Fatty acids, tall-oil, reaction products with 2-[(2-aminoethyl)amino]ethanol

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-09-25 to 2013-01-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD 439 and EU method B.46 in a GLP certified testing facility.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 microliter
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 microliter
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 microliter
- Concentration (if solution):

Duration of treatment / exposure:

35 minutes at 37°C

Number of replicates:

3 for each sample and control

Irritation / corrosion parameter:

% tissue viability

Remarks:

formation of formazan from MTT was determined being a measure of cell viability.

Run / experiment:

1

Value:

3.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

formation of formazan from MTT was determined being a measure of cell viability.

Run / experiment:

2

Value:

3.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

4.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value refers to % formazan production

Blank:

As blank, the optical density of isopropanol was measured in eight wells of the 96-wellplate.

Mean value:0.061

 

Table 1: Absorption values of the negative control, test item and positive control (OD at 570 nm)

Tissue no.	Measurement	Negative Control	400112	Positive Control
1	1	2.132	0.429	0.214
	2	2.151	0.425	0.211
2	1	2.044	0.440	0.215
	2	2.084	0.445	0.216
3	1	2.028	0.452	0.222
	2	2.044	0.469	0.225

 

From the measured absorption values, the mean of each tissue was calculated. From the mean absorption values the mean absorption of isopropanol (0.061) was subtracted. Mean and relative standard deviations (comparison of the three tissues) were also calculated.

Table 2: Mean absorption values subtracted by the mean absorption of the blank

Tissue no.	Negative Control	400112	Positive Control
1	2.081	0.366	0.152
2	2.003	0.382	0.155
3	1.975	0.400	0.163
Mean of the three tissues	2.020	0.383	0.157
Relative standard deviation of the three tissues	2.7%	4.4%	3.6%

 

Table 3: Mean absorption values of freeze killed tissues of negative control and test item (OD at 570 nm)  

Tissue no.	Negative Control	400112
1	0.093	0.380
2	0.087	0.400
3	0.088	0.399
Mean of the three tissues	0.090	0.393

 

The mean OD of the untreated freeze-killed tissue is subtracted from the mean OD of the treated freeze-killed tissue (= 0.304). This difference is then subtracted from the OD of the test item in the test. The calculated value is the “OD corrected”.

Corrected Values Viable Tissues

The absorption values of the negative and positive control and the corrected absorption values of the test item are given in the following table:

Table 4: Mean absorption values

Tissue no.	Negative Control	400112 (corrected)	Positive Control
1	2.081	0.062	0.152
2	2.003	0.078	0.155
3	1.975	0.096	0.163
Mean of the three tissues	2.020	0.079	0.157
Relative standard deviation of the three tissues	2.7%	21.3%	3.6%

 

Comparison of Formazan Production

For the test item and the positive control, the following percentage values of formazan production were calculated in comparison to the negative control:

Table 5: % Formazan production

Tissue no.	400112	Positive Control
1	3.1%	7.5%
2	3.9%	7.7%
3	4.8%	8.1%
Mean of % formazan production	3.9%	7.8%

 

 

Interpretation of results:

other: irritant according to EU regulation

Conclusions:

400112 is considered irritant in the Human Skin Model Test.

Executive summary:

To determine the Skin Irritation Potential of 400112 the Human Skin Model Test following EU-Method 8.46 resp. OECD 439 was conducted.

Three tissues of the human skin model EpiDerm™ were treated with the test item 400112 for 60 min. 30 µL of the liquid lest item were applied to each tissue (using a nylon mesh) and spread to match the tissue size (0.63 cm²). DPBS-buffer was used as negative control, 5% SDS-solution was used as positive control.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of 1.0 < mean OD < 2.5. The positive control showed clear irritating effects. Variation within tissues was acceptable for the positive and negative control (< 18%).

After the treatment with the test item 400112, the relative absorbance values were reduced to 3.9%. This value is well below the threshold for irritation potential (50%).

Therefore, 400112 is considered as irritant in the Human Skin Model Test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation, other

Remarks:

in vivo test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-10-05 to 2012-11-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD guideline 405 and EU method B.5 in a GLP-certified testing facility.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: 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:

One male albino New Zealand rabbit was supplied by the Elevage de Gérome (Quartier Labaste -F40260 Linxe).
Acclimatisation period: minimal 5-days
Age at the beginning of the test: 14 weeks
Body weight during the test: between 2.89 kg and 3.12 kg.
The animal was identified prior to inclusion in the test by means of a numbered ring on the edge of
one ear.
Housing: individual box installed in conventional air conditioned animal husbanding:
Temperature: 17 to 23°C
Relative humidity: 30 to 70%
Rate of air exchange: approximately between 10-15 changes per hour
Lighting: 12 h continuous light (07.00 to 19.00) and 12 h darkness.
Drinking water (tap-water from public distribution system) and foodstuff (SAFE- 112) were supplied freely.

Vehicle:

unchanged (no vehicle)

Controls:

other: the other eye of of the animal remained untreated serving as control

Amount / concentration applied:

0.1 mL of test item was used, as supplied.

Duration of treatment / exposure:

3 days

Observation period (in vivo):

3 days

Number of animals or in vitro replicates:

1

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

other: mean of scores at examinations after 1 h, 48 h and 72 h

Score:

3.7

Max. score:

4

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

other: mean of scores at examinations after 1 h, 48 h and 72 h

Score:

2

Max. score:

2

Remarks on result:

other: examination of "redness"

Irritation parameter:

iris score

Remarks on result:

other: The iris was not visible between day 0 (1 hour after instillation) and day 3.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

other: mean of scores at examinations after 1 h, 48 h and 72 h

Score:

4

Max. score:

4

Other effects:

White secretions requiring a physiological rinse was noted on days 2 and 3.
A brown coloration of the nictitating membrane was noted 1 hour after the test item application.
For ethical reasons, the animal was euthanized on day 3.

Animal n°	Time aftertreatment	CONJUNCTIVAE		IRIS	CORNEA
		CHEMOSIS	REDNESS	LESION	OPACITY
A2405	24h	3	2		4
	48h	4	2		4
	72h	4	2		4
TOTAL		11	6		12
Mean		3.7	2.0		4.0

Interpretation of results:

other: Category 1, causes serious eye damage according to EU regulation

Conclusions:

Based on the results of this in vivo test the test item 400112 must be classified according to Regulation (EC) No. 1272/2008 in category 1 "serious eye damage".

Executive summary:

The test item 400112 was instilled as supplied, into the eye of one New Zealand rabbit at the dose of 0.1 mL. The experimental protocol was established on the basis of the official method as defined in the OECD guideline No. 405 dated April 24^th, 2002 and the test method B.5 of the council regulation No. 440/2008.

The ocular reactions observed during the study have been moderate to severe and were not reversible.

For ethical reasons, the animal was euthanized on day 3.

In accordance with the Regulation (EC) No. 1272/2008, the test item must be classified in category 1 "causes serious eye damage".

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the in vitro skin irritation test, 400112 is must be classified in category 2 "Causes skin irritation".

Based on the results of the in vivo eye irritation test, 400112 must be classified in category 1 "causes serious eye damage" according to Regulation (EC) No. 1272/2008.