1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-C8-10 (even numbered) acyl derivs., hydroxides, inner salts

Endpoint summary

Description of key information

Skin irritation
-	Not irritating to skin; OECD TG 404, in vivo, rabbit, Type of coverage: semiocclusive, spray dried, 99.4% a.i. (RL1, GLP) read-across: C8-18 and C18 unsatd. AAPB
-	Not irritating to skin; OECD TG 404, in vivo, rabbit, Type of coverage: semiocclusive, 38.2%  a.i. in water (RL1, GLP) read-across: Formamidopropylbetaine



  

Eye irritation:
irritating to the eyes (Category 2); OECD TG 438, ICE with histopathology (RL1, GLP)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1994-12-06 to 1994-12-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

other: New Zealand Albino, Chbb: NZW (SPF)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Sex: male/female
- Age at study initiation: 3-5 month
- Weight at study initiation: 2.1 - 2.4 kg
- Housing: single housing
- Diet: ad libitum, laboratory rabbit diet Altromin 2123 and in addition hay (15 g daily)
- Water: ad libitum, tap water
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 3
- Humidity (%): 50 +/- 20
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

physiological saline

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 500 mg/patch, prepared to paste with 0.45 ml 0.9 % NaCl

Duration of treatment / exposure:

4 h

Observation period:

72 h
scorings 30-60 min, 24, 48 and 72 hours after test substance removal

Number of animals:

3

Details on study design:

PREPARATION OF ANIMALS
- An dorasal area of ca. 25 cm² was clipped 24 h before treatment.

TEST SITE
- Area of exposure: patches of 2.5 x 2.5 cm, loaded with 500 mg test substance prepared to paste with 0.45 ml 0.9 % NaCl/patch
- Type of wrap if used: cellulose patch (custom product from Beiersdorf AG, Hamburg, Germany), was held in place with a semi-occlusive bandage

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, washing with lukewarm tap water
- Time after start of exposure: 4 h

OBSERVATION
- Signs for erythema and edema were recorded 30-60 min, 24, 48, 72 h after removing of patches

SCORING SYSTEM:
- as stipulated by OECD 404 (Draize scheme)

Irritation parameter:

erythema score

Basis:

animal: #1, #3

Time point:

other: mean 24, 48, 72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

erythema score

Basis:

animal: #2

Time point:

other: mean 24, 48, 72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritation parameter:

edema score

Basis:

animal: #1, #2

Time point:

other: mean 24, 48, 72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

edema score

Basis:

animal: #3

Time point:

other: mean 24, 48, 72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

30 to 60 minutes after patch removal, 3/3 animals showed well defined erythema (score 2) and 1/3 animals each very slight oedema (score 1) or slight edema (score 2), 1/3 animals was free of edema at this observation. At the 24 hours scoring, erythema had lessened in 2/3 animals to score 1 and worsened in 1/3 animal to score 3. At the 24 hours scoring 2/3 animals showed an edema score 1. The only skin reaction still present at the 48 hours scoring, was a erythema score 2 in 1/3 animals. Fully reversibility in all animals was reached for erythema at the 72 hours scoring and for edema at the 48 hours scoring.

Table 1: Irritant/corrosive response data for each animal at each observation time up to removal of each animal from the test

Score at time point / Reversibility	Erythema	Edema
	Max. score: 4	Max. score: 2
30-60 min	2/2/2	1/2/0
24 h	1/3/1	1/1/0
48 h	0/2/0	0/0/0
72 h	0/0/0	0/0/0
Average 24h, 48h, 72h	0.33/1.67/0.33	0.33/0.33/0
Reversibility*)	c	c
Time for reversion (h)	48/72/48	48

*) Reversibility: c. = completely reversible; n.c.= not completely reversible; n. = not reversible

Interpretation of results:

GHS criteria not met

Conclusions:

Classification is based on erythema/eschar and oedema calculated as mean scores following grading at 24, 48 and 72 hours after removal of the test material, in consideration of reversibility.
No classification for skin irritation is justified for the "Coco AAPB (99.4 % dry solids)" according to CLP, EU GHS (Regulation (EC) No 1272/2008).

Executive summary:

In a primary dermal irritation study according to EU Method B.4 (1992) and OECD Guideline 404 (1992), 3 New Zealand White rabbits were dermally patch (2.5 x 2.5 cm²) exposed for 4 hours to 0.5 g of Coco AAPB (99.4 % dry solids) moistened with physiological saline. Test sites were covered with semi-occlusive dressing. Animals were then observed for 72 hours. Irritation was scored according to Draize as stipulated in the OECD guideline.

30 to 60 minutes after patch removal, 3/3 animals showed well defined erythema (score 2) and 1/3 animals each very slight edema (score 1) or slight edema (score 2), 1/3 animals was free of edema at this observation. At the 24 hours scoring, erythema had lessened in 2/3 animals to score 1 and worsened in 1/3 animal to score 3. At the 24 hours scoring 2/3 animals showed an edema score 1. The only skin reaction still present at the 48 hours scoring, was a erythema score 2 in 1/3 animals. Fully reversibility in all animals was reached for erythema at the 72 hours scoring and for edema at the 48 hours scoring.

In this study, Coco AAPB (99.4 % dry solids) is not a dermal irritant.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

09. May 2005 - 17. May 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Species: rabbit
Breed: New Zealand White, IVA: NZW
Breeder: Charles River, Extertal, Germany
Date of receipt: 2005-05-03
Sex: 1 male and 2 female
Body weights: 1805.3 g, 1905.8 g, 1949.1 g
Age: - 10 weeks
Identification: dyemarks and cage numbers
Diet: Altromin International, Lage, Germany, 2023 - 1425
Water: Drinking water for the animals consisted of normal tap water from municipal sources: Stadtische Werke Krefeld AG, Abt. 2 TGW, Krefeld, Germany. The composition of the water is regularly determined. The data are retained in the archive. The water was supplied to the animals ad libitum via drinking bottles with rubber stoppers and steel pipes.
Bedding material: Lignocel BK 10/20, Rettenmaier, Jagstzell, Germany
Husbandry: They were housed single in steel cages with a plastic bottom mould and a habitat of 5445 cm2 at the bottom and an overall height of 600 mm. A non-barrier system with air conditioning was used. The air conditioning had the following nominal values: Temperature: 20 ± 3 °C, Humidity: 30 -70 %. Climate control was run automatically. The lightening was in a 12-hour light/dark-cycle. Temperature and humidity were recorded continuously using a thermohygrometer, Lambrecht GmbH, Gottingen, Germany. The data were archived. There have been no deviations from nominal temperature and humidity values during the experiment.

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.5 mL of the test substance was applied to the left flank

Duration of treatment / exposure:

4 hours

Observation period:

Examination of the treated skin sites was done 1 hour, and approximately 24, 48, 72 hours as well as 7 days after removal of the test Substance.

Number of animals:

3

Details on study design:

Before the experiment was started, the animals had an acclimatization period of at least 5 days. The animals were clearly marked with dye and weighed. Food and drinking water was left for the animals ad libitum. 24 hours before dosing a part of the fur on both sides of the dorso-Iumbal region of each animal was removed by shearing with an electrical shaving machine exposing an area of skin approximately 10 x 15 cm. Care was taken to avoid abrading the skin and only animals with healthy intact skin were used in the experiment. Body weight was recorded immediately before test substance application and on the day of the final observation. As neither corrosive nor strong irritating effects to the skin have been expected the test substance was applied to all three animals for four hours at the same time. 0.5 mL of the test substance was applied to the left flank covered with a gauze pad (3 x 2 cm) which was held in place by strips of Blenderm (3M company, St. Paul, USA). The right flank
served as control region. Semi-occlusive dressings were bandaged with Acrylastic (Beiersdorf AG, Hamburg, Germany) and fixed with Leukoplast (Beiersdorf AG, Hamburg, Germany). Thus access by the animal to the patch and resultant ingestion of the test substance was prevented.
The test substance was removed by washing with tap water after 4 hours exposure time and the application areas were examined.

Irritation parameter:

erythema score

Basis:

animal: #1, #2, #3

Time point:

other: mean 24, 48, 72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal: #1, #2, #3

Time point:

other: mean 24, 48, 72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Skin Reactions: Only very slight erythema but no oedema was observed in one animal 1 h after removal of the test substance Formamidopropyldimethylbetain 50%ig. The numerical scores to the erythema and oedema formations are presented in the following table:

	Rabbit 504male					Rabbit 505 RV female					Rabbit 507 female
	1h	24h	48h	72h	7d	1h	24h	48h	72h	7d	1h	24h	48h	72h	7d
Erythema	0	0	0	0	0	0	0	0	0	0	1	0	0	0	0
Oedema	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Mean (24h-72h)		0 (E) 0 (Oe)					0 (E) 0 (Oe)					0 (E) 0 (Oe)

Interpretation of results:

GHS criteria not met

Conclusions:

Formamidopropyldimethylbetain (38.2% a.i.) is not irritating to the skin of rabbits. The mean grades of skin reactions at 24, 48 and 72 h reading correspond to values classified as not irritant by the Directive 67/548/EEC as well as GHS Regulation EC No 1272/2008.

Executive summary:

For labelling and classification purposes the test substance Formamidopropyldimethylbetain (38.2% a.i.), a yellowish clear liquid, was tested regarding its skin irritation potential. The potential to cause inflammatory or corrosive changes upon first contact with skin was

assessed by semi-occluded application of the test substance for four hours to the shorn skin of two female and one male rabbit, strain New Zealand White according to the DECO Guideline for the Testing of Chemicals, 404 "Acute Dermal Irritation/Corrosion". As neither corrosive nor strong irritating effects to the skin have been expected, the test substance was applied to all three animals for four hours at the same time. Dermal reactions at the treated skin sites were assessed 1 hour after removal of the test substance and approximately 24, 48, and 72 hours as well as 7 days later.

Systemic toxic symptoms after application were not observed at any time during the study. Body weight development of the animals was positive and within normal ranges. Only very slight erythema but no oedema was observed in one animal 1 h after removal of the

test substance. Especially between 24 and 72 hours no erythema and oedema were observed. The mean scores for erythema and oedema formation for the time period 24 h - 72 h were 0. Under the conditions of this study the test substance is non irritating and does not have to be classified for skin irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

adopted 26 July 2013

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: ROSS, spring chickens from poultry slaughterhouse v.d. Bor, Nijkerkerveen, the Netherlands
- Age at study initiation: Approximately 7 weeks old
- Weight at study initiation: body weight range approximately 1.5 2.5 kg

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.

Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus. Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes that showed opacity (score higher than 0.5), were unacceptably stained with fluorescein (score higher than 0.5), or showed any other signs of damage were rejected and were replaced.

After an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured once more to determine the zero reference value for corneal swelling calculations.

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg

Duration of treatment / exposure:

10 s

Observation period (in vivo):

eyes were examined at approximately 0, 30, 75, 120, 180 and 240 minutes after treatment

Number of animals or in vitro replicates:

negative control (physiological saline, 30 µL): 1
positive control (NaOH, 30 mg): 3
test group: 3

Details on study design:

REMOVAL OF TEST SUBSTANCE - Washing (if done): 20 mL saline
- Time after start of exposure: 10 s
SCORING SYSTEM: according to ICE classification criteria OECD TG 438
TOOL USED TO ASSESS SCORE: slit lamp microscope / fluorescein

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. Next, three corneas were treated with 30 mg test item. After an exposure period of 10 seconds, the corneal surface was rinsed thoroughly with 20 mL of isotonic saline at ambient temperature. After rinsing, each eye in the holder was returned to its chamber. The eyes were examined at 0, 30, 75, 120, 180 and 240 minutes after treatment. All examinations were performed with a slit-lamp microscope. Fluorescein retention was scored only at 30 minutes after treatment.
After the final examination, the test and control eyes were preserved in a neutral aqueous phosphate-buffered solution of 4% formaldehyde.
The tissues selected were embedded in paraffin wax, sectioned at 5 μm and stained with Periodic Acid-Schiff for histopathology examination. Ocular effects were evaluated using the endpoints of corneal thickness (swelling), corneal opacity and fluorescein retention.

Irritation parameter:

percent corneal swelling

Value:

14

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Value:

2.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

fluorescein retention score

Value:

2.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

After administration of the test item, a small lump of the test substance adhered to the cornea of one eye. After ca 5 minutes it was rinsed off by the saline drip.
The test item caused slight corneal swelling (14%), moderate or severe opacity (2.3) and moderate or moderate to severe fluorescein retention (2.2).
The one cornea with the prolonged exposure to the test item showed severe opacity and moderate to severe fluorescein retention.
The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate.
The positive control NaOH caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.
Microscopic examination of the corneas treated with the test item revealed very slight erosion of the epithelium.
Microscopic examination of the corneas treated with the negative control (saline) did not reveal any abnormalities. The positive control NaOH caused severe erosion of the epithelium, the majority of the nuclei of the stroma (inner region) not visible and necrosis of the endothelium.

Summary of results

	Maximum mean score for:			Irritation categories^1	Irritation Index^2	Classifications (EU-CLP^3/UN-GHS^4)
	Swelling %	Opacity	Fluorescein retention
Test item	14	2.3^5	2.2	II;III;III	104	2/2A
NaOH (positive control)	47	4.0^6	3.0	IV;IV;IV	187	1/1

 

1.    I = no effect; II = slight effect; III = moderate effect; IV = severe effect.

2.    Irritation Index = maximum mean corneal swelling + maximum mean opacity (x 20) + mean fluorescein score (x 20)

3.    EU-CLP: NC = not classified; Category 2 = Irritating to eyes; Category 1 = irreversible effects on the eye/serious damage to the eye. Regulation (EC) No 1272/2808 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2806.

4.    UN-GHS: NC = not classified; Category 2B = mild irritant, causes eye irritation; Category 2A = irritant, causes eye irritation; Category 1 = irreversible effects on the eye/serious damage to the eye. United Nations-Economic Commission for Europe (UN/ECE) (2003). Globally Harmonised System of Classification and Labelling of Chemicals (GHS).UN, New York and Geneva, 2007.

5.    a small lump of the test substance adhered to the cornea of one eye. After ca 5 minutes it was rinsed off by the saline drip

6.    Immediate iris constriction and severe loosening of epithelium

Individual histopathological findings

	Eye no.	Epithelium						Stroma			Endothelium
		Erosion	Necrosis	Vacuolation†			^Notes	Disorder of fibers	Pyknotic nuclei		Necrosis
				top	mid	low			outer region (adjacent to epithelium)	inner region (adjacent to endothelium)
Test item	1	½	-	-	-	-		-	-	-	-
	3	½	-	-	-	-		-	-	-	-
	5	½	-	-	-	-		-	-	-	-
NaOH (positive control)	2	3	-	-	-	-	‡	-	-	-	P
	4	3	-	-	-	-	‡	-	-	-	P
	6	3	-	-	-	-		-	-	-	P
Saline (negative control)	7	-	-	-	-	-		-	-	-	-

- = not observed; P = present; ½ = very slight; 1 = slight; 2 = moderate; 3 = severe; † = scored in the top/mid/low section of the epithelium; ‡ = majority of the nuclei of the stroma (inner region) not visible

 

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Based on the results from this Isolated Chicken Eye test, C8-10 Alkylamidopropyl betaine is considered to be irritating to the eyes (Category 2) under the experimental conditions described in this report.

Executive summary:

In an Isolated Chicken Eye (ICE) test according to OECD Guideline 438 (adopted 26 July 2013), the eye irritation potential of C8-10

Alkylamidopropyl betaine was assessed. In addition, the test included a negative control (saline) and a positive control (NaOH). Chicken eyes were obtained from slaughter animals used for human consumption.

The isolated chicken eyes were exposed to a single application of 30 mg 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.

After administration of the test item, a small lump of the test substance adhered to the cornea of one eye. After ca 5 minutes it was rinsed off by the saline drip.

The test item caused slight corneal swelling (14%), moderate or severe opacity (2.3) and moderate or moderate to severe fluorescein retention (2.2).

The one cornea with the prolonged exposure tothe test item showed severe opacity and moderate to severefluorescein retention. 

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

The positive control NaOH caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

Microscopic examination of the corneas treated with C8-10 Alkylamidopropyl betaine revealed very slight erosion of the epithelium.

Microscopic examination of the corneas treated with the negative control (saline) did not reveal any abnormalities. The positive control NaOH caused severe erosion of the epithelium, the majority of the nuclei of the stroma (inner region) not visible and necrosis of the endothelium.

Applying the classification criteria of the ICE, C8-10 Alkylamidopropyl betaine is classifed as Category 2 “Irritating to eyes”.

It is recognized that the prediction models adopted in the OECD Test Guideline 438 are for identifying i) chemicals inducing serious eye damage and ii) chemicals not requiring classification for eye irritation or serious eye damage. However, further support for identification of C8-10

Alkylamidopropyl betaine as a Category 2 irritant is derived from histopathology examination within this ICE test.

The effects observed in one cornea treated withthe test item demonstrates thatprolonged contact (mimicking exposure conditions of the Draize eye irritation test in rabbits) will dramatically increase the corneal effects.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No experimental data are on skin irritation available for the target substance C8-10 Alkylamidopropyl betaine. However, skin irritation studies are available for the closely related source substances C8-18 and C18 unsatd. AAPB and Formamidopropylbetaine. A justification for read-across is given below.

An in vitro eye irritation study (ICE, incl. histopathological assessment) is available for the target substance itself.

 

Skin irritation/corrosion

In a primary dermal irritation study according to EU Method B.4 (1992) and OECD Guideline 404 (1992), 3 New Zealand White rabbits were dermally patch (2.5 x 2.5 cm²) exposed for 4 hours to 0.5 g of C8-18 and C18 unsatd. AAPB (99.4 % dry solids) moistened with physiological saline. Test sites were covered with semi-occlusive dressing. Animals were then observed for 72 hours. Irritation was scored according to Draize as stipulated in the OECD guideline.

30 to 60 minutes after patch removal, 3/3 animals showed well defined erythema (score 2) and 1/3 animals each very slight edema (score 1) or slight edema (score 2), 1/3 animals was free of edema at this observation. At the 24 hours scoring, erythema had lessened in 2/3 animals to score 1 and worsened in 1/3 animal to score 3. At the 24 hours scoring 2/3 animals showed an edema score 1. The only skin reaction still present at the 48 hours scoring, was a erythema score 2 in 1/3 animals. Fully reversibility in all animals was reached for erythema at the 72 hours scoring and for edema at the 48 hours scoring.

In this study, C8-18 and C18 unsatd. AAPB (99.4 % dry solids) was not a dermal irritant.

 

The potential of Formamidopropylbetaine (38.2% a.i.) to cause inflammatory or corrosive changes upon first contact with skin was assessed by semi-occluded application of the test substance for four hours to the shorn skin of two female and one male rabbit, strain New Zealand White according to the OECD Guideline 404. As neither corrosive nor strong irritating effects to the skin have been expected, the test substance was applied to all three animals for four hours at the same time. Dermal reactions at the treated skin sites were assessed 1 hour after removal of the test substance and approximately 24, 48, and 72 hours as well as 7 days later.

Systemic toxic symptoms after application were not observed at any time during the study. Body weight development of the animals was positive and within normal ranges. Only very slight erythema but no oedema was observed in one animal 1 h after removal of the test substance. Especially between 24 and 72 hours no erythema and oedema were observed. The mean scores for erythema and oedema formation for the time period 24 h - 72 h were 0. Under the conditions of this study the test substance is not irritating and does not have to be classified for skin irritation.

 

Eye irritation/corrosion

In an Isolated Chicken Eye (ICE) test according to OECD Guideline 438 (adopted 26 July 2013), the eye irritation potential of C8-10 Alkylamidopropyl betaine was assessed. In addition, the test included a negative control (saline) and a positive control (NaOH). Chicken eyes were obtained from slaughter animals used for human consumption.

The isolated chicken eyes were exposed to a single application of 30 mg 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.

After administration of the test item, a small lump of the test substance adhered to the cornea of one eye. After ca 5 minutes it was rinsed off by the saline drip.

The test item caused slight corneal swelling (14%), moderate or severe opacity (2.3) and moderate or moderate to severe fluorescein retention (2.2).

The one cornea with the prolonged exposure to the test item showed severe opacity and moderate to severe fluorescein retention. 

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

The positive control NaOH caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

 

Microscopic examination of the corneas treated with C8-10 Alkylamidopropyl betaine revealed very slight erosion of the epithelium.

Microscopic examination of the corneas treated with the negative control (saline) did not reveal any abnormalities. The positive control NaOH caused severe erosion of the epithelium, the majority of the nuclei of the stroma (inner region) not visible and necrosis of the endothelium.

Applying the classification criteria of the ICE, C8-10 Alkylamidopropyl betaine is classifed as Category 2 “Irritating to eyes”.

It is recognized that the prediction models adopted in the OECD Test Guideline 438 are for identifying i) chemicals inducing serious eye damage and ii) chemicals not requiring classification for eye irritation or serious eye damage. However, further support for identification of C8-10 Alkylamidopropyl betaine as a Category 2 irritant is derived from histopathology examination within this ICE test.

The effects observed in one cornea treated with the test item demonstrate that prolonged contact (mimicking exposure conditions of the Draize eye irritation test in rabbits) will dramatically increase the corneal effects.

 

Although histopathology is not a mandatory endpoint for classification purposes, it has been recognised by OECD as potentially useful when a more complete characterization of corneal damage is needed (OECD 2011 and OECD, 2013b). In particular the OECD Guidance Document 160 (OECD, 2011) which is a supplement to the Test Guideline 438 was developed to promote the use of histopathology evaluation as an additional endpoint for ocular toxicity testing. This Guidance Document states that: “Histopathological evaluation may be useful for: (i) assessment of the histological damage of chemical classes or formulations that are not well characterized in these test methods; (ii) assisting with determination of a mode of action where it cannot be easily predicted; (iii) assisting with determination of the likelihood of delayed effects; (iv) evaluation of the depth of injury, which has been proposed as a measure of reversibility or irreversibility (Maurer et al. 2002); (v) further characterization of the severity or scope of the damage as needed (Harbell et al. 2006; ICCVAM 2010b; Maurer et al. 2002); (vi) assisting with discrimination of cases where the response falls along the borderline between two categories based on the test method decision criteria”.

 

A recent publication by the European Detergent Industry (Cazelleet al., 2014) established a set of defined histopathology criteria for use of histopathology as an additional endpoint to the standard prediction model in the ICE test method as encouraged by and for the reasons identified by OECD. Such histopathology criteria were found to be suitable to identify EU CLP / UN GHS Category 1 non-extreme pH detergent and cleaning products and to allow a better discrimination from Category 2 products. Since this recent publication focuses on non-extreme pH detergent and cleaning products the majority of which are surfactant-based, the histopathology criteria defined in this paper are considered to be relevant to interpretation of the histopathology evaluation in the ICE conducted on C8-10 Alkylamidopropyl betaine. On this basis, the histopathology results for C8-10 Alkylamidopropyl betaine confirmed this test substance as a non-Category 1 irritant, i.e. Category 2.

 

In accordance with REACH Annex XI, 1.4 confirmatory in vivo testing is scientifically not necessary when an adequate internationally validated and regulatory accepted in vitro study is provided and the results are adequate for the purpose of classification and labelling. Adequate and reliable documentation of the applied method is provided in the respective Robust Study Summary. To date, the ICE test is not accepted as a stand-alone test for evaluation of eye irritation but rather for identifying chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage. However, in combination with the use of histopathology as an additional endpoint to increase the robustness of identifying Category 1 irritants, testing of C8-10 Alkylamidopropyl betaine in animals is identified as not warranted. 

 

Summary

Based on interpolation of the results from the available studies on skin irritation conducted with the closely related source substances C8-18 and C18 unsatd. AAPB and Formamidopropylbetaine, the target substance C8-10 Alkylamidopropyl betaine is considered to be not irritating to skin.

C8-10 Alkylamidopropyl betaine was irritating to eyes, Category 2, in an in vitro eye irritation test.

 

There are no data gaps for the endpoint irritation/corrosion. No human data are available. However, there is no reason to believe that these results from rabbit would not be applicable to humans.

 

 

Justification for read-across

For details on substance identity and detailed toxicological profiles, please refer also to the general justification for read-across given at the beginning of the CSR and attached as pdf document to IUCLID section 13.

This read-across approach is justified based on structural similarities. The target and source substances contain the same functional groups. Thus a common mode of action can be assumed.

 

a. Structural similarity and functional groups

The target substance C8-10 Alkylamidopropyl betaine is a UVCB substance manufactured from fatty acids (C8 and C10) and N, N-dimethylpropylenediamine (DMAPA) and further reacted with sodium monochloroacetate.

The source substances C8-18 AAPB and C8-18 and C18 unsatd. AAPB are UVCB substances manufactured from natural fatty acids or oils and N, N-dimethylpropylenediamine (DMAPA) and further reacted with sodium monochloroacetate. As their origin is from natural sources, the used fatty acids may have a mixed slightly varying composition with an even numbered chain length from C8 to C18. Unsaturated C18 amounts may be included.

The source substance Formamidopropylbetaine is a monoconstituent substance manufactured from formic acid and N, N-dimethylpropylenediamine (DMAPA) and further reacted with sodium monochloroacetate.

 

b. Differences

Differences in chemical and other intrinsic properties of the target and source substances could potentially arise from the following facts:

-Different amounts of different carbon chain lengths (carbon chain length distribution):

Higher amounts of higher chain lengths and corresponding lower amounts of lower chain length lead to a rising average lipophilicity as can be seen from the increasing log Kow from Formamidopropylbetaine (log Kow: -3.3), C8-10 Alkylamidopropyl betaine (log Kow: 2.2), C12 AAPB (log Kow: 3.54), C8-18 AAPB (log Kow: 4.23).

- Different amounts of unsaturated fatty ester moieties:

The source substance C8-18 and C18 unsatd. AAPB contains considerable amounts of unsaturated C18 chains, which represents a worst case with respect to some toxicological endpoints, mainly local effects (e.g. irritation, sensitisation) and reactivity (HERA, 2002; Stillman, 1975; Aungst, 1989).

 

The provided structural similarities and impurity profiles support the proposed read-across hypothesis with high confidence.

 

 

Comparison of irritation data

 

Endpoints	Source substance	Target substance	Source substance
	C8-18 and C18 unsatd. AAPB	C8-10 Alkylamidopropyl betaine	Formamidopropylbetaine
Skin irritation, in vivo	WoE_RA_99.4% dry solids_Skin irritation / corrosion: 61789-40-0_8.1.1_HOECHST_1995_OECD404 OECD TG 404, in vivo, rabbit, Type of coverage: semiocclusive, spray dried, 99.4% a.i. not irritating   Reliability: 1 (reliable without restriction), GLP	No data, read-across	WoE_RA_LTOE 16399 Skin irritation / corrosion   OECD TG 404, in vivo, rabbit, Type of coverage: semiocclusive, 38.2% a.i. in water not irritating   Reliability: 1 (reliable without restriction), GLP
Eye irritation, in vitro	No data	key_Eye irritation. 73772-45-9 / 73772-46-0_8.2_Evonik_2014_OECD438   OECD TG 438, ICE with histopathology   irritating to the eyes (Category 2)   Reliability: 1 (reliable without restriction), GLP	No data
Eye irritation, in vivo	sup_RA_99.4% dry solids_Eye irritation: 61789-40-0_8.2.1_HOECHST_1995_OECD_405   OECD TG 405, in vivo, rabbit, spray dried, 99.4% a.i.   Category 1 (irreversible effects on the eye)   Reliability: 1 (reliable without restriction), GLP	sup_Eye irritation: 73772-45-9 / 73772-46-0_8.2._Evonik_1998_OECD 405_34.5%   OECD TG 405, in vivo, rabbit, 34.5%, aqueous solution   irritating to the eyes (Category 2)   Reliability: 1 (reliable without restriction), GLP	sup_RA_LTOE 16433 Eye irritation.001   OECD TG 405, in vivo, rabbit, 50% as manufactured   not irritating   Reliability: 1 (reliable without restriction), GLP

 

The source substances C8-18 and C18 unsatd. AAPB and Formamidopropylbetaine were not irritating to skin: In the study with C8-18 and C18 unsatd. AAPB, mean values of skin reactions at 24, 48 and 72 h were less than the cut-off values for classification i. e. < 2.3 (CLP) for erythema/eschar and edema in each individual animal. Also, all findings were fully reversible.

Formamidopropylbetaine was tested at 38.2% a.i.; the scores for erythema and edema were zero in all three animals.

The target substance C8-10 Alykalamidopropyl betaine was irritating to eye (Cat 2) in an in vitro ICE test including histopathological examination. Slight corneal swelling (14%), moderate or severe opacity (2.3) and moderate or moderate to severe fluorescein retention (2.2) and very slight erosion of the epithelium were observed.

Supporting data are available from an in vivo test conducted with 34.5% aqueous solution, also leading to classification as category 2.

The source substance C8-18 and C18 unsatd. AAPB caused irreversible effects to the eye, whereas the source substance Formamidopropylbetaine caused only minimal irritating effects not leading to classification.

With respect to eye irritation, the source substance C8-18 and C18 unsatd. AAPB represented a worst case, with decreasing irritation potential in the row C8-18 and C18 unsatd. AAPB > C8-10 Alkylamidopropyl betaine > Formamidopropylbetaine. Both extremes of this row were not irritating to skin, thus, an interpolation is applied, resulting also in non-classification of the target substance.

 

Quality of the experimental data of the analogues:

The available data are adequate and sufficiently reliable to justify the read-across approach.

The skin irritation studies were conducted according to (or comparable to) OECD Guideline 404 and are reliable (RL1).

The target substance was tested in a reliable (RL1) GLP-compliant study according to OECD TG 438 and OECD TG 405. The source substances were tested in reliable (RL1) GLP-compliant studies according to OECD TG 405.

The test materials used in the respective studies represent the source substance as described in the hypothesis in terms of substance identity and minor constituents.

Overall, the study results are adequate for the purpose of classification and labelling and risk assessment.

Conclusion

Based on structural similarities of the target and source substances as presented above and in more detail in the general justification for read across, it can be concluded that the available data on skin irritation from the source substances C8-18 and C18 unsatd. AAPB and Formamidopropylbetaine are also valid for the target substance C8-10 Alkylamidopropyl betaine.

C8-18 and C18 unsatd. AAPB is a worst case model substance, as it contains short chain fatty acid moieties as well as unsaturated moieties, fatty acid amounts which might have some increasing influence on the irritating potential.

The read-across is further supported by the results obtained in the eye irritation studies: A decreasing irritation potential in the row C8-18 and C18 unsatd. AAPB > C8-10 Alkylamidopropyl betaine > Formamidopropylbetaine was demonstrated.

It is assumed that this trend also applies to skin irritation potential. Both extremes of this row (C8-18 and C18 unsatd. AAPB and Formamidopropylbetaine) were not irritating to skin. Thus, also the target substance (located in the middle of this row) is expected to be not irritating to skin.

References

Aungst, 1989.Structure/Effect Studies of Fatty Acid Isomers as Skin Penetration Enhancers and Skin Irritants. Pharmaceutical Research, March 1989, Volume 6, Issue 3, pp 244-247

HERA, 2002: Fatty Acid Salts – Human Health Risk Assessment

Stillman et al., 1975. Relative irritancy of free fatty acids of different chain length. Contact Dermatitis. 1975;1(2):65-9

Justification for classification or non-classification

Skin irritation

Based on reliable, adequate and relevant data, C8-10 Alkylamidopropyl betaine does not need to be classified for skin irritation according to regulation (EC) 1272/2008.

 

Eye irritation

Based on reliable, adequate and relevant data, C8-10 Alkylamidopropyl betaine is classified as Category 2 (Irritating to eyes) according to regulation (EC) 1272/2008 and labelled with H319: Causes serious eye irritation and the signal word “Warning”.