Registration Dossier

Administrative data

Description of key information

Skin irritation/Corrosion

Two skin irritation / corrosion studies are available onSodium 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). The key study is an in-vivo study performed in 1987 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 Coco iminodiglycinate (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 withCAS no2060541-51-5)all showing consistent results.

 

Eye irritation/corrosion

Only in-vitro eye irritation tests are availableon 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). 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. 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 all 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:
29 April 1987 - 6 May 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study is performed under GLP and according to OECD 404.
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: David Percival Ltd., Moston, Sandbach, Cheshire, U.K. and Nottingham University, School of Agriculture, Sutton Bonington, Leicestershire, U.K.
- Age at study initiation: 12-16 weeks
- Weight at study initiation: 2.40 -3.12 kg
- Housing: Individually housed in suspended metal cages.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21
- Humidity (%): 55-60
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 29 April 1987 - 6 May 1987
Type of coverage:
semiocclusive
Preparation of test site:
other: clipped
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
0.5ml
Duration of treatment / exposure:
4 hours
Observation period:
7 days
Number of animals:
3
Details on study design:
TEST SITE
- Area of exposure: 2.5x2.5 cm
- % coverage: no data
- Type of wrap if used: A quantity of 0.5ml of the test material was introduced under a 2.5 cm x 2.5 cm gauze patch and placed i n position on the shorn skin. The patch was secured i n position over the test material with a strip of surgical adhesive tape (BLENDERN: approximate size 25 m x 40 mm). To prevent the animals from interfering w i t h the patches the trunk of each I rabbit was wrapped in an elasticated corset (TUBIGRIP).

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Any residual test material removed by gentle swabbing with cotton wool soaked in distilled water.
- Time after start of exposure: 4 hours

SCORING SYSTEM: According to Draize,
Scoring system Erythema and oedema
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 edema(barely perceptible)
2 Slight edema(edges of the area well defined by definite raising)
3 Moderate edema(raised approximately 1mm)
4 Severe edema( raised more than 1 mm and extending beyond the area of exposure)
Irritation parameter:
erythema score
Basis:
mean
Time point:
other: 24-48-72 hours
Score:
0.53
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
edema score
Basis:
mean
Time point:
other: 24-48-72 hours
Score:
0
Max. score:
4
Reversibility:
fully reversible within: 1 day
Irritant / corrosive response data:
Very slight erythema was noted at all test sites one hour after removal of the wrapping and at the 24-hour observation but persisted at only one test site at the 48 and 72-hour observations. Very slight oedema was noted at all test sites one hour after removal of the wrapping but at only one test site at the 24-hour observation. All test sites appeared normal on day seven.

Summary of dermal lesions (following 4-h application)

no.

Effect

Hour

Days after application

Mean score erythema

24/48/72 h

Mean score oedema

24/48/72 h

1

1

2

3

7

31

Erythema/ eschar

Oedema

1

1

1

0

0

0

0

0

0

0

0.3

0.0

68

Erythema/ eschar

Oedema

1

1

1

0

0

0

0

0

0

0

0.3

0.0

90

Erythema/ eschar

Oedema

1

1

1

0

1

0

1

0

0

0

1.0

0.0

Mean

0.53

0.0

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
The test material was found to be non-i rritant to rabbit skin according to GHS.
Executive summary:

A skin irritation/corrosion study was carried out in order to assess the irritancy potential of a test material following a single 4-hour, semi-occluded application to the intact rabbit skin. The study was designed to comply with the recommendations of the OECD Guidelines for Testing of Chemicals (1981) No. 404 "Acute Dermal Irritation/Corrosion" and was performed under GLP. Very slight erythema was noted at all test sites one hour after removal of the wrapping and at the 24-hour observation but persisted at only one test site at the 48 and 72-hour observations. Very slight oedema was noted at all test sites one hour after removal of the wrapping but at only one test site at the 24-hour observation. All test sites appeared normal on day seven. The test material was found to be non-i rritant to rabbit skin according to GHS.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2016-06-13 - 2016-06-17
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 (undiluted)
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:
91.6
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 mixture of 10 µl test item per 2 mL MTT medium showed reduction of MTT as compared to the solvent. The mixture turned blue/purple.

For quantitative correction of results, the part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using killed tissues. Therefore, three tissues were treated with the test item (KT) and with the negative control DPBS (KU), respectively. NSMTT was calculated relative to the negative control of living tissues (NK) according to the following formula:

NSMTT [%] = [(ODKT- ODKU)/ODNK] * 100

Mean ODKT= 0.067

Mean ODKU= 0.080

Mean ODNK= 0.776

The calculated NSMTT was ≤ 30% (-1.76%) relative to the negative control of livingepidermisand could therefore be used for determination of the true MTT metabolic conversion (TODTT) of the test item treated living tissues (TM) according to the following formula:

TODTT= ODTM- (ODKT- ODKU)

The mixtures of 10 µL of the test item per 90 µL aqua dest. and per 90 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSClivingequaled 0%.

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:

In the present study Sodium oleylamphopolycarboxyglycinate was applied topically to the EPISKIN-SMätissue for 15 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects 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:
June 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was performed accosring to US federal methods which are comparable to the OECD guideline and the study was performed under GLP. Observations were made for 72 hours although effects were not fully reversed at that time.
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.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Principles of method if other than guideline:
The animals were observed for 72 hours although the effects in one of the animals had not fully reversed at that time.
GLP compliance:
yes
Specific details on test material used for the study:
Appearance: clear orange liquid
Container: screw capped plastic bottle
Name: Ampholak 7TX
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: recognised breeders
- Age at study initiation: no data
- Weight at study initiation: 2.0-2.3 kg
- Housing: in grid bottom metal cages, not mentioned if the housing was individual
- Diet (e.g. ad libitum): A commercially ( available antibiotic free pelleted rabbit diet (Product Ref. 680, Dalgety-Spillera Limited), ad libitum
- Water (e.g. ad libitum): Mains drinking water via automatic drinking nozzles in each cage were available ad libitum
- Acclimation period: No data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-20
- Humidity (%): 70-90
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: June 1985

Female animals were used
Vehicle:
unchanged (no vehicle)
Controls:
other: untreated eye of the animals
Amount / concentration applied:
0.1 ml
Duration of treatment / exposure:
A single dose observed for 72 hours
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
6
Details on study design:
REMOVAL OF TEST SUBSTANCE
not done

SCORING SYSTEM:
Scoring system eye irritation

Cornea: degree of density (area most dense taken for reading)
0 No ulceration or opacity
1 Scattered or diffuse areas of opacity (other than slight dulling of normal lustre), details of iris clearly visible
2 Easily discernible translucent areas, details of iris slightly obscured
3 Nacrous areas, no details of iris visible, size of pupil barely discernible
4 Opaque cornea, iris not discernible through the opacity

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

Iris
0 Normal
1 Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia or injection, any of these or combination of any thereof, iris still reacting to light (sluggish reaction is positive)
2 No reaction to light, haemorrhage, gross destruction (any or all of these)

Conjunctivae
Redness (refers to palpebral and bulbar conjunctivae cornea and iris)

0 Blood vessels normal
1 Some blood vessels definitely hyperaemic
(injected)
2 Diffuse, crimson colour, individual vessels not easily discernible
3 Diffuse, beefy red

Chemosis (lids and/or nictitating membranes)
0 No swelling 0
1 Any swelling above normal (includes nictitating membranes)
2 Obvious swelling with partial eversion of lids
3 Swelling with lids about half-closed
4 Swelling with lids more than half-closed

Discharge
0 No discharge
1 Any amount different from normal (does not include small amounts observed in inner canthus of normal animals)
2 Discharge with moistening of the lids and hairs just adjacent to lids
3 Discharge with moistening of the lids and hairs a considerable area around the eye


TOOL USED TO ASSESS SCORE: Examination was canfined to a macroscopic observation and aids such as a binocular loupe, slit lamp or fluorescsin,staining were not employed.
Irritation parameter:
conjunctivae 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
Irritant / corrosive response data:
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 of the group. Moderate conjunctival irritation was maintained by the one rabbit throughout the study.

Rabbit No and sex

Region of the eye

Hours after instillation

Average 24-48-72 hours

24

48

72

840-f

Cornea

Degree of opacity

0

0

0

0

Area

0

0

0

0

Iris

0

0

0

0

Conjunctivae

Redness

0

0

0

0

Chemosis

0

0

0

0

Discharge

0

0

0

0

842-f

Cornea

Degree of opacity

0

0

0

0

Area

0

0

0

0

Iris

0

0

0

0

Conjunctivae

Redness

0

0

0

0

Chemosis

0

0

0

0

Discharge

0

0

0

0

843-f

Cornea

Degree of opacity

0

0

0

0

Area

0

0

0

0

Iris

0

0

0

0

Conjunctivae

Redness

0

0

0

0

Chemosis

0

0

0

0

Discharge

0

0

0

0

845-f

Cornea

Degree of opacity

0

0

0

0

Area

0

0

0

0

Iris

1

0

0

0.3

Conjunctivae

Redness

1

1

1

1

Chemosis

4*

3

3

3.3

Discharge

2

2

2

2

846-f

Cornea

Degree of opacity

0

0

0

0

Area

0

0

0

0

Iris

0

0

0

0

Conjunctivae

Redness

0

0

0

0

Chemosis

0

0

0

0

Discharge

0

0

0

0

847-f

Cornea

Degree of opacity

0

0

0

0

Area

0

0

0

0

Iris

0

0

0

0

Conjunctivae

Redness

0

0

0

0

Chemosis

0

0

0

0

Discharge

0

0

0

0

*Blistering of conjunctival-membranes

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 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 vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
12 January 1993
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Study performed according to in vitro method. Not validated, regulatory acceptance if the outcome is severly irritating. 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:
no 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: chicken eggs
Strain:
other: Ross
Details on test animals or tissues and environmental conditions:
Day 10 fertilised chicken eggs of the Ross variety were obtained from Suffolk Sovereign Hatcheries, Eye, Suffolk on the morning of test commencement. The eggs were transported in a constant temperature carrier to minimise heat loss. The eggs were maintained in a humidified incubator at 39ºC until later that day.
Vehicle:
unchanged (no vehicle)
Controls:
other: positive (glacial acetic acid) and negative (sterile water) control
Amount / concentration applied:
200 µl
Duration of treatment / exposure:
20 seconds
Observation period (in vivo):
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.
Number of animals or in vitro replicates:
3 eggs
Details on study design:
Preparation of CAMs:
A lin e 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 CAM beneath, using fine forceps. Excess water was poured off. Only undamaged, well formed CAMs were used i n tests.

All materials were tested in triplicate, as supplied, and applied directly to the CAM surface

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The membranes were washed away by irrigation with warm sterile water.
- Time after start of exposure: 20 seconds

SCORING SYSTEM:

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.

SCORE, RESPONSES
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, Coagulation
Heam - haemorrhage
TCL - terminal capillary leakage

Interpretation
Mean points scored, Grade
0, non-irri tant
0.1-0.9, no significant effect
1.0-1.9, mildly irritant
2.0-4.9, moderately irritant
>5.0 severely irritant


TOOL USED TO ASSESS SCORE: None
Irritation parameter:
other: overall mean irritation score
Run / experiment:
The Hen's Egg Chorioallantoic Membrane Test for Irritation Potential
Value:
4
Remarks on result:
positive indication of irritation
Remarks:
Max score: 10.0
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 5 minutes
Score:
4
Max. score:
10
Reversibility:
other: Not applicable
Irritant / corrosive response data:
Based on a mean score of 4.0 (see below), the test compound is considered a moderate eye irritant in vitro

egg no 4: light injection; score 3

egg no 5: light injection; score 6

egg no 6: light injection; score 3

mean score: 4.0

Conclusions:
Based on the results of this CAM test, Ampholak 7TX would be a moderate eye irritant in vitro and considered 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 7TX would be a moderate irritant in vitro and 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
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 [Coco iminodiglycinate (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 withCAS no2060541-51-5)], 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 no2098351-38-1) 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 withCAS no2060541-51-5) 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 no2060541-47-9). 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, 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)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 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)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.