fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide

Endpoint summary

Description of key information

Skin irritation:

The skin irritation property of the registration substance, sodium N-lauroyl glutamate, is derived based on the read-across to sodium N-cocoyl glutamate and sodium N-cocoyl glycinate.

The registration substance and the read-across sources are amides of fatty acids and amino acids and can be characterized as "N-fatty acyl amino acids", of which endogeous occurence and metabolism are known. Based on the comparable chemical structure, comparable phys-chem data and expected comparable metabolism, these compound are likely exhibit comparable toxicity profiles.

Sodium N-cocoyl glutamate is investigated for its skin irritation property according to the OECD 404. 0.5 ml of the test material was applied to the rabbit skin semi occusively for 4 hours. Slight erythema was found in two animals out of three and these effect were reverseible. Sodium N-cocoyl glutamate is not skin irritating.

Sodium N-cocoyl glycinate is investigated for its skin irritation property according to the OECD 404. 0.5 ml of the test material was applied to the rabbit skin semi occusively for 4 hours. Well-defined and mild erythea and edema were found in all treated animals and these effects were reversible. Sodium N-cocoyl glycinate is not skin irritating.

Sodium N-cocoyl glycinate was investigated for its skin irritation property according to the OECD 439. 10 mg of test compound was applied topically to the EPISKIN-SM tissue for 15 minutes followed by a 42 hour post incubation period and immediate determination of cytotoxic effects via MTT reduction assay. No significant effect on the cell viability was obtained. Sodium N-cocoyl glycinate is not skin irritating.

Likewise the registration substance, sodium N-lauroyl glutamate is considered as not skin irritating.

Eye irritation:

The eye irritation property of the registration substance, sodium N-lauroyl glutamate, is derived based on the read-across to sodium N-cocoyl glutamate and sodium N-cocoyl glycinate.

The registration substance and the read-across sources are amides of fatty acids and amino acids and can be characterized as "N-fatty acyl amino acids", of which endogeous occurence and metabolism are known. Based on the comparable chemical structure, comparable phys-chem data and expected comparable metabolism, these compound are likely exhibit comparable toxicity profiles.

Sodium N-cocoyl glutamate was investigated for its eye irritation property according to the OECD 405. 0.1 ml of the test material was applied into the eye of rabbits. Three animals were used. In all treated animals clear irritating effects were found and these effects were reversible within 14 days. It is an eye irritant.

Sodium N-cocoyl glycinate was investigated for its eye irritation property according to the Guideline OECD 405. The test material, 25% sodium N-cocoyl glycinate, was applied to eyes of rabbits. Three animals were used. In all treated animals clear irritating effects were found and these effects were reversible within 14 days. It is an eye irritant.

Sodium N-cocoyl glycinate was investigated for its eye irritation property according to the Guideline OECD 437. The test material was consisted of 68% sodium N-cocoyl glycinate and 20% sodium chloride. The obtained results were indicative of severely irritating effect of the test material. However, due to the high salt content, it is not clear whether the obtained results is to be applied specifically for the sodium N-cocoyl glycinate.

Likewise, the registration substance, sodium N-lauroyl glutamate is considered as an eye irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2012-02-28 to 2012-03-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EU Method B.46 (Skin Irritation)

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

EPISKIN-SM (TM) reconstructed human epidermis model (SkinEthic)

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 mg + 5 µL aqua dest.

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 +/- 1 h

Number of replicates:

three

Irritation / corrosion parameter:

% tissue viability

Value:

112

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

 

Name	Negative Control			Positive Control			Test Item
Tissue	1	2	3	1	2	3	1	2	3
absolute OD550	1.325	1.338	1.225	0.144	0.135	0.138	1.471	1.394	1.475
	1.260	1.311	1.196	0.143	0.131	0.144	1.408	1.386	1.418
blank-corrected OD550	1.282	1.295	1.182	0.102	0.092	0.095	1.428	1.351	1.432
	1.217	1.268	1.153	0.100	0.089	0.101	1.365	1.343	1.375
mean OD550of the duplicates (blank-corrected)	1.250	1.282	1.168	0.101	0.090	0.098	1.397	1.347	1.404
total mean OD550of 3 replicate tissues (blank-corrected)	1.233^*			0.096			1.383
SD OD550	0.06			0.01			0.04
relative tissue viabilities [%]	101.4	103.9	94.7	8.2	7.3	8.0	113.3	109.3	113.8
mean tissue viability [%]	100.0			7.8^**			112.1
SD tissue viability [%]^***	4.8			0.4			2.5
CV [% viability]	4.8			5.6			2.2

^*          Corrected mean OD₅₅₀of the negative control corresponds to 100% absolute tissue viability.

^**      mean relative tissue viability of the three positive control tissues is£ 40%

^***     the standard deviation (SD) obtained from the three concurrently tested tissues is < 18%

Interpretation of results:

GHS criteria not met

Conclusions:

Sodium N-cocoyl glycinate was investigated for its skin irritation property according to the Guideline OECD 439. It is not skin irritating.

Executive summary:

Sodium N-cocoyl glycinate was investigated for its skin irritation property according to the OECD 439. 10 mg of test compound was applied topically to the EPISKIN-SM tissue for 15 minutes followed by a 42 hour post incubation period and immediate determination of cytotoxic effects via MTT reduction assay. No significant effect on the cell viability was obtained. Sodium N-cocoyl glycinate is not skin irritating.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989

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:

New Zealand White

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

0.5 ml

Duration of treatment / exposure:

4h

Observation period:

7 d

Number of animals:

3

Irritation parameter:

erythema score

Basis:

mean

Remarks:

observations in three animals

Time point:

24/48/72 h

Score:

0.56

Max. score:

1

Reversibility:

fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

mean

Remarks:

observations in three animals

Time point:

24/48/72 h

Score:

0

Max. score:

0

Table: Skin irritation scores
Observation Time	Erythema			Oedema
	Animal 1	Animal 2	Animal 3	Animal 1	Animal 2	Animal 3
1h	1	0	1	0	0	0
24h	1	0	1	0	0	0
48h	1	0	1	0	0	0
72h	1	0	0	0	0	0
7 day	0	0	0	0	0	0
Mean of 24/48/72h	1	0	0.67	0	0	0

Interpretation of results:

GHS criteria not met

Conclusions:

Sodium N-cocoyl glutamate was investigated for its skin irritation property according to the Guideline OECD 404. It is not irritating.

Executive summary:

Sodium N-cocoyl glutamate was investigated for its skin irritation property according to the OECD 404. 0.5 ml of the test material was semi occusively applied to the clipped skin area of rabbits for 4 hours. Erythema of score 1 was found in two animals (out of three) which disappeard within 7 days. No oedema was found. Sodium N-cocoyl glutamate is not skin irritating.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

assessment report

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands BV
- Age at study initiation: 14 weeks (males, females)
- Weight at study initiation: 2.2 kg (male); 2.5 kg (females)
- Housing: individually in stainless steel cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10 - 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hour cycle dark / light

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL / animal
- Concentration (if solution): undiluted

Duration of treatment / exposure:

4 hour

Observation period:

14 days

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: approx. 10 cm x 10 cm
- % coverage: 100
- Type of wrap if used: surgical gauze patch

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 4 hours

SCORING SYSTEM: Irritation scoring according to Council Regulation (EC) No 440/2008

Irritation parameter:

erythema score

Basis:

mean

Remarks:

all animals

Time point:

24/48/72 h

Score:

1.67

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

animal #1

Remarks:

mean

Time point:

24/48/72 h

Score:

1.33

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

animal #2

Remarks:

mean

Time point:

24/48/72 h

Score:

1.67

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

animal #3

Remarks:

mean

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

mean

Remarks:

all animals

Time point:

24/48/72 h

Score:

1.11

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #1

Remarks:

mean

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #2

Remarks:

mean

Time point:

24/48/72 h

Score:

1.33

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #3

Remarks:

mean

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible

Other effects:

Scaling noted

Table: Skin irritation scores
Observation Time	Erythema			Oedema
	Animal 1	Animal 2	Animal 3	Animal 1	Animal 2	Animal 3
1h	2	2	2	1	2	1
24h	2	2	2	1	2	1
48h	1	2	2	1	1	1
72h	1	1	2	1	1	1
7 day	0	1	0	0	0	0
10 day	0	0	0	0	0	0
14 day	0	0	0	0	0	0
Mean of 24/48/72h	1.33	1.67	2	1	1.33	1

Interpretation of results:

GHS criteria not met

Conclusions:

Sodium N-cocoyl glycinate was investigated for its skin irritation property according to the Guideline OECD 404. It is not skin irritating.

Executive summary:

Sodium N-cocoyl glycinate was investigated for its skin irritation property according to the OECD 404. 0.5 ml of the test material was semi occusively applied to the clipped skin area of rabbits for 4 hours. Three animals were treated. Mild erythema and edema was found in all treated animals and these effects disappeard within 14 days. Sodium N-cocoyl glycinate is not skin irritating.

Reference 4

Endpoint:

skin irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Justification for read-across is provided in chapter 13 as separate statement

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands BV
- Age at study initiation: 14 weeks (males, females)
- Weight at study initiation: 2.2 kg (male); 2.5 kg (females)
- Housing: individually in stainless steel cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10 - 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hour cycle dark / light

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL / animal
- Concentration (if solution): undiluted

Duration of treatment / exposure:

4 hour

Observation period:

14 days

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: approx. 10 cm x 10 cm
- % coverage: 100
- Type of wrap if used: surgical gauze patch

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 4 hours

SCORING SYSTEM: Irritation scoring according to Council Regulation (EC) No 440/2008

Irritation parameter:

erythema score

Basis:

mean

Remarks:

all animals

Time point:

24/48/72 h

Score:

1.67

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

animal #1

Remarks:

mean

Time point:

24/48/72 h

Score:

1.33

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

animal #2

Remarks:

mean

Time point:

24/48/72 h

Score:

1.67

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

animal #3

Remarks:

mean

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

mean

Remarks:

all animals

Time point:

24/48/72 h

Score:

1.11

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #1

Remarks:

mean

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #2

Remarks:

mean

Time point:

24/48/72 h

Score:

1.33

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #3

Remarks:

mean

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible

Other effects:

Scaling noted

Table: Skin irritation scores
Observation Time	Erythema			Oedema
	Animal 1	Animal 2	Animal 3	Animal 1	Animal 2	Animal 3
1h	2	2	2	1	2	1
24h	2	2	2	1	2	1
48h	1	2	2	1	1	1
72h	1	1	2	1	1	1
7 day	0	1	0	0	0	0
10 day	0	0	0	0	0	0
14 day	0	0	0	0	0	0
Mean of 24/48/72h	1.33	1.67	2	1	1.33	1

Interpretation of results:

GHS criteria not met

Conclusions:

The skin irritation property of the registration substance is assessed based on the read-across to Sodium N-cocoyl glycinate.
Sodium N-cocoyl glycinate was investigated for its skin irritation property according to the Guideline OECD 404. It is not skin irritating.
Likewise the registration substance is not skin irritating.

Executive summary:

 

The skin irritation property of the registration substance is assessed based on the read-across to Sodium N-cocoyl glycinate.

Sodium N-cocoyl glycinate was investigated for its skin irritation property according to the OECD 404. 0.5 ml of the test material was semi occusively applied to the clipped skin area of rabbits for 4 hours. Three animals were treated. Mild erythema and edema was found in all treated animals and these effects disappeard within 14 days. Sodium N-cocoyl glycinate is not skin irritating.

Likewise, the registration substance is not skin irritant. 

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:

supporting study

Study period:

1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

0.1 ml

Duration of treatment / exposure:

single application

Observation period (in vivo):

14 days

Number of animals or in vitro replicates:

3

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

observations in three treated animals

Time point:

24/48/72 h

Score:

1.78

Max. score:

3

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Basis:

mean

Remarks:

observations in three treated animals

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

observations in three treated animals

Time point:

24/48/72 h

Score:

2.89

Max. score:

3

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

observation in three treated animals

Time point:

24/48/72 h

Score:

2.44

Max. score:

3

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Eye Irritation Scores
Reading time	Animal	Opacity	Iris	Conjunctivae
				Redness	Chemosis
1h	1	^d	1	2	3
	2	^d	1	2	2
	3	^d	1	2	2
24 hours	1	1	1	3	3
	2	1	1	3	2
	3	1	1	3	3
48 hours	1	2	1	3	2
	2	2	1	3	3
	3	2	1	3	3
72 hours	1	2	1	2	1
	2	3	1	3	2
	3	2	1	3	3
7 day	1	0	0	0	0
	2	1^v	1	2	1
	3	0	1	1^c	1
14 day	1	0	0	0	0
	2	0	0	0^c	0
	3	0	0	0^c	0
d = dulling of the normal lustre of the corneal surface v = vascularization of the cornea c = convulted eyelids

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Sodium N-cocoyl glutamate was investigated for its eye irritation property according to the Guideline OECD 405. It is eye irritating.

Executive summary:

Sodium N-cocoyl glutamate was investigated for its eye irritation property according to the OECD 405. 0.1 ml of the test material was applied into the eye of rabbits. The obtained mean scores in three treated animals were 1.8 for corneal opacity, 1.0 for iris lesion, 2.9 for redness of conjuctivae and 2.4 for chemosis of conjuctivae. All the effects were reversible within 14 days. Sodium N-cocoyl glutamate is eye irritating.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2012-02-21 to 2012-05-25

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

unsuitable test system

Remarks:

Comments on the reliability of the obtained results with regard to hazard assessment of sodium cocoyl glycinate: - The test material contained 20% NaCl. - The applied test design did not include the treatment of the test system only with 20% NaCL. - The applied test system (ex-vivo) is a closed system and therefore is likely to be more susceptible to unspecific physical effects such as osmotic effect. Although the applied test system is to be regarded as valid for the test material, it may not valid for sodium cocoyl glycinate.

Qualifier:

according to guideline

Guideline:

other: OECD Guidline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants), adopted: 7 September 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und dLebensmittelsicherheit, München, Germany)

Vehicle:

physiological saline

Amount / concentration applied:

The test item was diluted with physiological saline 0.9% NaCl to gain a 20% concentration.

Duration of treatment / exposure:

750 microL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method).
After 4 hours ± 5 minutes incubation at 32 +/- 1 °C either the test substance or the control substance was removed.

Details on study design:

Test System

Preparation of the Corneas:
The assay uses isolated corneas obtained as a by-product from an abattoir from freshly slaughtered animals
(from Attenberger Fleisch GmbH & Co. KG).
On the test day, fresh eyes were collected from the slautherhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories.
Immediately after arrival of the eyes, cornea preparation was initiated.
The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera.
The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders
(MC2, Clermont, France) with the endothelial side against the O-ring of the posterior chamber, they had been visually
examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top
of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red)
containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were
incubated for one hour at 32 +/- 1 °C in a water bath.

Calibration of the Opacitometer:
The opacitometer had been switched on 15 min before the calibration procedure was started. Empty cornea holders were
placed into the opacitometer and the readout was adjusted to zero using the “BAL”-turning knob. For calibration the polyester
foil no. 1 was introduced into the test chamber and the readout was adjusted to 75 using the “CAL”-turning knob. To test the
linearity of the measurement, two additional calibration foils, polyester foil no. 2 and polyester foil no. 3, were measured.
For these, the opacitometer was supposed to display 150 and 225, respectively (± 3%). If this had not been the case,
the calibration procedure would have had to be repeated. The calibration procedure was performed before each test
and was documented in the raw data.

Treatment of the Corneas:
After the equilibration period, the medium was removed from both chambers and replaced with fresh Complete RPMI.
An initial opacity measurement was performed on each of the corneas using an opacitometer (MC2, Clermont, France).
Three corneas with opacity readings approximately equivalent to the median opacity of all corneas were selected as
negative-control corneas. The opacity of each cornea was read against an air-filled chamber and recorded. Corneas
that have an initial opacity reading above 7 units were not dosed. The medium was removed from the anterior chamber
and replaced with the test item or control.
750 microL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method).
After 4 hours ± 5 minutes incubation at 32 +/- 1 °C either the test substance or the control substance was removed
and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance,
the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI
and an opacity measurement was performed.
After the opacity measurement the medium was removed from both chambers of the holder. The posterior chamber was
refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and
the corneas were incubated for 90 minutes at 32 +/- 1 °C. Then the medium from the posterior chamber was removed and
its optical density at 490 nm (OD490) was determined, using a spectrophotometer.

Test Groups:
3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive control treated with imidazole 20% in physiological saline 0.9% NaCl
The BCOP assay is considered to be valid if the in vitro score obtained with the positive control falls within the two
standard deviations of the current historical mean.

Evaluation of Results:
The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading.
These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas.
The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.
The mean OD490 for the blank wells were calculated. The mean blank OD490 was subtracted from the OD490 of each well (corrected OD490).
Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500),
were taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test article
and the positive control were calculated by subtracting the average corrected OD490 of the negative control corneas from the corrected
OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490
The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for
that treatment condition.
The following formula was used to determine the in vitro score:
In vitro score = mean opacity value + (15 x mean OD490 value)

Irritation parameter:

in vitro irritation score

Value:

242.15

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Table2:  Opacity

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1	Negative	4	5	1
2	Control	4	4	0
3		4	5	1
MV		4.00	4.67	0.67
4	Positive	5	229	224	223.33
5	Control	5	168	163	162.33
6		4	216	212	211.33
MV		4.67	204.33	199.67	199.00
7	Test Item	3	187	184	183.33
8		3	283	280	279.33
9		3	172	169	168.33
MV		3.00	214.00	211.00	210.33

 

Table3:  Permeability

Cornea No.	Test Item	OD490	Corrected OD490 Value
1	Negative	0.020
2	Control	0.016
3		0.034
MV		0.023
4	Positive	2.114	2.091
5	Control	2.006	1.983
6		1.944	1.921
MV		2.021	1.998
7	Test Item	2.146	2.123
8		2.140	2.117
9		2.148	2.125
MV		2.145	2.121

 

Table4:  In VitroIrritation Score

Cornea No.	Test Item	Corrected Opacity Value	Corrected OD490 Value	IVIS
1	Negative	1.00	0.020
2	Control	0.00	0.016
3		1.00	0.034
MV		0.67	0.023	1.02
4	Positive	223.33	2.091
5	Control	162.33	1.983
6		211.33	1.921
MV		199.00	1.998	228.97
7	Test Item	183.33	2.123
8		279.33	2.117
9		168.33	2.125
MV		210.33	2.121	242.15

 

Interpretation of results:

study cannot be used for classification

Conclusions:

Sodium N-cocoyl glycinate was investigated for its eye irritation property according to the Guideline OECD 437. The obtained IVIS score was indicative of severely irritating property. However, due to the high salt content of the test material (20% sodium chloride), it is not clear whether the obtained results is to be applied specifically for the sodium N-cocoyl glycinate.

Executive summary:

Sodium N-cocoyl glycinate was investigated for its eye irritation property according to the Guideline OECD 437. The test material was consisted of 68% sodium N-cocoyl glycinate and 20% sodium chloride. Three bovine corneas were treated with 750 µl test material for 4 hours. The mean opacity score of 199 and mean permeability score of 2.12 were obtained, the corresponding mean IVIS score being 242. However, due to the high salt content, it is not clear whether the obtained results is to be applied specifically for the sodium N-cocoyl glycinate.

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study according to GLP

Reason / purpose for cross-reference:

assessment report

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands BV
- Age at study initiation: 15 weeks (male), 16 weeks (females)
- Weight at study initiation: 2.4 kg (male), 2.5 - 2.6 kg (females)
- Housing: individually in stainless steel cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10 - 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hour dark / light cycle

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 24 hours

SCORING SYSTEM: numerical scoring system according Council Regulation (EC) 440/2008

TOOL USED TO ASSESS SCORE: fluorescein, diagnostic-lamp

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

all animals

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no signs of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no signs of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no signs of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Remarks:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no signs of irritation

Irritation parameter:

iris score

Basis:

mean

Remarks:

all animals

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no signs of irritation

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no signs of irritation

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no signs of irritation

Irritation parameter:

iris score

Basis:

animal #3

Remarks:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no signs of irritation

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

all animals

Time point:

24/48/72 h

Score:

1.77

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal #1

Remarks:

mean

Time point:

24/48/72 h

Score:

1.67

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal #2

Remarks:

mean

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal #3

Remarks:

mean

Time point:

24/48/72 h

Score:

1.67

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

all animals

Time point:

24/48/72 h

Score:

1.77

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

mean

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

mean

Time point:

24/48/72 h

Score:

1.33

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #3

Remarks:

mean

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible

Eye Irritation Scores
Reading time	Animal	Opacity	Iris	Conjunctivae
				Redness	Chemosis
1h	1	^d	1	2	3
	2	^d	1	2	2
	3	^d	1	2	2
24 hours	1	1	1	3	3
	2	1	1	3	2
	3	1	1	3	3
48 hours	1	2	1	3	2
	2	2	1	3	3
	3	2	1	3	3
72 hours	1	2	1	2	1
	2	3	1	3	2
	3	2	1	3	3
7 day	1	0	0	0	0
	2	1^v	1	2	1
	3	0	1	1^c	1
14 day	1	0	0	0	0
	2	0	0	0^c	0
	3	0	0	0^c	0
d = dulling of the normal lustre of the corneal surface v = vascularization of the cornea c = convulted eyelids

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Sodium N-cocoyl glycinate was investigated for its eye irritation property according to the Guideline OECD 405. It induced adverse effects in eyes that were reversible within 14 days. It is irritating to eyes.

Executive summary:

Sodium N-cocoyl glycinate was investigated for its eye irritation property according to the Guideline OECD 405. The test material, 25% sodium N-cocoyl glycinate, was applied to eyes of rabbits. Three animals were used. In all treated animals clear irritating effects were found and these effects were reversible within 14 days. It is an eye irritant.

Reference 4

Endpoint:

eye irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study according to GLP

Justification for type of information:

Justification for read-across is provided in chapter 13 as separate statement

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands BV
- Age at study initiation: 15 weeks (male), 16 weeks (females)
- Weight at study initiation: 2.4 kg (male), 2.5 - 2.6 kg (females)
- Housing: individually in stainless steel cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10 - 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hour dark / light cycle

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 24 hours

SCORING SYSTEM: numerical scoring system according Council Regulation (EC) 440/2008

TOOL USED TO ASSESS SCORE: fluorescein, diagnostic-lamp

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

all animals

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no signs of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no signs of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no signs of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Remarks:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no signs of irritation

Irritation parameter:

iris score

Basis:

mean

Remarks:

all animals

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no signs of irritation

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no signs of irritation

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no signs of irritation

Irritation parameter:

iris score

Basis:

animal #3

Remarks:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no signs of irritation

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

all animals

Time point:

24/48/72 h

Score:

1.77

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal #1

Remarks:

mean

Time point:

24/48/72 h

Score:

1.67

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal #2

Remarks:

mean

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal #3

Remarks:

mean

Time point:

24/48/72 h

Score:

1.67

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

all animals

Time point:

24/48/72 h

Score:

1.77

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

mean

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

mean

Time point:

24/48/72 h

Score:

1.33

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #3

Remarks:

mean

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible

Eye Irritation Scores
Reading time	Animal	Opacity	Iris	Conjunctivae
				Redness	Chemosis
1h	1	^d	1	2	3
	2	^d	1	2	2
	3	^d	1	2	2
24 hours	1	1	1	3	3
	2	1	1	3	2
	3	1	1	3	3
48 hours	1	2	1	3	2
	2	2	1	3	3
	3	2	1	3	3
72 hours	1	2	1	2	1
	2	3	1	3	2
	3	2	1	3	3
7 day	1	0	0	0	0
	2	1^v	1	2	1
	3	0	1	1^c	1
14 day	1	0	0	0	0
	2	0	0	0^c	0
	3	0	0	0^c	0
d = dulling of the normal lustre of the corneal surface v = vascularization of the cornea c = convulted eyelids

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The eye irritation property of the registration substance is assessed based on the read-across to Sodium N-cocoyl glycinate.
Sodium N-cocoyl glycinate was investigated for its eye irritation property according to the Guideline OECD 405. It induced adverse effects in eyes that were reversible within 14 days. It is irritating to eyes.
Likewise, the registration substance is irritating to eyes.

Executive summary:

The eye irritation property of the registration substance is assessed based on the read-across to Sodium N-cocoyl glycinate.
Sodium N-cocoyl glycinate was investigated for its eye irritation property according to the Guideline OECD 405. It induced adverse effects in eyes that were reversible within 14 days. It is irritating to eyes.
Likewise, the registration substance is irritating to eyes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Justification for classification or non-classification

The skin irritation property of the registration substance sodium N-lauroyl glutamate is derived based on the read-across to Sodium N-cocoyl glutamate and Sodium N-cocoyl glycinate. No skin irritating property can be reliably derived for the registration substance and therefore no classification is assigned.

The eye irritation property of the registration substance sodium N-lauryl glutamate is derived based on the read-across to Sodium N-cocoyl glutamate and Sodium N-cocoyl glycinate. Sodium N-lauroyl glutamate is expected to be an eye irritant. It should be classified as Category 2 based on GHS criteria.