Overbased reaction products of distilled tall oil and magnesium hydroxide’

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

other: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 30 March 2010 and 05 April 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in Section 13

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

skin irritation / corrosion, other

Remarks:

In Vitro

Type of information:

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

Adequacy of study:

key study

Study period:

The study was performed between 30 March 2010 and 05 April 201

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see "Remark"

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP condition

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in Section 13

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: EU Guideline Testing of Chemicals B46

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Draft Test Guideline

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

human skin model reconstituted human epidermis model

Source species:

human

Cell type:

other: reconsituted human epidermal cells

Justification for test system used:

ECVAM recommended in vitro method. Also EU Guideline B46

Vehicle:

unchanged (no vehicle)

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Three

Type of coverage:

other: Applied topically to reconstiyuted human epidermal cells

Vehicle:

unchanged (no vehicle)

Irritation parameter:

other: Viability of cells

Basis:

other: Mean viability of cells

Time point:

other: Day 6

Reversibility:

other: Not applicable

Remarks on result:

other: See relative mean viability below.

Irritant / corrosive response data:

The relative mean viability of the test material treated tissues was 93.1% after a 15 minute exposure.

Other effects:

no

Interpretation of results:

other: Not irritating

Remarks:

expert judgment

Conclusions:

The test material was considered to be Non-Irritant (NI)

Executive summary:

Introduction:

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKINTM reconstituted human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 -[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 hour post-exposure incubation period is also determined for test materials which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.

Methods:

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for approximately 42 hours. At the end of the postexposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm. Data are presented in the form of percentage viability (MTT reduction in the test material treated tissues relative to negative control tissues).

Results:

The relative mean viability of the test material treated tissues was 93.1% after a 15-minute exposure.

Quality criteria:

The quality criteria required for acceptance of results in the test were satisfied. Conclusion:  The test material was considered to be Non-Irritant (NI).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EU Guideline Testing of Chemicals B46

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Draft Test Guideline

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids C18-(unsaturated) lithium salts

EC Number:

700-426-3

Cas Number:

502962-81-4

Molecular formula:

Since this is a complex mixture of constituents no single formula can be assigned

IUPAC Name:

Fatty acids C18-(unsaturated) lithium salts

Details on test material:

Sponsor's identification : Fatty acids C18-(unsaturated) lithium salts
Description : off white solid
Batch number : SF 173
Date received : 21 January 2010
Expiry date : 21 January 2012
Storage conditions : room temperature in the dark

In vitro test system

Test system:

human skin model

Remarks:

reconstituted human epidermis model

Source species:

human

Cell type:

other: reconsituted human epidermal cells

Justification for test system used:

ECVAM recommended in vitro method. Also EU Guideline B46

Vehicle:

unchanged (no vehicle)

Duration of treatment / exposure:

15 MINUTES

Duration of post-treatment incubation (if applicable):

42 hrs

Number of replicates:

Three

Test system

Type of coverage:

other: Applied topically to reconstiyuted human epidermal cells

Vehicle:

unchanged (no vehicle)

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

no

Amount / concentration applied:

TEST MATERIAL

- The test Material was applied neat.

- Amount(s) applied (volume or weight with unit):
Approximately 10 mg of the test material was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 µl of sterile distilled water to improve contact between the solid test material and the epidermis.

- Concentration (if solution):
The test material was used as supplied.

VEHICLE
No vehicle used

Duration of treatment / exposure:

15 Minutes & 42 hour post exposure incubation

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

TEST SITE
- Area of exposure:
Approximately 10 mg of the test material was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 µl of sterile distilled water to improve contact between the solid test material and the epidermis.

- % coverage:
The test material was applied topically to the corresponding tissues ensuring uniform covering.

- Type of wrap if used:
None used

REMOVAL OF TEST SUBSTANCE
- Washing (if done):
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing PBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test material.

- Time after start of exposure:
15 Minutes post exposure

SCORING SYSTEM:
Quantitative MTT Assessment (percentage tissue viability)
For the test material the relative mean tissue viabilities obtained after the 15 minute treatment followed by the 42 hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:

mean OD540 of test material / mean OD540 of negative control x 100 = Relative mean tissue viability (percentage of negative control)

Classification of irritation potential is based upon relative tissue viability following the 15 minute exposure period followed by the 42 hour post-exposure incubation period according to the following:

Mean tissue viability is ≤50% : Irritant (I) R38

Mean tissue viability is >50% : Non-Irritant (NI)

Results and discussion

In vivo

Results

Irritation parameter:

other: Viability of cells

Basis:

mean

Remarks:

Viability of cells

Time point:

other: Day 6

Reversibility:

other: Not Applicable

Remarks on result:

other: See relative mean viability below.

Irritant / corrosive response data:

The relative mean viability of the test material treated tissues was 93.1% after a 15 minute exposure.

Other effects:

No

Any other information on results incl. tables

RESULTS

Direct MTT Reduction

An assessment found the test material was able to directly reduce MTT. Therefore, an additional procedure using water-killed tissues was performed during the determination of skin irritation potential. The test material was adequately rinsed from the tissues, however the results obtained showed that during rinsing no degree of interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

Test Material, Positive Control Material and Negative Control Material

The individual and mean OD₅₄₀ values, standard deviations and tissue viabilities for the test material, negative control material and positive control material are given in Table 1. The mean viabilities and standard deviations of the test material and positive control, relative to the negative control are also given in Table 1.

The relative mean viability of the test material treated tissues was 93.1% after a 15 -minute exposure.

The qualitative evaluation of tissue viability is given in Table 2.

Following the 15-minute exposure the test material treated tissues appeared blue/white which was considered indicative of viable tissue.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was ≤40% relative to the negative control treated tissues and the standard deviation value of the percentage viability was ≤20%. The positive control acceptance criterion was therefore satisfied.

The mean OD₅₄₀ for the negative control treated tissues was ≥0.6 and the SD value of the percentage viability was ≤20%. The negative control acceptance criterion was therefore satisfied.

Table 1 : Mean OD₅₄₀ Values and Percentage Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material	OD540of tissues	Mean OD540of triplicate tissues	±SDof OD540	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Material	1.016	0.962	0.002	105.6	100*	5.4
	0.958			99.6
	0.913			94.9
Positive Control Material	0.068	0.064	0.009	7.1	6.7	0.9
	0.054			5.6
	0.070			7.3
Test Material	0.877	0.896	0.021	91.2	93.1	2.2
	0.919			95.5
	0.892			92.7

SD=    Standard deviation

*=     The mean viability of the negative control tissues is set at 100%

Table 2 : Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material	Tissue 1	Tissue 2	Tissue 3
Negative Control Material	-	-	-
Positive Control Material	++	++	++
Test Material	-	-	-

MTT visual scoring scheme
-          =         blue tissue (viable)
+         =         blue/white tissue (semi-viable)
++       =         tissue is completely white (dead

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

The test material was considered to be Non-Irritant (NI)

Executive summary:

Introduction: The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN^TM reconstituted human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 -[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 hour post-exposure incubation period is also determined for test materials which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.

Methods:

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for approximately 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm.

Data are presented in the form of percentage viability (MTT reduction in the test material treated tissues relative to negative control tissues).

Results: 

The relative mean viability of the test material treated tissues was 93.1% after a 15-minute exposure.

Quality criteria: 

The quality criteria required for acceptance of results in the test were satisfied.

Conclusion:  The test material was considered to be Non-Irritant (NI).