Registration Dossier

Administrative data

Description of key information

Skin irritation (in vitro): irritating (OECD439, EPISKIN)
Eye irritation (in vitro): irritating (OECD438, ICE)
Respiratory irritation: not irritating

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:
15-21 November 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD439 and under GLP conditions.
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: Method B.46 of Commission Regulation 440/2008/EC (In vitro skin irritation)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guidelines for the Testing of Chemicals No. 439 "In Vitro Skin Irritation" (adopted 22 July 2010)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
human
Strain:
other: In vitro skin model (EPISKIN standard model)
Details on test animals and environmental conditions:
THE EPISKIN MODEL
- Supplier: SkinEthic Laboratories, Nice, France
- After receiving the EPISKIN, the tissue was pre-incubated overnight in maintenance medium at 37°C, 5% CO2 in air.

ENVIRONMENTAL CONDITIONS: all incubations, with the exception of the test substance incubation of 15 minutes at room temperature:
- Temperature (°C): 37
- 5% CO2 in air
- Photoperiod: dark
Type of coverage:
other: not applicable
Preparation of test site:
other: The test item was applied topically to the corresponding tissues ensuring uniform covering.
Vehicle:
unchanged (no vehicle)
Controls:
other: POSITIVE CONTROL: Sodium Dodecyl Sulphate (SDS), prepared as a 5% w/v aqueous dilution. NEGATIVE CONTROL: Dulbecco's Phosphate Buffered Saline (DPBS) with Ca++ and Mg++, used as supplied
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL
- Concentration (if solution): Undiluted substance

POSITIVE CONTROL: Sodium Dodecyl Sulphate (SDS), prepared as a 5% w/v aqueous dilution
To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip. After 7 minutes contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period.

NEGATIVE CONTROL: Dulbecco's Phosphate Buffered Saline (DPBS) with Ca++ and Mg++, used as supplied
Duration of treatment / exposure:
Exposure: 15 minutes
Incubation: 42 hours
Number of animals:
A total of 9 tissues were used: Triplicate tissues were treated with: test substance, positive control or negative control respectively.
Details on study design:
TEST SITE: EXPOSURE OF THE TISSUES
After receiving the EPISKIN, the tissue was pre-incubated overnight in maintenance medium at 37°C, 5% CO2 in air.

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 DPBS with Ca++ and Mg++.
- Time after start of exposure: 15 minutes.

SCORING SYSTEM: The principle of the assay was based on the measurement of cytotoxicity (irritancy) in reconstructed human epidermal cultures following topical exposure to the test item by means of the colourimetric MTT reduction assay. Cell viability was 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 item treated tissue relative to the negative controls.

TOOL USED TO ASSESS SCORE: The amount of extracted formazan was determined spectrophotometrically at 540 nm in duplicate (without a reference filter) using the Anthos 2001 microplate reader.
Irritation / corrosion parameter:
other:
Value:
10.8
Remarks on result:
other:
Remarks:
Basis: mean. Max. score: 100.0. Reversibility: other: Not applicable. Remarks: SD: ± 2.9%. (migrated information)
Irritation / corrosion parameter:
other:
Value:
0.101
Remarks on result:
other:
Remarks:
Basis: mean (of triplicate tissue). Max. score: 0.934. Reversibility: other: Not applicable. Remarks: SD: ± 0.027. (migrated information)
Irritant / corrosive response data:
The test item was considered to be irritant.
Other effects:
No other effects observed

The positive control had a mean cell viability of 5.5% after 15 minutes exposure. The negative control was set at 100%.

The standard deviation of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The relative mean tissue viability after 15 minutes treatment with Cashmeran compared to the negative control tissue was 10.8%. Since the mean relative tissue viability for Cashmeran was below 50% after 15 minutes treatment, Cashmeran is considered to be irritant. It is concluded that this test is valid and that Cashmeran is irritant in the in vitro skin irritation test under the experimental conditions described in this report.
Executive summary:

This report describes the ability of Cashmeran to induce skin irritation on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-TM)). The possible skin irritation potential of Cashmeran was tested through topical application for 15 minutes. The study procedures described in this report were based on the OECD Guidelines for the Testing of Chemicals No. 439 "In Vitro Skin Irritation" (adopted 22 July 2010) and Method B.46 of Commission Regulation (EC) No. 440/2008/EC.

Skin tissue was treated with Cashmeran by topical application of 10 µL Cashmeran. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with Cashmeran compared to the negative control tissue was 10.8%.

Since the mean relative tissue viability for Cashmeran was below 50% after 15 minutes treatment Cashmeran is considered to be irritant. The positive control had a mean cell viability of 5.5% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

Finally, it is concluded that this test is valid and that Cashmeran is irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study performed according to OECD 438 and under GLP conditions for indicating severe eye damage. The test is, however, not fully validated for severe eye irritation but the results are sufficiently adequate to conclude on the eye irritation effect of the substance
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD guideline 438 ("Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants")
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
other: Chicken
Strain:
other: ROSS, spring chickens
Details on test animals or tissues and environmental conditions:
TEST ANIMALS (eye-donors)
- Source: Poultry slaughterhouse v.d. Bor, Amersfoortseweg 118, Nijkerkerveen, The Netherlands
- Age at study initiation: 7 weeks old
- Weight at study initiation: approx. 1.5-2.5 kg
Vehicle:
unchanged (no vehicle)
Controls:
other: saline (negative control) and 5% benzalkonium chloride (BAC, positive control)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 30 uL
- Concentration: Undiluted (100%)
Duration of treatment / exposure:
10 seconds
Observation period (in vivo):
240 minutes (examination at 0, 30, 75, 120, 180 and 240 minutes after treatment)
Number of animals or in vitro replicates:
Number of eyes used:
- Test group: 3
- Positive control: 3
- Negative control: 1
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: Rinsing with 20 mL saline
- Time after start of exposure: 10 seconds

SCORING SYSTEM: Corneal thickness and corneal opacity were scored at different time points, while fluorescein retention was scored only at 30 minutes after exposure. These endpoint were related to 4 classes of eye irritation (not; slightly; moderately; severely irritating) and finally reconciled to the classification criteria as applicable in EU-CLP, in accordance with the decision criteria in OECD438. Additionally, morphological and microscopic effects were examined.

TOOL USED TO ASSESS SCORE: Slit lamp microscope.

HISTOPATHOLOGY: After treatment, all eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde and corneas were embedded in parraffin wax, sectioned and stained. Histopathological examination was not performed, but the microscopic slides were filed in the archive.
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: Not relevant (see remarks)
Score:
110
Max. score:
240
Reversibility:
other: Not relevant
Remarks on result:
other: Irritation index = maximum mean corneal swelling + maximum mean opacity (x20) + mean fluorescein score (x20)
Irritant / corrosive response data:
Cashmeran caused moderate swelling (20%) of the cornea, moderate or moderate to severe corneal opacity (2.2) and moderate or moderate to severe fluorescein retention (2.3). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control eyes showed severe corneal effects and demonstrated the suitability and sensitivity of the ICE to detect severe eye irritants.
Other effects:
Wrinkling of the epithelium followed by erosion/loosening of the top-layer of the epithelium were observed in the test substance group. Severe loosening of the epithelium was observed in the positive control group.

Please find in the table below the summary results.

Test material

Maximum score for:

Irritation categories

Irritation index

Classification (EU-CLP

Swelling %

Opacity

Fluorescein retention

Cashmeran

20

2.2

2.3

III;III;III

110

2

Saline (negative control)

0

0.0

0.0

Not applicable, one eye tested

BAC 5% (w/v) (positive control)

27

3.0

3.0

III;IV;IV

147

1

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the observed irritation index for Cashmeran and according to the EU-CLP classification schemes of the Isolated Chicken Eye Test, Cashmeran should be classified as irritating and Category 2: “Irritating to eyes”.
Executive summary:

The eye irritating potential of Cashmeran was determined in an isolated chicken eye test performed according to OECD guideline 438 and under GLP conditions.

Cashmeran caused moderate swelling (20%) of the cornea, moderate or moderate to severe corneal opacity (2.2) and moderate or moderate to severe fluorescein retention (2.3). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. Furthermore, the positive control eyes showed severe corneal effects and demonstrated the suitability and sensitivity of the ICE to detect severe eye irritants. The irritation index for Cashmeran as defined in the OECD guideline was 110 out of a maximum of 240.

Based on the irritation index for Cashmeran the substance does not need to be classified as severely irritating but according to the EU-CLP classification schemes of the ICE, the substance should be classified as irritating and Category 2: “Irritating to eyes”.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

The ability of Cashmeran to induce skin irritation was determined in a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-TM)). The possible skin irritation potential of Cashmeran was tested through topical application for 15 minutes. The study procedures described in this report were based on the OECD Guidelines for the Testing of Chemicals No. 439 "In Vitro Skin Irritation" (adopted 22 July 2010) and Method B.46 of Commission Regulation (EC) No. 440/2008/EC.

Skin tissue was treated with Cashmeran by topical application of 10 µL Cashmeran. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with Cashmeran compared to the negative control tissue was 10.8%.

Since the mean relative tissue viability for Cashmeran was below 50% after 15 minutes treatment Cashmeran is considered to be irritant. The positive control had a mean cell viability of 5.5% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

Finally, it is concluded that this test is valid and that Cashmeran is irritant in the in vitro skin irritation test under the experimental conditions described in this report.

The eye irritating potential of Cashmeran was determined in an isolated chicken eye test performed according to OECD guideline 438 and under GLP conditions.

Cashmeran caused moderate swelling (20%) of the cornea, moderate or moderate to severe corneal opacity (2.2) and moderate or moderate to severe fluorescein retention (2.3). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. Furthermore, the positive control eyes showed severe corneal effects and demonstrated the suitability and sensitivity of the ICE to detect severe eye irritants. The irritation index for Cashmeran as defined in the OECD guideline was 110 out of a maximum of 240.

Based on the irritation index for Cashmeran and according to the EU-CLP classification schemes of the ICE, the substance should be classified as irritating.

For respiratory irritation mostly human data are used for the assessment because no suitable in vitro or in vivo tests are available that can identify respiratory irritation (REACH guidance R.7.2.3). There are no human data such as indicated in R7.2.3 the ECHA guidance that indicate respiratory reactions of the substance e.g. from consumer experience or occupational exposure. Despite the skin/eye irritation potential, the substance is not corrosive or severely damaging (eye) which minimizes the respiratory irritation hazard (REACH guidance: 7.2.1.2). Also the substance has a low vapour pressure (1 Pa) and viscosity higher than water (31 versus 1 mPa.s), which minimises the exposure via the inhalation route.


Justification for selection of skin irritation / corrosion endpoint:
The one in vitro study available is sufficiently adequate to cover the skin irritation and corrosion potential.

Justification for selection of eye irritation endpoint:
The one in vitro study available is adequate to cover the eye irritation and corrosion potential.

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: irritating

Justification for classification or non-classification

Cashmeran needs to be classified as irritating to the skin, eyes in accordance with the criteria outlined in Annex VI of 67/548/EEC (R36, R38) and Annex I of 1272/2008/EC (H315, H319).

Cashmeran does not need to be classified for respiratory tract irritation because it is not a corrosive or severely irritating substance with the criteria outlined in Annex VI of 67/548/EEC and Annex I of 1272/2008.