1,1'-dithiobis[hexahydro-2H-azepin-2-one]

Endpoint summary

Description of key information

1) Wingenroth, 2011, in vitro, skin corrosion, reconstructed human epidermis, non-corrosive
2) Wingenroth, 2011, in vitro, skin irritation, reconstructed human epidermis, non-irritant
3) Wingenroth, 2011, in vitro, eye irritation, human epithelial cornea model (SkinEthicTMHuman Corneal Epithelial Model (HCE)), irritating
4) Wingenroth, 2011, in vitro, eye irritation, Hen’s Egg Test-Chorioallantoic Membrane (HET-CAM) Test, not corrosive

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:

2011-03-09 - 2011-03-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well-documented GLP-Guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline 439 - In Vitro Skin Irritation Reconstructed Human Epidermis Test Method

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: no data

Source strain:

other: not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

Reconstructed tissues
The experiment was carried out on a reconstructed human epidermis EST-1000 (CellSystems, St. Katharinen, Germany).
The tissue equivalents were shipped in 24 well cell culture plates on Agarose supplemented with maintenance medium (Kit contents EST-1000; CellSystems, Cat.-No.CS-1001). Inserts were of 0.6 cm2 size. All tests were performed in triplets.

Adaptation to cell culture conditions
Inserts with EST-1000 reconstructed human epidermis (0.6 cm2) were packed under sterile conditions and were shipped refrigerated on supplemented Agarose. Upon arrival, 6 well culture plates were pre-filled with 1ml of fresh and cool maintenance medium. The reconstructed tissues were placed into the prepared cell culture plates (1 insert/well) and were adapted to the recommended tissue culture conditions (5% CO2, 37°C, max humidity) afterwards for at least 6 hours before use. In case of cultivation of the skin equivalents for more than 24 hours, a daily medium change is required by aspirating the medium and replacing it by 1ml new maintenance medium (37°C) for each well.

Environmental conditions
The environmental conditions in the incubator were standardised as follows:
Incubator temperature: 37 ± 2° C
CO2 gas concentration: 5 %
Humidity: maximum
Occasional deviations from these conditions occurred e.g. as a result of opening the incubators door, but this has no apparent effect on the course or outcome of the study. All Incubation steps were performed in a CO2 atmosphere incubator (Heraeus, Osterode-Germany).

Test item formulation
Test item was used undiluted, i.e. 100% concentration.

Application of the test material and incubation
For testing of chemical induced irritation the EST-1000 inserts were exposed to 30 mg of the test item for 20 min (RT; three inserts per period of incubation time). 0.9% NaCl or 5% SDS (each 30 µl) treated epidermal models were used as negative and positive controls, respectively (determination in triplicates)

Determination of cell viability (MTT)
After the exposure period of 20 minutes the inserts were washed carefully in PBS. After a post – exposure incubation of 42h in the incubator MTT reduction was performed. For viability testing the inserts were placed in new 24 well plates containing 300 µl of MTT solution (37°C, 1 mg/ml in MTT-assay medium, delivered by Cell Systems®). The tissues were incubated for about 3 hours under cell culture conditions (5% CO2, 37°C, max humidity). The extraction of blue formazan was performed in Isopropanol (24 well plates, 2 ml per insert) on a vertical shaker (2 hours). For determination of cell viability the absorption of the Isopropanol-extracts were measured in duplicates at 570 nm in an automatic reader (EL808, Bio-Tek; 96 well format, 200 µl).
Data acquisition and evaluation were done with "Gen5" (software by Bio-Tek).
The MTT reduction assay is the most frequently used assay for the determination of cell viability. The assay depends on the intracellular capacity of living cells to chemically reduce the yellow 3-[4,5-Dimethythiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (Sigma, Deisenhofen-Germany) to blue formazan crystals. The test has shown to give accurate and reproducible results in various laboratories and has practically been modified for accurate analysis of cell viability in three dimensional skin models.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg

NEGATIVE CONTROL
Physiological saline solution (0.9 % NaCl, 30µl)

POSITIVE CONTROL
SDS (sodium dodecyl sulfate) solution (5%, 30µl)

Duration of treatment / exposure:

20 min

Number of replicates:

triplicates

Species:

other: in vitro test: The experiment was carried out on a reconstructed human epidermis EST-1000

Strain:

other: reconstructed human epidermis EST-1000

Details on test animals or test system and environmental conditions:

not applicable as an in vitro test was performed.

The tissue equivalents were shipped in 24 well cell culture plates on Agarose supplemented with maintenance medium. Inserts were of 0.6 cm² size. All tests were performed in triplets.
Inserts with EST-1000 reconstructed human epidermis (0.6 cm²) were packed under sterile conditions and were shipped refrigerated on supplemented Agarose. Upon arrival, 6 well culture plates were pre-filled with 1mL of fresh and cool maintenance medium. The reconstructed tissues were placed into the prepared cell culture plates (1 insert/well) and were adapted to the recommended tissue culture conditions (5% CO2, 37°C, max humidity) afterwards for at least 6 hours before use. In case of cultivation of the skin equivalents for more than 24 hours, a daily medium change is required by aspirating the medium and replacing it by 1mL new maintenance medium (37°C) for each well.

The environmental conditions in the incubator were standardised as follows:
Incubator temperature: 37 ± 2° C
CO2 gas concentration: 5 %
Humidity: maximum
Occasional deviations from these conditions occurred e.g. as a result of opening the incubators door, but this has no apparent effect on the course or outcome of the study. All Incubation steps were performed in a CO2 atmosphere incubator (Heraeus, Osterode-Germany).

Type of coverage:

open

Preparation of test site:

other: no preparation needed, beside the acclimatisation of the inserts to the tissue culture conditions.

Vehicle:

unchanged (no vehicle)

Controls:

other: no animals were used as this is an in vitro test. Physiological saline solution (0.9 % NaCl, 30 µl) was used as negative control. A SDS (sodium dodecyl sulphate) solution (5%, 30 µl) was used as positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg per insert
- Concentration (if solution): 100 % (undiluted)
- Temperature: room temperature

Duration of treatment / exposure:

20 min

Observation period:

reading was performed immediately after the end of exposure

Number of animals:

not applicable as an in vitro test was performed.
3 inserts were used for each incubation time.

Details on study design:

TEST SITE
- Area of exposure: 0.6 cm² (whole insert)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): washed carefully with phosphate buffered saline
- Time after start of exposure: immediately after end of exposure

SCORING SYSTEM:
For the current test, an irritation potential of a test materials according to UN GHS category 2 is predicted if the mean relative tissue viability of three individual tissues exposed to the test substance is less than or equal (≤ ) to 50% of the mean viability of the negative controls.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value

Value:

101.17

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none stated

Table 1: Summary of results

Compound	Cell viability [%]	Classification*
Caprolactam disulphide	101.17	Non Irritant (NI)
Positive control	1.34	Irritant (I)
Negative control	100.00	Non irritant (NI)

*: Classification was done in accordance with EU Test Method B.46.

Table 2: Tabular summary of the results

 

Sample No.	Test item	OD mean*	StdDev	% Viability
1-3	Negative control NaCl 0.9%	1.97	0.14	100.00
4-6	Positive control SDS 5%	0.03	0.00	1.34
7-9	Caprolactam disulphide	1.99	0.33	101.17

* 6 values

 Table 3: Classification criteria

In vitro result	In vivo prediction
mean tissue viability < 50	Irritant (I)
mean tissue viability > 50	Non-irritant (NI)

(I):           Irritant, R38 = Skin irritation category 2, H315

(NI):        Non – irritant = no skin irritation category

Interpretation of results:

GHS criteria not met

Conclusions:

The study was performed according to the OECD Guideline 439, EU Method B.46 without deviations and considered to be of the highest quality (reliability Klimisch 1). The vehicle fulfilled validity criteria of the test system. Caprolactam disulphide was shown not to produce any significant cell viability reduction. The test material was considered to be not irritating under the conditions of this test.

Executive summary:

Caprolactam disulphide was subject to an in vitro test to evaluate its irritating properties (Wingenroth, 2011). This study was performed in 2011, according to OECD TG 439 as well as according to EU Test Method B.46, both without deviations. The experiment was carried out on a reconstructed human epidermis EST-1000 for detection of topically applied skin irritants with the test item caprolactam disulphide. This approach was done, applying the sequential testing strategy of OECD testing guideline 404, which allows the use of validated in vitro methods for the determination of skin corrosion /irritation. A 100% concentration was tested on the skin/epidermal equivalents in triplets. For the determination of time related cytotoxic effects the incubation periods were 20 min (30mg per insert plus 30µL 0.9% NaCl to moisten and ensure good contact with the skin; three replicates). After the exposure period of 20 minutes, followed by a 42 hours post-treatment incubation period, the cell viability was measured to be101.17%in the MTT (Methylthiazoletetrazolium) conversion assay. The results of the concurrent negative control (NC, 0.9% NaCl) and positive control (PC, 5% SDS) demonstrated the viability (NC) and sensitivity (PC) of the test model.

Thus, the results show that caprolactam disulphide is considered to have no skin irritation category, as no significant impact on cell viability after test item exposure was evident.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

2011-03-30 - 2011-03-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well-documented GLP study

Principles of method if other than guideline:

The HCE model is currently involved in the eye irritation validation conducted by the COLIPA following ECVAM guidelines. This study is expected to end late this year and results published early 2012. The HCE is produced and commercialized by SkinEthic since 2000 - more than 11 years -, and is the only model made from human corneal cells. The model is routinely used by the major Cosmetic and Pharmaceutical companies, and has already been prevalidated in 2004. Furthermore, this model is recognized as the model of choice and scientifically relevant as documented by several publications.

GLP compliance:

yes (incl. QA statement)

Species:

other: corneal epithelial tissue (mucosa) without a stratum corneum

Strain:

other: SkinEthicTM Human Corneal Epithelial Model (HCE); SkinEthic, France

Details on test animals or tissues and environmental conditions:

not applicable as an in vitro test was performed.

The experiment was carried out on reconstituted human ocular epithelia (SkinEthicTM Human Corneal Epithelial Model (HCE); SkinEthic, France).
The model used for this study is a corneal epithelial tissue (mucosa) without a stratum corneum. The ultra-structure (tissue morphology and thickness) is similar to the corneal mucosa of the human eye.

The tissue equivalents were shipped in 24 well cell culture plates on semi solid agar’s medium. The scope of supply contains Maintenance Medium for incubation. Inserts were of 0.5 cm² size. All tests were performed in triplets.

HCE inserts (0.5 cm²) were packed under sterile conditions and were shipped on semi solid agar’s medium. Upon receipt each insert was transferred from the packaging plate to 6 well culture plates containing 1 mL of fresh maintenance medium per well. The HCE inserts were incubated for at least 2 hours (5% CO2, 37°C, max humidity). Afterwards a media change was performed and the HCE inserts were continuing adapted overnight to the recommended tissue culture conditions (5% CO2, 37°C, max humidity). In case of cultivation of the skin equivalents for more than 24 hours, a daily medium change is required by aspirating the medium and replacing it by 1 mL new maintenance medium (37°C) for each well.

The environmental conditions in the incubator were standardised as follows:
Incubator temperature: 37 ± 2° C
CO2 gas concentration: 5 %
Humidity: maximum
Occasional deviations from these conditions occurred e.g. as a result of opening the incubators door, but this has no apparent effect on the course or outcome of the study. All Incubation steps were performed in a CO2 atmosphere incubator (Heraeus, Osterode-Germany).

The viability of the cells in the model must be sufficiently high to be able to accurately discriminate between the positive and negative control substances. Cell viability is measured by the amount of MTT reduction, i.e. an OD value following exposure to the negative control substance or the test item.

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable as an in vitro test. Phosphate Buffered Saline (PBS, 30 µl) was used as negative control. 1H-1,2,4-Triazole-3-thiol (30 mg, plus 30 µl PBS to moisten and ensure good contact with the skin) was used as positive control in three replicates.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg
- Concentration (if solution): 100 % (undiluted)

Duration of treatment / exposure:

60 min

Number of animals or in vitro replicates:

not applicable as an in vitro test was performed.
3 inserts per period of incubation time were used.

Details on study design:

For testing of chemically induced eye irritation the HCE inserts were exposed to 30 mg of the test item for 60 min (RT; three inserts per period of incubation time). PBS (30 µL) or 1H-1,2,4-Triazole-3-thiol (30 mg) treated epidermal models were used as negative and positive controls, respectively, in triplicates.

Determination of cell viability (MTT)
After the exposure period of 60 minutes the inserts were washed carefully with PBS. After a post-exposure incubation of 16h in the incubator MTT reduction assay was performed. For viability testing the inserts were placed in new 24 well plates containing 300 µL of MTT solution (37°C, 0.5 mg/mL in Maintenance medium). The tissues were incubated for about 3 hours under cell culture conditions (5% CO2, 37°C, max humidity). The extraction of blue formazan was performed in Isopropanol (24 well plates, 1.5 mL per insert) on a vertical shaker (for at least 2 hours). For determination of cell viability per insert the absorption of the Isopropanol-extracts were measured in duplicates at 570 nm in an automatic reader (EL808, Bio-Tek; 96 well format, 200 µL).

Data acquisition and evaluation were done with "Gen5" (software by Bio-Tek).
The MTT reduction assay is the most frequently used assay for the determination of cell viability. The assay depends on the intracellular capacity of living cells to chemically reduce the yellow 3-[4,5-Dimethythiazol-2-yl]-2,5-diphenyl-tetrazolium bromide to blue formazan crystals. The test has shown to give accurate and reproducible results in various laboratories and has practically been modified for accurate analysis of cell viability in three dimensional skin models.

REMOVAL OF TEST SUBSTANCE
- Washing (if done):
- Time after start of exposure:

SCORING SYSTEM:
Cytotoxicity indices (viability decrease measured by MTT)
A test substance is predicted to be an ocular irritant if the mean relative tissue viability (%) exposed to the test substance is ≤ 50%.

Irritation parameter:

other: % cell viability

Run / experiment:

mean

Value:

38.32

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

No other effects were reported.

Table 1: Summary of results

Compound	Cell viability [%]	Evaluation
Test item	38.32	irritant
Positive control	3.80	irritant
Negative control	100.00	non-irritant

Table 2: Tabular summary of the results 

Sample No.	Test item	OD mean*	StdDev	% Viability
1-3	Negative control  PBS	0.81	0.01	100.00
4-6	Positive control 1H-1,2,4-Triazole-3-thiol	0.03	0.02	3.80
7-9	Test item	0.31	0.03	38.32

* 6 values

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The study was performed according to COLIPA following ECVAM guidelines and considered to be of the highest quality (reliability Klimisch 1). The negative and positive control fulfilled validity criteria of the test system. Caprolactam disulphide was shown to produce any significant cell viability reduction. The test material was considered to be irritating to eyes under the conditions of this test.

Executive summary:

Caprolactam disulphide was subject to an in vitro test to evaluate its ocular irritation properties to the eyes after topical exposure of the test item (Wingenroth, 2011). This study was performed in 2011, according to Colipa following ECVAM guidelines. The experiment was carried out on a human epithelial cornea model for detection of eye irritants with the test item caprolactam disulphide. When cultivated at the air-liquid interface in a chemically defined medium, the immortalized human cornea epithelial cells from the cell line HCE reconstruct a corneal epithelial tissue (mucosa), without a stratum corneum, ultra-structurally (tissue morphology and thickness) similar to the corneal mucosa of the human eye.

The model used is standardised and commercially available (SkinEthic^TMHuman Corneal Epithelial Model (HCE)). A 100% concentration was tested in triplets. A test substance is predicted to be an ocular irritant if the mean relative tissue viability (%) exposed to the test substance is ≤ 50%. For the determination of time related cytotoxic effects the incubation periods were 60 min (30mg per insert plus 30µL 0.9% NaCl to moisten and ensure good contact; three replicates). After the exposure period of 60 minutes, followed by a 16 hours post-treatment incubation period, the cell viability was measured to be 38.32% in the MTT (Methylthiazoletetrazolium) conversion assay. The results of the concurrent negative control (NC, PBS) and positive control (PC, 1H-1,2,4 -Triazole-3 -thiol) demonstrated the viability (NC) and sensitivity (PC) of the test model.

Thus, the results show that caprolactam disulphide is considered to be irritating to eyes under the conditions of this test method.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

Caprolactam disulphide was subject to an in vitro test to evaluate its corrosive properties. This study for predicting non-specific, corrosive potentials of compounds by using reconstructed human skin (RHS) was performed in 2011, according to OECD TG 431 as well as with EC guideline (amending Council Directive 67/548/EEC, B.40 Skin corrosion) without deviations. The experiment was carried out on a reconstructed human epidermis EST-1000 for detection of topically applied skin corrosives with the test item caprolactam disulphide.

Corrosive skin effects of substances are defined as irreversible damage of skin; namely, visible necrosis through the epidermis and into the dermis, following the application of a test substance for up to four hours. In vivo corrosive reactions are typified by ulcers, bleeding, and bloody scabs. A 100% concentration was tested on the skin/ epidermal equivalents in triplets. For the determination of time related cytotoxic effects the incubation periods were 3 min and 60 min, respectively. To check the reliability of this test procedure a blinded positive/negative control study is conducted at regular intervals in the lab. This reliability check is always performed by two technicians, two laboratories in parallel using the same batches of the control test items. The test item was applied at a 100% concentration, i.e. 25 mg per insert. (plus 50 µL 0.9% NaCl to moisten and ensure good contact with the skin).The MTT (methylthiazoletetrazolium) method has determined the following values of viability after 3 min or after 60 min of incubation: 92.41% and 37.71%, respectively. Thus, the results show that no corrosive property of the test item was determined by the assay used.

Caprolactam disulphide was subject to an in vitro test to evaluate its irritating properties (Wingenroth, 2011). This study was performed in 2011, according to OECD TG 439 as well as according to EU Test Method B.46, both without deviations. The experiment was carried out on a reconstructed human epidermis EST-1000 for detection of topically applied skin irritants with the test item caprolactam disulphide. This approach was done, applying the sequential testing strategy of OECD testing guideline 404, which allows the use of validated in vitro methods for the determination of skin corrosion /irritation. A 100% concentration was tested on the skin/epidermal equivalents in triplets. For the determination of time related cytotoxic effects the incubation periods were 20 min (30mg per insert plus 30 µL 0.9% NaCl to moisten and ensure good contact with the skin; three replicates). After the exposure period of 20 minutes, followed by a 42 hours post-treatment incubation period, the cell viability was measured to be 101.17% in the MTT (methylthiazoletetrazolium) conversion assay. The results of the concurrent negative control (NC, 0.9% NaCl) and positive control (PC, 5% SDS) demonstrated the viability (NC) and sensitivity (PC) of the test model.

Thus, the results show that caprolactam disulphide is considered to have no skin irritation category, as no significant impact on cell viability after test item exposure was evident. This is sufficient for classification and labelling as the acute dermal toxicity study also showed no toxicity of the test substance up to 2000 mg/kg bw.

Eye irritation:

Caprolactam disulphide was subject to an in vitro test to evaluate its ocular irritation properties to the eyes after topical exposure of the test item (Wingenroth, 2011). This study was performed in 2011, according to Colipa following ECVAM guidelines. The experiment was carried out on a human epithelial cornea model for detection of eye irritants with the test item caprolactam disulphide. When cultivated at the air-liquid interface in a chemically defined medium, the immortalised human cornea epithelial cells from the cell line HCE reconstruct a corneal epithelial tissue (mucosa), without a stratum corneum, ultra-structurally (tissue morphology and thickness) similar to the corneal mucosa of the human eye.

The model used is standardised and commercially available (SkinEthic^TMHuman Corneal Epithelial Model (HCE)). A 100% concentration was tested in triplets. A test substance is predicted to be an ocular irritant if the mean relative tissue viability (%) exposed to the test substance is ≤ 50%. For the determination of time related cytotoxic effects the incubation periods were 60 min (30mg per insert plus 30µL 0.9% NaCl to moisten and ensure good contact; three replicates). After the exposure period of 60 minutes, followed by a 16 hours post-treatment incubation period, the cell viability was measured to be 38.32% in the MTT (methylthiazoletetrazolium) conversion assay. The results of the concurrent negative control (NC, PBS) and positive control (PC, 1H-1,2,4 -triazole-3-thiol) demonstrated the viability (NC) and sensitivity (PC) of the test model.

Thus, the results show that caprolactam disulphide is considered to be irritating to eyes under the conditions of this test method.

Caprolactam disulphide was subject to an in vitro test to evaluate its ocular irritation properties to the eyes after topical exposure of the test item to the chorioallantoic membrane (Wingenroth, 2011). This study was performed in 2011, according to ICCVAM Test Method Evaluation Report: Appendix G: ICCVAM Recommended Protocol for Future Studies Using the Hen’s Egg Test-Chorioallantoic Membrane (HET-CAM) Test method, November 2006. Hen`s egg tests are used to determine the potential irritant/corrosive property of a test compound using an in vitro alternative to the Draize methodology (OECD405). The extra-embryonal blood vessel system of the incubated chicken egg is used to observe the acute toxicological effects of irritant / corrosive test substances. This test was performed by using the chorioallantoic membrane (HET-CAM, 8d incubation) of chicken eggs. After the application of the test item blood vessels and albumen were examined and scored for the following irritant effects: vasodilation, slight haemorrhage; vessel lysis, strong haemorrhage; blood coagulation, albumen coagulation for a period of 300 seconds. 300 mg of the test item was applied per egg directly on the chorioallantoic membrane. The positive (SDS) and the negative (NaCl) control showed the expected results and proved thereby the validity of the test system. The test item was identified as "non-irritant" to the chorioallantoic membrane (Irritation Score(IS) = 1) under the conditions of this assay.

These results show that even though the irritating effects of caprolactam disulphide were clearly shown in the experiments using the human epithelial cornea model, the HETCAM test was negative. Based on the negative result obtained in the HET-CAM test, the test item is not considered as a substance causing severe damage to eyes according to Chapter R.7a of ECHA's Guidance on Information Requirements and Chemical Safety Assessment. Therefore the eye irritating potential of the test item is not extreme and therefore a classification as a category 2 eye irritant is reasonable.

Justification for selection of skin irritation / corrosion endpoint:
well documented GLP-guideline study according to OECD TG 439 as well as according to EU Test Method B.46, both without deviations.

Justification for selection of eye irritation endpoint:
well documented GLP-study according to Colipa following ECVAM guidelines

Effects on eye irritation: irritating

Justification for classification or non-classification

Skin irritation:

According to the European regulation (EC) No 1272/2008 the test material does not meet the criteria for classification and will not require labelling as irritating to the skin.

Eye irritation:

The values of the cell viability obtained in the human corneal epithelial model were slightly below 50 % and show therefore that eye irritation is to be expected. However, the results of the HETCAM-test did not show positive reactions. Taking these results into account, it is reasonable to classify the substance as a Category 2 irritant for eyes. In summary, the test material does meet the criteria for classification according to the European regulation (EC) No 1272/2008 and will require labelling as irritating to eyes (Category 2, H319).