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Diss Factsheets

Administrative data

Description of key information

In vitro Skin Irritation (EU Method B.46: RHE Model Test): not irritating 
RA-S CAS 112-84-8, OECD 404, rabbit, 4h semiocclusive: not irritating
In vitro Eye Irritation (Reconstructed Human Corneal Epithelium Model): not irritating
In vivo Eye Irritation, OECD 405: not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study. Reliability was changed from "1" to "2" according to the ECHA guidance document "Practical guide 6: How to report read-across and categories".
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: The Broekman Institute, Someren, The Netherlands
- Age at study initiation: 10, 13 and 17 weeks
- Weight at study initiation: 2303, 2605 and 2853 g
- Housing: Individually in a plastic cage with a perforated floor
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: At least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21
- Humidity (%): 50-60
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
semiocclusive
Preparation of test site:
shaved
Vehicle:
water
Controls:
other: The right flank served as untreated control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g
- Concentration (if solution): Moistened with 0.5 mL water

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.5 mL
Duration of treatment / exposure:
4h
Observation period:
1, 24, 48 and 72h
Number of animals:
3
Details on study design:
TEST SITE
- Area of exposure: 6 cm²
- Type of wrap if used: The test substance was spread on 6 cm² patch of Metalline (Lobmann, Germany) and mounted on permeable tape (Micropore, 3M, St. Paul, USA). This was applied to the left flank of each animal, the right flank being covered with the same dressing without test substance. Finally, the animals were wrapped in flexible bandage (Coban, 3M, St. Paul, USA)

REMOVAL OF TEST SUBSTANCE
- Washing: The remaining test substance was removed, using a dry tissue and subsequently a tissue moistened with tap-water.
- Time after start of exposure: 4h

SCORING SYSTEM: Draize
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no effects
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no effects
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no effects
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no effects
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no effects
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no effects
Irritant / corrosive response data:
The test substance caused only very slight erythema in all three animals, 60 minutes after removal of the dressings.
Other effects:
No signs of systemic intoxication were observed in any of the rabbits.

Based on these results the test substance should be considered as not-irritating to the skin. According to the criteria of the CLP regulation the test substance does not have to be classified as irritating to the skin.

Interpretation of results:
other: CLP criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
CLP: not classified
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Principles of method if other than guideline:
EPISKIN reconstituted human epidermis model: 15 min treatment followed by 42 hours post-exposure incubation period.
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
other: reconstituted human epidermis
Cell source:
other: adult human-derived epidermal keratinocytes
Source strain:
other: not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 15 min

SCORING SYSTEM: not applicable, measurement of cytotoxicity by colourimetric MTT reduction assay
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ± 2 mg
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours post-exposure incubation period
Number of replicates:
triplicate tissues
Irritation / corrosion parameter:
% tissue viability
Remarks:
Test substance
Run / experiment:
mean of 3 runs
Value:
117.4
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
Negative control
Run / experiment:
mean of 3 runs
Value:
100
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Irritation / corrosion parameter:
% tissue viability
Remarks:
Positive control
Run / experiment:
mean of 3 runs
Value:
7.3
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Other effects / acceptance of results:
The test material was considered to be Non-Irritant (NI).

The test material did not directly reduce MTT.

Mean OD540 values and percentage viabilities for the negative control material, positive control material and test material:

Material

OD540of tissues

Mean OD540if triplicate tissues

± SD of OD540

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative control

0.784

0.834

0.045

94.0

100*

5.4

0.847

101.6

0.871

104.4

Positive control

0.069

0.061

0.016

8.3

7.3

1.9

0.071

8.5

0.043

5.2

Test material

1.079

0.979

0.087

129.4

117.4

10.4

0.925

110.9

0.933

111.9

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

The relative mean viability of the test material treated tissues was 119.2% after a 15 -minute exposure, the test material treated tissues appeared blue which was considered indicative of viable tissue.

The relative mean tissue viability for the positive control treated tissues was ≤ 40% relativ to the negative control, 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.

Conclusion:

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

Interpretation of results:
other: CLP criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
CLP: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
Only 2 animals tested
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
Only 2 animals tested
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd, Hillcrest, Belton, Loughborough, UK
- Age at study initiation: 12 to 20 weeks
- Weight at study initiation: 2.01 and 2.88 kg
- Housing: individually in suspended cages
- Diet: 2030 Teklad Global Rabbit diet (Harlan Teklad, Blackthorn, Bicester, Oxon, UK), ad libitum
- Water: mains drinking water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23
- Humidity (%): 30 - 70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Controls:
other: untreated eyes served as controls
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 ml (corrsponding to approximately 51 mg, measured by gently compacting the required volumen into an adapted syringe)
Duration of treatment / exposure:
single application without washing
Observation period (in vivo):
1, 24, 48 and 72 hours
Number of animals or in vitro replicates:
2
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): no

SCORING SYSTEM: Draize

TOOL USED TO ASSESS SCORE: light source from a standard ophthalmoscope
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no effects
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no effects
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: no effects
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: no effects
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritant / corrosive response data:
No corneal or iridial effects were observed during the relevant observation period of the study. Moderate corneal irritation was only noted in both treated eyes one hour after treatment. Minimal conjunctival irritation was noted at the 24-hour observation. Both treated eyes appeared normal at the 48-hour observation.

Although only 2 animals have been tested the study is fully acceptable for assessment, as even a positive irritation response in a third animal would not lead to a classification according to the criteria of the OECD guidelines for studies performed with 3 animals. Therefore, testing of a third animal would have not increased validity of the study for regulatory purposes and was omitted due to animal welfare reasons.
Other effects:
Both animals showed expected gain in body weight during the study.

Although only 2 animals were tested the study is fully acceptable for assessment from a regulatory point of view, as even contribution of a third animal demonstrating most severe irritation reactions would have no impact on final classification according to the criteria for eye irritation tests conducted with 3 animals.

Interpretation of results:
other: CLP criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
CLP: not classified
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail; performed under GLP.
Qualifier:
no guideline available
Principles of method if other than guideline:
In vitro determination of the eye irritation potential with the SkinEthic Reconstructed Human Corneal Epithelium model.
GLP compliance:
yes (incl. QA statement)
Species:
human
Strain:
other: transformed human keratinocytes of the HCE cell line
Details on test animals or tissues and environmental conditions:
not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable, in vitro
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg
Duration of treatment / exposure:
10 min
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
not applicable, in vitro
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 10 minutes

SCORING SYSTEM: not applicable, colorimetric determination of cell viability in treated tissues relative to control tissues
Irritation parameter:
mean percent tissue viability 
Remarks:
Test substance
Run / experiment:
mean
Value:
102.9
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The quality criterion required for the acceptance of results in the test was satisfied.

The test material was not able to directly reduce MTT.

Assessment of eye irritation potential:

The mean OD540 values and mean viabilities for each treatment group are given in the following table.

Assessment of Eye Irritation Potential – Viability of RHC Tissues:

Material

Mean Tissue Viability

Mean OD540

Viability (%)

Negative Control

1.014

0.979

100*

0.943

Positive Control

0.263

0.247

25.2

0.230

Test Material

1.067

1.007

102.9

0.947

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

The relative mean viability of the test material treated tissues after a 10-minute exposure was 102.9%.

It was considered unnecessary to proceed with tissue histopathology.

The test material and negative control material treated tissues appeared blue which was considered to be indicative of viable tissue. The positive control tissues appeared appeared blue/white which was considered to be indicative of semi-viable tissue.

The quality criterion required for the acceptance of results in the test was satisfied.

Conclusion:

According to the protocol followed the test material was considered to be a Non-Irritant (NI).

Interpretation of results:
other: CLP criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
CLP: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

There is an in vitro skin irritation study available for Amides, C16-C18 (even numbered) (Warren, 2010) conducted according to EU Method B.46. This is a validated method that has been accepted by the authorities in the light of animal welfare for the assessment of skin irritating potential. Therefore, it was chosen as key study. The study was performed with the EPISKIN Reconstituted Human Epidermis Model. The human epidermal cultures were topically treated with the test substance for 15 minutes, followed by a 42-hour post-exposure incubation period. Thereafter cell viability in the culture was assessed by means of the colourimetric MTT reduction assay. In this experimental setting the test substance had no adverse effects on cell viability in the human epidermal culture. Therefore, the substance was considered to be non-irritating to the skin in vitro under the conditions of the test.

As additional support for the in vitro data, although not necessary from a regulatory standpoint, read-across data from an in vivo skin irritation test according to OECD Guideline 404, performed in 3 animals with erucamide (CAS 112-84-5), a structurally related substance, is included and chosen as additional key study (Daamen, 1988).

Erucamide is the fatty acid amide resulting from the amidation of erucic acid. Erucic acid is a mono-unsaturated fatty acid with a carbon chain consisting of 22 carbon atoms with a double bond at position 13 (omega-9) of the carbon chain (cis-docos-13-enoic acid). Comparable to stearamide, the main constituent of Amides C16-C18 (even numbered), it is not classified in Annex VI of Regulation (EC) No 1272/2008, and does not have to be self-classified according to the available experimental data. Erucic acid is also present in various oils and fats which are part of our diets (20-40% in oils from mustard seeds and up to 50% in original high erucic rapeseed oil, <2% in low erucic acid rapeseed oil; Beare-Rogers, 2001) and will also be broken down into shorter-chain fatty acids in the process of beta-oxidation. One of the most famoust uses is in Lorenzo's oil, a 4:1 mixture of the triglyceride forms of oleic and erucic acid, i.e. an investigational drug used for the treatment of adrenoleukodystrophy, for which an U.S. Patent was established.

In this skin irritation study the animals were exposed for 4 hours under semiocclusive conditions. Only slight erythema 1 hour after removal of the dressing was observed, which was completely reversible within 24 hours, and no signs of systemic toxicity were observed. Based on these results the structurally related substance was considered as not irritating. Therefore, Amides, C16-C18 (even numbered) does not have to be considered as irritating to the skin in vivo, either.

There is data for eye irritation available from an in vitro and an in vivo study performed with Amides, C16-C18 (even numbered).

The in vivo study was conducted in compliance with OECD Guideline 405, although only 2 rabbits were included in the test (Bradshaw, 2010). Nevertheless this study was fully acceptable from a regulatory point of view, as even the contribution of a third animal demonstrating severe irritation reactions would have had no impact on the final classification according to the criteria for eye irritation tests with 3 animals. Therefore, this approach is reasonable considering animal welfare aspects, and the study was chosen as key study. No corneal or iridial effects were observed during the 72-hour observation period of the study. Moderate corneal irritation was noted in the treated eyes of both animals one hour after treatment which had completely reversed until the 24-hour reading. No corneal or iridial effects were observed during the observation period relevant for assessment. Minimal conjunctival irritation in form of erythema and edema was observed at the 24-hour reading; both eyes apperared normal at the 48-hour reading. Due to the lack of any effects after 72 hours the study was terminated then. Considering the mild character and the complete reversibility within 72 hours the test substance was considered to be not irritating to the eyes.

This view is supported by an in vitro study which had been performed using the SkinEthic Reconstructed Human Corneal Epithelium Model and which was chosen as additional key study (Warren, 2010). The cells of the tissue cultures were exposed to the test substance for 10 minutes. Following a 3-hour incubation period with MTT, cell viability was determined using the colourimetric MTT reduction assay. Based on the lack of effects on cell viability the test substance was considered to be a Non-Irritant in vitro, as well, in accordance with the in vivo assay.

Justification for classification or non-classification

According to the criteria of Regulation (EC) No 1272/2008 the test substance does not have to be classified as irritating to skin or irritating to the eyes.