Reaction products of fatty acid chlorides, C8-12 (even numbered) with glycine and sodium hydroxide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-02-21 to 2012-05-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und dLebensmittelsicherheit, München, Germany)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium cocoyl glycinate (SCG) [INCI]

IUPAC Name:

Sodium cocoyl glycinate (SCG) [INCI]

Details on test material:

Chemical name: Glycine, N-coco acyl derivs., sodium salts

Test system

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)

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

242

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Hostapon SG (70% Sodium N-Cocoyl Clycinate) is eye damaging according to the results obtained in BCOP study.

Executive summary:

Hostapon SG (70% N-cocoyl glycinate) was investigated for its eye irritation property according to the Guideline OECD 437. The obtained IVIS of 242.15 is indicative that Hostapon SG is eye damaging.