Reaction mass of 2-O-α-L-Rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid and α-L-Rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-11-23 to 2017-11-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Remarks:

Skin irritation study

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-04-17 - 2018-05-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Remarks:

Skin Corrosion study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline for the Testing of Chemicals No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 640/2012, L 193, Part B.46. “In vitro Skin Irritation: Reconstructed Human Epidermis Test Method” 06-Jul-2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 2-8 °C; in closed packaging

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Source strain:

other: not applicable

Justification for test system used:

Justification for the Selection of the Test System: This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.
Justification for the Selection of the Test Method: This test method is able to detect chemicals that cause skin irritation, i.e. produce reversible damage to the skin and allows for hazard identification in accordance with UN GHS “Category 2”. Depending on the regulatory framework it can also be used to identify non-classified chemicals.

Vehicle:

unchanged (no vehicle)

Remarks:

25 µL of sterile DPBS were applied to the epidermal surface in order to improve the contact between the powder and the epidermis.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
The test was carried out with the reconstituted three-dimensional human skin model EpiDerm (MatTek). This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell). The EpiDerm epidermis model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum analogous to patterns found in vivo.
- Tissue batch number(s): Keratinocyte strain 00267
- Production date: 2018-04-25

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature / 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface, this process also occurred sequentially, e.g. in one-minute intervals. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper.
- Observable damage in the tissue due to washing: none stated

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ml
- Incubation time: 3 h ± 5 min at 37 ± 1 °C, 5.0% CO2, humidified to 95%.
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not required, no MTT interference

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with DPBS. The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 (UN GHS “Category 2”), if the tissue viability after exposure and post-incubation is less or equal to 50%. Further testing is required to resolve between UN GHS categories 1 and 2 and decide on the final classification of the test substance. The test substance may be considered as non-irritant to skin in accordance with UN GHS “No Category” if the tissue viability after exposure and post-treatment incubation is more than 50%.
Prediction Model
Mean Tissue Viability
(% negative control) Prediction I / NI
≤ 50 % Irritant (I): UN GHS “Category 2”
> 50 % Non-Irritant (NI): UN GHS “No Category”

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg + 25 µL DPBS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL 5% sodium dodecyl sulfate solution
- Concentration (if solution): 5%

Duration of treatment / exposure:

60 ± 1 min incubation time (35 ± 1 min in incubator)

Duration of post-treatment incubation (if applicable):

The plates were post-incubated at 37 ± 1°C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing 0.9 mL fresh assay medium and incubated for additional 18 ± 2 h.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

3.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean Relative Tissue Viability [%]: 3.2%

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: not stated
- Direct-MTT reduction: no
- Colour interference with MTT: no

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

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Testing was performed via a GLP OECD 439 guideline study on the registered substance itself. The present in vitro method is recommended in a tiered testing approach, it was used as subsequent testing to an in vitro skin corrosion test, which revealed that the test item was non-corrosive to the skin.
The validity criteria are met, making the results sufficiently reliable to assess the irritating potential of the test item to the skin. The present in vitro method allows the identification of irritating chemical substances and mixtures. After the treatment, the mean value of relative tissue viability was reduced to 3.3%. This value is well below the threshold for skin irritation (50%). Hence, the test item is therefore classified as “irritant” in accordance with UN GHS “Category 2”.

Executive summary:

In the present study the skin irritant potential of 'Decanoic acid, 3-[[6-deoxy-2-O-(6-deoxy-a-L-manno-pyranosyl)-a-L-mannopyranosil-1-(carboxymethyl)octyl ester, mixture with 1-(carboxymethyl) octyl 3-[(6-deoxy-a-L-mannopyranosyl)oxy]decanoate' was analysed according to OECD 439 under GLP. The EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 60 min exposure and 42 h post-incubation period and compared to those of the concurrent negative controls.

The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

The mixture of 25 mg of the test item per 300 µl aqua dest. showed colouring detectable by unaided eye-assessment. Therefore, the absorption of the chemical in water was measured in the range of 570 ± 30 nm. No absorption was measured in the relevant range.

The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 50% (3.3%) after 60 min treatment and 42 h post-incubation.

Conclusion: In this study under the given conditions the test item showed irritant effects. The relative mean tissue viability after 60 min of exposure and 42 h post-incubation was ≤ 50%. The test item is therefore classified as “irritant” in accordance with UN GHS “Category 2”.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS: "In vitro Skin Corrosion: Human Skin Model Test"

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt fuer Gesundheit und Lebensmittelsicherheit (05.06.2015)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-O-α-L-Rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid and α-L-Rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid

EC Number:

604-610-3

Cas Number:

147858-26-2

IUPAC Name:

Reaction mass of 2-O-α-L-Rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid and α-L-Rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

other: human

Justification for test system used:

The EpiDerm Skin Model is a well established organotypic, three-dimensional model of the human epidermis and is used for in vitro experiments since many years. It is known for its similarity for human skin.
The EpiDerm human skin model has been validated by ECVAM for the discrimination between corrosive and non-corrosive chemicals.

Vehicle:

water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm, EPI-200, MatTek
- Tissue batch number(s): 25860

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: PBS, 20 washing steps

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ml
- Incubation time: 3 h
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- N. of replicates : 2
- Method of calculation used:
Non-specific MTT reduction calculation (NSMTT):
NSMTT [%] = [(ODKT - ODKU) / ODNK] × 100

ODKU: negative control untreated killed tissues OD (mean)
ODKT: test item treated killed tissues OD (mean)
ODNC: negative control living tissues OD (mean)

If non-specific MTT reduction is < 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) of the test item treated living tissues (TM) was calculated for each treatment period according to the following formula:

TODTT = ODTM – (ODKT – ODKU)

ODTM: test item treated living tissues




NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- 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 3 minutes exposure is greater than or equal to 50 % and 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%.]

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

25 mg of test item moistened with 25 µl water.

Duration of treatment / exposure:

3 min and 60 min

Duration of post-treatment incubation (if applicable):

3 h

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min, mean of 2 replicates

Value:

70

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min, mean of 2 replicates

Value:

76.5

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Table 1. Results of 3 min Experiment

Name	Negative Control		Test Item		Positive Control
Tissue	1	2	1	2	1	2
Absolute OD570	1.870	2.052	1.353	1.346	0.159	0.199
	1.841	2.084	1.371	1.428	0.166	0.207
	1.899	2.041	1.383	1.450	0.170	0.211
OD570- Blank Corrected	1.826	2.008	1.308	1.301	0.114	0.154
	1.796	2.039	1.327	1.384	0.121	0.163
	1.854	1.997	1.338	1.405	0.126	0.167
Mean OD570 of 3 Aliquots (Blank Corrected)	1.825	2.015	1.324	1.363	0.120	0.161
SD OD570 of 3 Aliquots	0.029	0.031	0.028	0.055	0.025	0.025
Total Mean OD570of 2 Replicate Tissues (Blank Corrected)	1.920*		1.344		0.141
SD OD570of 2 Replicate Tissues	0.134		0.028		0.029
Mean Relative Tissue Viability [%]	100.0		70.0		7.3
Coefficient Of Variation [%]***	7.0		2.1		20.5

* corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.

*** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%.

Table 2. Results of 60 min Experiment

Name	Negative Control		Test Item		Positive Control
Tissue	1	2	1	2	1	2
Absolute OD570	2.054	1.879	1.612	1.421	0.151	0.104
	2.031	2.017	1.601	1.498	0.156	0.103
	2.009	1.979	1.592	1.500	0.162	0.107
OD570- Blank Corrected	2.009	1.835	1.567	1.377	0.107	0.059
	1.987	1.972	1.557	1.453	0.111	0.058
	1.964	1.934	1.547	1.455	0.117	0.062
Mean OD570of 3 Aliquots (Blank Corrected)	1.987	1.914	1.557	1.428	0.112	0.060
SD OD570of 3 Aliquots	0.022	0.068	0.026	0.047	0.025	0.025
Total Mean OD570of 2 Replicate Tissues (Blank Corrected)	1.950*		1.493		0.086
SD OD570of 2 Replicate Tissues	0.052		0.091		0.037
Mean Relative Tissue Viability [%]	100.0		76.5		4.4**
Coefficient Of Variation [%]***	2.6		6.1		42.6

* corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.

** mean relative tissue viability of the 60 min positive control < 15%,

*** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%.

Table 3. Test Acceptance Criteria

	Value	Cut off	pass/fail
Mean Absolute OD57onmNK (3 min Experiment)	1.965	0.8 < NK < 2.8	pass
Mean Absolute OD570nmNK (60 min Experiment)	1.995	0.8 < NK < 2.8	pass
Mean Relative Tissue Viability [%] of PC (60 min experiment)	4.4	< 15%	pass
CV[%](in the range of 20 - 100% viability)	2.1 - 7.0	< 30%	pass

Table 4. Historical Data

	Mean	SD	n
OD570of NK (3 min Experiment)	1.895	0.313	10
OD570of NK (60 min Experiment)	1.867	0.261	11
Relative Tissue Viability [%] of PC (60 min experiment)	6.1	1.99	11
CV [%](in the range of 20 - 100% viability)	7.8	7.5	31

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

The test item is classified as "non-corrosive".

Conclusions:

The test item 'Decanoic acid, 3-[[6-deoxy-2-O-(6-deoxy-a-L-manno-pyranosyl)-a-L-mannopyranosil-1-(carboxymethyl)octyl ester, mixture with 1-(carboxymethyl)octyl 3-[(6-deoxy-a-L-mannopyranosyl)oxy]decanoate' (CAS 147858 -26 -2) is not considered as corrosive to skin.
This in vitro method allows the identification of corrosive and non-corrosive substances and mixtures in accordance with UN GHS and was used as a first study in a top-down irritation / corrosion testing approach. In consequence, a subsequent skin irritation study was performed to distinguish whether the substance needs to be classified as skin irr. Cat. 2 or whether no classification acc. Regulation 1272/2008 is required.

Executive summary:

The potential of the test item to induce skin corrosion was analysed by using the three-dimensional human skin model EpiDerm, comprising a reconstructed epidermis with a functional stratum corneum.

In the present study 'Decanoic acid, 3-[[6-deoxy-2-O-(6-deoxy-a-L-manno-pyranosyl)-a-L-mannopyranosil-1-(carboxymethyl)octyl ester, mixture with 1-(carboxymethyl)octyl 3-[(6-deoxy-a-L-mannopyranosyl)oxy]decanoate' was applied topically to the EpiDerm tissue for 3 min and 60 min followed by immediate determination of cytotoxic effects via MTT reduction assay. Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after both treatment periods had been compared to the corresponding negative control tissues.

The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was ≥ 50% (70%) after 3 min treatment and ≥ 15% (76.5%) after 60 min treatment.

The controls confirmed the validity of the study. The mean OD570 of the two negative control tissues was ≥ 0.8 and ≤ 2.8 for each exposure period. The mean relative tissue viability (% negative control) of the positive control was < 15% (4.4%) after 60 min treatment. The coefficient of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of all dose groups was ≤ 30% (2.1% - 7.0%).

Therefore, the test item Rhamnolipid mixture (CAS 147858 -26 -2) is not considered as corrosive to skin.