Glycine, N-methyl-, N-coco acyl derivs.

Endpoint summary

Description of key information

Skin irritation (OECD 404), rabbit: corrosive

Eye irritation (in vitro and in vivo): Data waiving

As the registered substance is classified for its skin corrosion properties as Skin Corr. 1B (H314), no eye irritation studies have to be conducted according to Regulation (EC) No. 1907/2006 (REACH), Annexes VII and VIII, Item 8.2, Column 2.

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:

key study

Study period:

2004-09-21 to 2004-10-20

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

GLP compliance:

yes

Species:

rabbit

Strain:

other: SPF albino rabbit of the stock Chbb:HM

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Source: Charles River Deutschland GmbH, Kißlegg, Germany
- Age at study initiation: 56 weeks
- Weight at study initiation: 2400 g
- Housing: individual female rabbit was caged in a polyphenyl ether (PPE) cage
- Diet: pelleted Altromin 2123, Lage, Lippe, ad libitum
- Water: (tap/filtered) water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 55 ± 15
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

water

Controls:

other: untreated site of the same animal served as control

Amount / concentration applied:

0,5 mL

Duration of treatment / exposure:

Initial test: 3 min and 1 h

Observation period:

Initial test: 28 days
Reading time points: 1, 24, 48, 72 h and 7, 14, 21 and 28 days

Number of animals:

1 female

Details on study design:

TEST SIDE:
- Area of exposure: 6.25 cm² on the right side of the back
- Type of wrap: The treated skin was covered with adhesive Gothaplast tape (2.5 cm)

REMOVAL OF TEST SUBSTANCE:
- Washing: Residual test material was removed with mild soap and lukewarm water.
- Time after start of exposure: 3 min, 1h

SCORING SYSTEM:
Draize scoring system

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

other: 1 h

Score:

4

Max. score:

4

Reversibility:

fully reversible

Remarks:

after 28 days

Remarks on result:

other: skin has been washed 1 h p.a. due to severe immediate effects

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

4

Max. score:

4

Reversibility:

fully reversible

Remarks:

after 28 days

Remarks on result:

other: skin has been washed 1 h p.a. due to severe immediate effects

Irritation parameter:

edema score

Basis:

animal #1

Time point:

other: 1 h

Score:

3

Max. score:

4

Reversibility:

fully reversible

Remarks:

after 28 days

Remarks on result:

other: skin has been washed 1 h p.a. due to severe immediate effects

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

fully reversible

Remarks:

after 28 days

Remarks on result:

other: skin has been washed 1 h p.a. due to severe immediate effects

Irritant / corrosive response data:

- The animal showed a very slight erythema on the middle and posterior right test field immediately after the application, 1h and 24 h after the termination of exposure a severe erythema to eschar formation and a moderate oedema was observed in the middle right test field as well as a severe erythema to eschar formation and a slight oedema on the posterior right test field.
- 48 h after the termination of exposure the animal showed a severe erythema to eschar formation as red-brownish discoloration and a severe oedema reaching widely beyond the patch area of the middle and posterior right test field.
- 72 h after the termination of exposure a severe erythema to eschar formation and a severe oedema were observed on the middle and posterior right test field of the animal.
- 7 days after the termination of exposure the animal showed a markedly elevated, brownish crust of scales being partially dropped off on the whole area of the middle and posterior right test field.
- 14 days after the termination of exposure granulation tissue was observed underneath the dropped off crust of scales on the middle and posterior right test field.
- 21 days after the termination of exposure the affected skin areas of the middle and posterior test field of the animal were healed almost completely.
- 28 days after the termination of exposure no pathological changes were macroscopically discernible anymore at the animal

Individual erythema and oedema scores

Animal No./ weight in kg	Exposure	Test field	Erythema formation					Oedema formation
			0	1	24	48	72	0	1	24		48	72
1 / 2,4	3 min	anterior ri.	0	0	0	0	0	0	0		0	0	0
	1 h	median ri.	1	4	4	4	4	0	3		3	4a)	4
	Instead of 4 h, stop after approx.. 1 h	posterior ri.	1	4	4	4	4	0	2		2	4a)	4

Remark 4a): Red-brownish discoloration of the patch area and oedema reaching beyond the patch area widely

Interpretation of results:

other: Skin corr. 1B, H314. Classification according to Regulation (EC) No. 1272/2008 (CLP/EU GHS).

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-12-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

GLP compliance:

yes

Species:

human

Strain:

other: human-derived epidermal keratinocytes (EPI-200)

Details on test animals or test system and environmental conditions:

TEST SKIN MODEL
- Source: MatTek Corporation, MA, USA

TEST METHOD
The model represents a reconstructed three-dimensional skin model based on normal human-derived epidermal keratinocytes which have been cultured to form a multilayered epidermis including basal, spinous and granular layers, and a multi-layered stratum corneum. Irritant materials are identified by their ability to penetrate the stratum corneum and to damage the underlaying cell layers which is determined through a decrease in cell viability as determined by MTT reduction assay.

ADAPTATION TO CELL CULTURE CONDITIONS
Upon receipt, tissues were pre-incubated in assay medium for 1 h (37 °C, 5% CO2) before use. After a medium exchange an amount of 50 µL per insert of the test and reference items were applied onto the surface of the skin models so that they were covered completely. The incubation times were 3 min and 1 h.

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37
- CO2 gas concentration (%): 5
- Humidity: maximum

Type of coverage:

other: open in vitro system

Preparation of test site:

other: intact reconstructed human epidermis

Vehicle:

water

Controls:

other: concurrent control tissues treated with the vehicle served as negative controls, positive controls were exposed to 8N KOH

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL
- Concentration (if solution): 100%

VEHICLE
- Amount(s) applied: 50 µL
- Lot/batch no: 0274

POSITIVE CONTROL SUBSTANCE:
- Positive control substance: KOH, 8N
- Amount(s) applied: 50 µL

Duration of treatment / exposure:

3 min and 1 h

Observation period:

Not applicable

Number of animals:

The test was performed in duplicates for each test or control group and treatment period (3 and 60 min).

Details on study design:

TEST SITE
- Area of exposure: 0.6 cm²

REMOVAL OF TEST SUBSTANCE
- The test item was gently rinsed off from the skin models and the tissues were incubated in MTT solution.
- Time after start of exposure: 3 and 60 min

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed. Therefore, tissues were incubated in pre-warmed MTT solution for 3 h at 37 °C and 5% CO2. After aspiration of the MTT solution, tissues were washed 3 times in phosphate buffered saline followed by tissue drying. Extraction of the formazan product was carried out in isopropanol. The optical density was measured at 570 nm wave length in a plate spectrophotometer.

Irritation / corrosion parameter:

other: cell viability (%)

Run / experiment:

3 min

Value:

100

Remarks on result:

other: Basis: mean value of negative controls (distilled water)

Irritation / corrosion parameter:

other: cell viability (% of negative control)

Run / experiment:

3 min

Value:

24

Remarks on result:

other: Basis: mean value of positive controls (8 N KOH)

Irritation / corrosion parameter:

other: cell viability (% of negative control)

Run / experiment:

3 min

Value:

36

Remarks on result:

other: Basis: mean value of the test item

Irritation / corrosion parameter:

other: cell viability (%)

Run / experiment:

1 h

Value:

100

Remarks on result:

other: Basis: mean value of negative controls (distilled water)

Irritation / corrosion parameter:

other: cell viability (% of negative control)

Run / experiment:

1 h

Value:

12

Remarks on result:

other: Basis: mean value of positive controls (8 N KOH)

Irritation / corrosion parameter:

other: cell viability (% of negative control)

Run / experiment:

1 h

Value:

6

Remarks on result:

other: Basis: mean value of the test item

Other effects / acceptance of results:

In comparison to the untreated control (NC) for the test item there was measured a cell viability of 35 percent and 6 percent after a 3 min and 1 h application, respectively. Based on the results of the study the test item should be classified as corrosive.

Table 1: OD values (570 nm) measured for each test sample

Exposure time	Test substances	Tissue	Optical density (OD)					Cell viability % untreated control
			Value 1	Value 2	Value 3	Mean	Mean Tissue 1+2
3 min	Negative control (NC) = distilled water	1	2.007	1.993	2.010	2.003	2.031	100
		2	2,067	2,022	2,074	2,058
	PERLASTAN C	1	0.637	0.617	0.627	0.627	0.731	36
		2	0.852	0.822	0.833	0.836
	Positive control (PC) = 8 N KOH	1	0.545	0.527	0.533	0.535	0.485	24
		2	0.438	0.431	0.436	0.435
1 h	Negative control (NC) = distilled water	1	1.841	1.848	1.853	1.847	1.755	100
		2	1.687	1.640	1.658	1.662
	PERLASTAN C	1	0.127	0.123	0.121	0.124	0.112	6
		2	0.104	0.097	0.098	0.100
	Positive control (PC) = 8 N KOH	1	0.168	0.163	0.191	0.174	0.213	12
		2	0.251	0.250	0.252	0.251

Interpretation of results:

other: Skin corr. 1, H314. Classification according to Regulation (EC) No. 1272/2008 (CLP/EU GHS).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

Two studies addressing the skin irritation/corrosion potential are available with the registered substance Glycine, N-methyl-, N-coco acyl derivs (CAS 68411-97-2).

In the reliable skin irritation study performed according to OECD TG 404 and in compliance with GLP one female Albino rabbit was exposed to 0.5 mL of undiluted test substance onto the clipped skin for 3 and 60 min via semi-occlusive dressing (Frey-Tox, 2004). Skin reactions were evaluated according to the Draize scoring system 1, 24, 48, 72 h and 7, 14, 21 and 28 days post-application. Since only a slight erythema was observed immediately after 60 min post application, the 4 h exposure was started. One hour post application the animal showed severe erythema (grade 4) and edema (grade 2) formation, in order that the 4 h exposure was immediately terminated and a confirmatory test was precluded. 24 to 72 hours post application severe erythema (grade 4) and edema (grade 4) were still observed. Thus, mean erythema and edema scores over 24, 48 and 72 h of 4 and 3 were calculated, respectively. In spite of the reduced exposure of 1 h time and severe skin reactions the observation period was extended up to 28 days. Within this period severe skin reactions could be revealed and no pathological changes were discernible anymore. Based on the study results and according to EU classification criteria, the test substance is classified as corrosive to the skin.

 

In a further study, the in vitro skin corrosion properties of Glycine, N-methyl-, N-coco acyl derivatives (CAS 68411-97-2) were tested in a study performed according to OECD TG 431 under GLP conditions using the EpiDermTM, reconstructed three-dimensional human epidermis (EPI-200) model (Frei-Tox, 2005). In the study, the tissues were treated with the neat test material for exposure periods of 3 and 60 min. Positive and negative controls were included in the study. The corrosive potential of the test material was predicted from the relative mean tissue viabilities compared to the mean viability of the negative control tissues. The positive and negative control substance gave the expected results. The relative mean tissue viabilities of the test compound were 36% after 3 min and 6% after 1 h. Thus, in the described test Glycine, N-methyl-, N-coco acyl derivatives (CAS 68411-97-2) is considered to be corrosive to the skin.

Taken together, the available data on the registered substance indicate that Glycine, N-methyl-, N-coco acyl derivatives (CAS 68411-97-2) is corrosive to the skin.

 

Eye irritation

Regulation (EC) No. 1907/2006 (REACH) stipulates in its Annexes VII and VIII that tests for eye irritation / severe eye damage do not need to be conducted if the registered substance is classified as skin corrosion. Since Glycine, N-methyl-, N-coco acyl derivs (CAS 68411-97-2) is classified as Skin Corr. 1B as a result of adequate in vitro and in vivo data, testing for eye irritation / severe eye damage is deemed not suitable and necessary. As a result of the skin corrosion properties, contact by any route of exposure to the substance must be avoided and effects of the substance on eyes are avoided as well.

Justification for classification or non-classification

The available data on skin irritation / corrosion of Glycine, N-methyl-, N-coco acyl derivs (CAS 68411-97-2) meet the criteria for classification as Skin Corr. 1B (H314) according to Regulation (EC) No. 1272/2008 (CLP).

No data on eye irritation / severe eye damage are available. Studies addressing the eye irritation / severe eye damage potential are not required due to the classification of the registered substance as skin corrosive (Skin Corr. 1B). In consequence, classification of Glycine, N-methyl-, N-coco acyl derivs (CAS 68411-97-2) as Eye Damage 1 (H318) is implicit.