Fatty acids, tall-oil, reaction products with pentaethylenehexamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012/13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

April 13, 2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 22, 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, tall-oil, reaction products with pentaethylenehexamine

EC Number:

288-927-9

EC Name:

Fatty acids, tall-oil, reaction products with pentaethylenehexamine

Cas Number:

85940-40-5

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

Fatty acids, tall-oil, reaction products with pentaethylenehexamine

Test material form:

liquid: viscous

Details on test material:

viscous liquid, UVCB

In vitro test system

Test system:

human skin model

Remarks:

EpiDerm™ 200

Source species:

human

Cell type:

other: human-derived epidermal keratinocytes

Cell source:

other: commercially available kit (EpiDerm™ 200)

Source strain:

other: not applicable

Details on animal used as source of test system:

not applicable

Justification for test system used:

Based on the results of ECVAM (European Center for Validation of Alternative Methods) funded validation studies, it was concluded by the ECVAM Scientific Advisory Committee that the EpiDerm™ human epidermis model is suitable to be used for distinguishing between corrosive and non-corrosive chemicals (ECVAM: ESAC statement on the application of the EpidermTM human skin model for skin corrosivity testing of 14-15 Mar 2000) as well as between irritant and non-irritant chemicals (ECVAM: ESAC statement on the scientific validity of in-vitro tests for skin irritation testing of 5 Nov 2008).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ 200
- Tissue batch number(s): not specified
- Production date: not specified
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: 25 Sep 2012

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 min at room temperature or 1 h at 37 °C (corrosion test); 25 min at room temperature followed by 35 min at 37 °C (irritation test)
- Temperature of post-treatment incubation (if applicable): 42 h at 37 °C (irritation test)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 1 washing step, volume not specified
- Observable damage in the tissue due to washing: not specified
- Modifications to validated SOP: not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL assay medium
- Incubation time: 3 h
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm (OD570)
- Filter: no reference filter (not further specified)
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: not specified
- Barrier function: not specified
- Morphology: not specified
- Contamination: not specified
- Reproducibility: These historical control data demonstrate the reproducibility of results and robustness of the procedures.

NUMBER OF REPLICATE TISSUES: 2 (corrosion test); 3 (irritation test)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues (if applicable): Epi-200 tissue was killed by freezing at –20 °C
- N. of replicates: 1
- Method of calculation used: In case of direct reduction of MTT by the test substance, the OD570 values measured in the freeze-killed control tissues (KC) would be used to correct the mean OD570 of the testsubstance treated tissues (mean OD570 KC corrected). Since killed tissue might still have a residual enzyme activity that is able to produce some formazan net OD570 KC is calculated by subtracting the OD570 KC of the NC from the OD570 KC of the test substance. In case the net OD570 KC is greater than 0.1 it is subtracted from the respective mean OD570 to result in the mean OD570 KC corrected. The mean OD570 KC corrected represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 (corrosion test); 2 (irritation test)

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
- The test substance is considered to be irritating to skin if the viability is less than or equal to 50%.
- The test substance is considered to be non-irritating to skin if the viability is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

other: MTT-reduction control (De-ionized water or test substance (corrosion test); PBS or test substance (irritation test)

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL (corrosion test); 30 µL (irritation test)
- Concentration (if solution): undiluted test substance

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL (corrosion test); 30 µL (irritation test)
- Concentration (if solution): not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL (corrosion test); 30 µL (irritation test)
- Concentration (if solution): 8 n (corrosion test); 5 % (irritation test)

Duration of treatment / exposure:

Corrosion test: 3 min at room temperature or 1 h at 37 °C

Irritation test: 25 min at room temperature followed by 35 min at 37 °C

Duration of post-treatment incubation (if applicable):

Irritation test: 42 hours post-incubation period at 37 °C

Number of replicates:

see above

Test system

Vehicle:

unchanged (no vehicle)

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Corrosion test, 3 min exposure

Value:

113

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Corrosion test, 1 h exposure

Value:

82

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Irritation test, 1st test run

Value:

65

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Irritation test, 2nd test run

Value:

99

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: not reported
- Direct-MTT reduction: The test substance was able to reduce MTT directly. Therefore an additional MTT reduction control (KC) was introduced (performed with the corrosion test and the 2nd test run of the irritation test). As the MTT-reduction control was used for calculation of the 1-hour exposure in the corrosion test, an influence of the test substance due to direct MTT reduction had to be excluded for the irritation test. Therefore a 2nd test run of the irritation test was performed with an additional MTT reduction control. The ability of direct MTT reduction did not impair the study result as demonstrated by the concurrently performed exposure of control tissues inactivated by freezing used during the 2nd test run.
- Colour interference with MTT: not reported

DEMONSTRATION OF TECHNICAL PROFICIENCY: Based on the historical data, a profound experience with the assay is assumed

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values: see tables 4 and 5

Any other information on results incl. tables

Table 1: Results of the corrosion test

		Exposure: 3 min				Exposure: 1 hour
Test substance		tissue 1	tissue 2	KC	mean	tissue 1	tissue 2	KC	mean*
NC	mean OD570	1.902	1.691	0.254	1.797	2.253	2.118	0.250	2.185
	viability [% of NC]	105.9	94.1	-	100	103.1	96.9	-	100
Test substance	mean OD570	2.041	2.010	0.335	2.025	1.943	1.950	0.412	1.785
	viability [% of NC]	113.6	111.9	-	113	88.9	89.2	-	82
PC	mean OD570	0.185	0.277	-	0.231	0.110	0.106	-	0.108
	viability [% of NC]	10.3	15.4	-	13	5.0	4.9	-	5

* The mean for the test substance after 1 hour exposure is given after KC-correction.

The result of the KC did not indicate an increased MTT reduction at the exposure period of 3 minutes (difference to KC of NC is not greater than 0.1). Thus the KC was not used for viability calculation for this exposure time.

Table 2: Results of the irritation test - 1st test run

Test substance		tissue 1	tissue 2	tissue 3	mean	SD
NC	mean OD570	2.295	2.144	2.121	2.187
	viability [% of NC]	105.0	98.1	97.0	100	4.33
Test substance	mean OD570	1.367	1.174	1.700	1.414
	viability [% of NC]	62.5	53.7	77.7	65	12.16
PC	mean OD570	0.059	0.063	0.065	0.062
	viability [% of NC]	2.7	2.9	2.9	3	0.13

 

 

Table 3: Results of the irritation test - 2nd test run

Test substance		tissue 1	tissue 2	tissue 3	KC	mean	SD
NC	mean OD570	2.404	2.415	2.401	0.057	2.407
	viability [% of NC]	99.9	100.3	99.8	-	100	0.29
Test substance	mean OD570	2.347	2.414	2.384	0.067	2.381
	viability [% of NC]	97.5	100.3	99.0	-	99	1.38
PC	mean OD570	0.096	0.090	0.105	-	0.097
	viability [% of NC]	4.0	3.7	4.4	-	4	0.32

The result of the KC did not indicate an increased MTT reduction (difference to KC of NC is not greater than 0.1). Thus the KC was not used for viability calculation.

Table 4: Historical control data - Corrosion test

Historical Range of NC
OD570
Exposure Time	Historical Period	Mean OD	SD	Mean + 2 SD	Mean - 2 SD
3 minutes	Feb 2010 - Aug 2012	1.901	0.200	2.30	1.50
60 minutes	Feb 2010 - Aug 2012	1.867	0.203	2.27	1.46
Historical Range of PC
OD570
Exposure Time	Historical Period	Mean OD	SD	Mean + 2 SD	Mean - 2 SD
3 minutes	Feb 2010 - Aug 2012	0.361	0.087	0.54	0.19
60 minutes	Feb 2010 - Aug 2012	0.155	0.048	0.25	0.06
Viability (%)
Exposure Time	Historical Period	Mean %	SD	Mean + 2 SD	Mean - 2 SD
3 minutes	Feb 2010 - Aug 2012	19.16	4.73	28.62	9.69
60 minutes	Feb 2010 - Aug 2012	8.47	2.82	14.12	2.82

Table 5: Historical control data - Irritation test

Historical Range of NC
OD570 Historical Period	Mean OD	SD	Mean + 2 SD	Mean - 2 SD
Feb 2010 - Nov 2012	2.060	0.281	2.62	1.50

 

Historical Range of PC
OD570 Historical Period	Mean OD	SD	Mean + 2 SD	Mean - 2 SD
Feb 2010 - Nov 2012	0.111	0.052	0.22	0.01

 

Viability (%)
Historical Period	Mean %	SD	Mean + 2 SD	Mean - 2 SD
Feb 2010 - Nov 2012	5.5	2.47	10.45	0.56

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the observed results and applying the evaluation criteria it was concluded, that the test item does not show a skin irritation potential in the EpiDerm™ skin corrosion/irritation test under the test conditions chosen.