiglycinate revealed very slight erosion and very slight or slight vacuolation (top region) of the epithelium.

Microscopic examination of the corneas treated with Sodium oleylamphopolycarboxyglycinate revealed very slight erosion and slight vacuolation (top region) of the epithelium.

According to the AISE histopathology criteria, upgrading of the classifications of the samples to Category 1 on basis of the histopathology of the corneas is not required. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control Benzalkonium Chloride 5% revealed slight, moderate or severe erosion and very slight or slight vacuolation (top, mid or low region) of the epithelium and the epithelium partly detached from the basement membrane.

Interpretation of results:
study cannot be used for classification
Conclusions:
The in vitro Isolated chicken eye test is considered to over-predict the actual eye irritation potential of the substance shown in the rabbit tests. An explanation could be the lack of possibility to evaluate the reversibility of the effects seen in in vitro studies.
Executive summary:

Sodium cocoamphopolycarboxyglycinate (Amines, N-(3-aminopropyl)-N’-C12-18-alkyltrimethylenedi-, N-(carboxymethyl)derivs., sodium salts with CAS number 2060541-51-5), Coco iminodiglycinate (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS number 2098351-38-1) and Sodium oleylamphopolycarboxyglycinate (1,3-Propanediamine, N1-(3-aminopropyl)-N3-[3-[(9Z)-9-octadecen-1-ylamino]propyl]-, N-(carboxymethyl) derivs., sodium salts with CAS number 2060541 -49 -1) were evaluated neat for eye irritation potential in the Isolated Chicken Eye (ICE) test. The isolated chicken eyes were exposed to a single application of 30 μL for 10 seconds followed by a 20 mL saline rinse. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells. In addition, histopathology of the corneas was performed.

Sodium cocoamphopolycarboxyglycinate (Amines, N-(3-aminopropyl)-N’-C12-18-alkyltrimethylenedi-, N-(carboxymethyl)derivs., sodium saltsCAS number with 2060541-51-5), Coco iminodiglycinate (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS number 2098351-38-1)and Sodium oleylamphopolycarboxyglycinate (1,3-Propanediamine, N1-(3 -aminopropyl)-N3-[3-[(9Z)-9-octadecen-1-ylamino]propyl]-, N-(carboxymethyl) derivs., sodium salts with CAS number 2060541 -49 -1) caused corneal effects leading to a Category 2/2B classification, consisting of very slight or slight corneal swelling (range of mean scores 8-12%), slight-to-moderate or moderate opacity (range mean scores 1.5-2.0) and slight-to-moderate fluorescein retention (mean score 1.5). The opacity and fluorescein were observed as focal areas. Microscopic examination of the corneas treated with Sodium cocoamphopolycarboxyglycinate revealed very slight erosion with slight necrosis of the epithelium. Microscopic examination of the corneas treated with Coco iminodiglycinate revealed very slight erosion and very slight or slight vacuolation (top region) of the epithelium. Microscopic examination of the corneas treated with Sodium oleylamphopolycarboxyglycinate revealed very slight erosion and slight vacuolation (top region) of the epithelium.

According to the AISE histopathology criteria, upgrading of the classifications of the samples to Category 1 on basis of the histopathology of the corneas is not required.

The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. Microscopic examination did not reveal any abnormalities. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight, moderate or severe erosion and very slight or slight vacuolation (top, mid or low region) of the epithelium and the epithelium partly detached from the basement membrane.

Applying the classification criteria of the ICE, the following irritation classifications can be assigned:

Sodium cocoamphopolycarboxyglycinate (Amines, N-(3-aminopropyl)-N’-C12-18-alkyltrimethylenedi-, N-(carboxymethyl)derivs., sodium saltsCAS number:2060541-51-5), Coco iminodiglycinate (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts withCAS number 2098351-38-1)and Sodium oleylamphopolycarboxyglycinate (1,3-Propanediamine, N1-(3 -aminopropyl)-N3-[3-[(9Z)-9-octadecen-1-ylamino]propyl]-, N-(carboxymethyl) derivs., sodium salts withCAS number 2060541 -49 -1)

- Category 2B:“Mild irritant/causes eye irritation” (UN-GHS classification);

- Category 2:“Irritating to eyes” (EU-CLP classification).

Although there is no current regulatory acceptance to use this result to classify a substance as eye irritation Category 2/2B), the proposed classifications are deemed appropriate.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation / corrosion

The available data on this endpoint is consistent within the group. Reliable in-vivo skin corrosion/irritation studies are available on the two substances with the shortest alkyl chain [Sodium cocoamphopolycarboxyglycinate (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium saltswith CAS no2098351-38-1) and Sodium cocoamphopolycarboxyglycinate (Amines, N-(3-aminopropyl)-N’-C12-18-alkyltrimethylenedi-, N-(carboxymethyl)derivs., sodium salts with CAS no 2060541-51-5)], as well as the substance with the longest alkyl chain [Sodium oleylamphopolycarboxyglycinate (1,3-Propanediamine, N1-(3-aminopropyl)-N3-[3-[(9Z)-9-octadecen-1-ylamino]propyl]-, N-(carboxymethyl) derivs., sodium saltswith CAS no 2060541-49 -1)], showing no skin irritating properties. This data is backed up with in-vitro studies on the smallest (shortest alky, lowest number amine and carboxymethylated groups,CAS no 2098351-38-1) as well as the biggest structure (longest alkyl-unsaturated, highest number amine and carboxymethylated groups, CAS no 2060541-49-1) within the amphoteric, glycinate group. Interpolation of the data within the group is therefore possible. Both the in-vitro data as well as the in-vivo skin irritation tests indicates low skin irritating properties. All the tests are performed on the technical products (a.i. 40 % aqueous solutions). The neat substances may potentially have more irritating properties. This would be in line with the QSAR models estimating the skin irritating properties on the pure substances to be mildly irritating. But as the substances exclusively are produced, handled and marketed as water based solutions, it is not considered relevant to test the neat substances.

 

Eye irritation / corrosion

Two in-vivo eye irritation studies are available on two of the amphoteric, glycinate substances: Sodium cocoamphopolycarboxyglycinate (Amines, N-(3-aminopropyl)-N’-C12-18-alkyltrimethylenedi-, N-(carboxymethyl)derivs., sodium salts with CAS no 2060541-51-5) and Sodium tallowamphopolycarboxyglycinate (Amines, N-[3-[(3-aminopro yl)amino]propyl]-N’-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, N-(carboxymethyl) derivs., sodium salts with CAS no 2060541-47-9). The effects seen, such as redness in conjunctiva and cornea, were reversible and mild, therefore not generating any classification. Using this data for read across within the group would be extrapolating data to the extremes of the group. In order to support this read across in-vitro eye irritation studies on all the four substances were performed, both in EpiOcular and Isolated Chicken Eye test. Also the in-vitro tests results were consistent within the group, indicating a mild to irritating results. There is no possibility to evaluate the reversibility of the effects seen in in-vitro studies. The in-vitro tests EpiOcular and Isolated chicken eye tests are considered to over-predict the actual eye irritation potential of the substance shown in the rabbit tests. The effects in the in-vitro studies are mild, and do not indicate Cat 1 classification for eye irritation. Currently in-vivo data is required in order to classify eye irritation Cat 2. The weight of evidence from the available eye irritation data on this group of substances indicates no classification for eye irritation.         

Justification for classification or non-classification

Skin

Based on the above mentioned information, Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts (CAS no:2098351-38-1) and the other three substances within the amphoteric, glycinate substane group, are not to beclassified according to Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 and is considered “not irritating” to skin.

 

Eye

The conclusion from the available studies is that the substance Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts (CAS no:2098351-38-1) and the other three substances within the amphoteric, glycinate substane group, are not to be classified according to Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 and is considered “not irritating” to eye.