pentasodium 3,7,11-tris(carboxylatomethyl)-15-(C12-18)-alkyl-3,7,11,15-tetraazaheptadecane-1,17-dioate

Endpoint summary

Description of key information

Skin irritation/Corrosion

One skin irritation / corrosion study is available onSodium cocoamphopolycarboxyglycinate(Amines, N-(3-aminopropyl)-N’-C12-18-alkyltrimethylenedi-, N-(carboxymethyl)derivs., sodium salts with CAS no 2060541 -51 -5). The key study is an in-vivo study performed in 1993 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 the available in-vivo and in-vitro data on the other two substances within the group Sodium cocoamphopolycarboxyglycinate (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 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 no 2060541-49-1) all showing consistent results.

 

Eye irritation/corrosion

Sodium cocoamphopolycarboxyglycinate(Amines, N-(3-aminopropyl)-N’-C12-18-alkyltrimethylenedi-, N-(carboxymethyl)derivs., sodium saltswith CAS no 2060541-51-5) has been tested both in-vivo and in-vitro studies. The available in-vivo study, the key study, was performed in 1993 under GLP. A single instillation of the test material 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. Also the other in-vivo study available on the Sodium tallowamphopolycarboxyglycinate (Amines, N-[3-[(3-aminopropyl)amino]propyl]-N’-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, N-(carboxymethyl) derivs., sodium salts with CAS no 2060541-47-9) indicated no eye irritation.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

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 September 1993 - 19 September 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed under GLP and according to OECD guideline.

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Froxfield (U.R.) Ltd., Petersfield, Hampshire, England
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 2.2-2.6 kg
- Housing: housed individually in metal cages with perforated floors
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: yes, time not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19
- Humidity (%): 30-70
- Air changes (per hr): 19
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 14 September 1993 - 19 September 1993

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:

6 days

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: 25 mm x 25 mm gauze pad to one intact skin site on each animal.
- % coverage: no data
- Type of wrap if used: Each treatment site was covered with "Elastoplast" elastic adhesive dressing for four hours.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure period, the semi-occlusive dressing and gauze pad were removed and the treatment site was washed with warm water (30°C to 40°C) to remove any residual test substance. The treated area was blotted dry with absorbent paper.
- Time after start of exposure: 4 hours

SCORING SYSTEM: 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:

1

Max. score:

4

Reversibility:

fully reversible within: 6 days

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 24-48-72 hours

Score:

0.2

Max. score:

4

Reversibility:

fully reversible within: 2 days

Irritant / corrosive response data:

Well defined erythema wjth very slight oedema was seen in all three animals on Day 1. These reactions gradually ameliorated and had resolved completely by Day 6.

Other effects:

There were no signs of toxicity or ill health in any rabbit during the observation period.

Summary of dermal lesions (following 4-h application)

Animal no.	Effect	Hour	Days after application					Mean score erythema 24/48/72 h	Mean score oedema 24/48/72 h
		1/2	1	2	3	4	5
2457	Erythema/ eschar Oedema	2 1	1 0	1 0	1 0	1 0	0 0	1	0
2458	Erythema/ eschar Oedema	2 1	1 1	1 0	1 0	1 0	0 0	1	0.3
2459	Erythema/ eschar Oedema	2	1 1	1 0	1 0	1 0	0 0	1	0.3
Mean								1	0.2

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

A single semi-occlusive application of the test material to intact rabbit skin for four hours elicited slight to well-defined dermal reactions. All reactions had resolved by Day 6. The composition of the test material is estimated to be 40% active ingredient (incl NaCl) and 60 % water. Amines, N-(3-aminopropyl)-N’-C12-18-alkyltrimethylenedi-, N-(carboxymethyl)derivs., sodium salts (CAS no 2060541-51-5) is therefre not classified as irritating to skin under GHS.

Executive summary:

A study was performed to assess the in-vivo skin irritation potential of the test material consisting with a composition of 40% active ingredient (incl NaCl) and 60 % water. The method followed was that described in EEC Methods for the determination of toxicity, Annex to Directive 92/69/EEC (OJ No. L383A, 29.12.92), Part B, Method B.4. Acute toxicity (skin irritation). Three rabbits were each administered a single dermal dose of 0.5 ml of the test substance and observed for six days. A single semi-occlusive application of the active ingredient to intact rabbit skin for four hours elicited slight to well-defined dermal reactions in all three animals. All reactions had resolved by Day 6. Based on the result, test substance Amines, N-(3-aminopropyl)-N’-C12-18-alkyltrimethylenedi-, N-(carboxymethyl)derivs., sodium salts (CAS no 2060541-51-5) is not classified as irritating to skin under GHS.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 September 1993 - 4 October 1993.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Only available as final draft report.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Froxfield Rabbits
- Age at study initiation: 10-14 weeks
- Weight at study initiation: 2.3-3.2 kg
- Housing: individually in metal cages with perforated floors
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: yes, time not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19
- Humidity (%): 30-70
- Air changes (per hr): 19
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: 20 September 1993 - 4 October 1993.

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated eye of the animal

Amount / concentration applied:

0.1 ml

Duration of treatment / exposure:

One single instillation was performed and the animals were obsreved for 7 days after instillation.

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

3

Details on study design:

The test substance was not removed from the eye.

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

TOOL USED TO ASSESS SCORE: handheld light

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: 24-48-72 hours

Score:

1.1

Max. score:

4

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

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: 24-48-72 hours

Score:

0.9

Max. score:

4

Reversibility:

fully reversible within: 5 days

Irritant / corrosive response data:

Corneal opacites developed in two animals. Iridial inflammation was seen in one animal one hour after instillation only. A diffise crimson colouration of the conjunctivae was seen in two animals. Considerable swelling of the conjunctivae with partial eversion of the eyelids was seen in all three animals. The eyes were normal 3 or 7 days after instillation.

Other effects:

There were no signs of toxicity or ill health in any rabbit during the observation period.

Rabbit No and sex	Region of the eye		Hours after instillation						Average 24-48-72 hours
			1	24	48	72	96	5-7(days)
2450-m*	Cornea		0	0	0	0	0	0	0
	Iris		0	0	0	0	0	0	0
	Conjunctivae	Redness	1	1	1	0	0	0	0.6
		Chemosis	2	1	1	0	0	0	0.6
2463-m	Cornea		1	1	1	0	0	0	0.6
	Iris		0	0	0	0	0	0	0
	Conjunctivae	Redness	2	2	1	1	1	0	1.3
		Chemosis	2	1	1	1	1	0	1
2464-m	Cornea		0	2	2	1	1	0	1.6
	Iris		1	0	0	0	0	0	0
	Conjunctivae	Redness	2	2	2	2	2	0	2
		Chemosis	2	1	1	1	1	0	1

* Pilot animal

Conclusions:

A single instillation of the test material 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 test material is estimated to be 40% active ingredient (incl NaCl) and 60 % water. The active ingredient Amines, N-(3-aminopropyl)-N’-C12-18-alkyltrimethylenedi-, N-(carboxymethyl)derivs., sodium salts (CAS no 2060541-51-5) is therefre not classified as irritating to eyes.

Executive summary:

A study was performed to assess the eye irritation potential of the test material 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). The composition of the test material is estimated to be 40% active ingredient (incl NaCl) and 60 % water. Three rabbits were each administered a single ocular dose of 0.1 ml of the test material and observed for seven days after instillation. The active ingredient is Amines, N-(3-aminopropyl)-N’-C12-18-alkyltrimethylenedi-, N-(carboxymethyl)derivs., sodium salts (CAS no 2060541-51-5). A single instillation of the test material 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 active ingredient Amines, N-(3-aminopropyl)-N’-C12-18-alkyltrimethylenedi-, N-(carboxymethyl)derivs., sodium salts (CAS no 2060541-51-5) is therefre not classified as irritating to eyes.

Reference 2

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

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.

Reference 3

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

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 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 cocoamphopolycarboxyglycinate(Amines, N-(3-aminopropyl)-N’-C12-18-alkyltrimethylenedi-, N-(carboxymethyl)derivs., sodium salts withCAS no2060541-51-5) 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 substanceSodium cocoamphopolycarboxyglycinate(Amines, N-(3-aminopropyl)-N’-C12-18-alkyltrimethylenedi-, N-(carboxymethyl)derivs., sodium salts withCAS no2060541-51-5)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.