butyl 4-hydroxycyclohexanecarboxylate

Endpoint summary

Description of key information

The substance did not show a skin irritating potential in an in vitro skin irritation test using a reconstructed human skin model.

The eye irritating potential of the substance was investigated in two in vitro tests, i.e. a BCOP and an ICE test. No corrosive or severe irritating effects have been observed in the BCOP test. From the ICE test, it is concluded that the substance is irritating to the eyes (Category 2, sub-category 2B).

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:

2013-03-18 to 2013-07-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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:

other: SOP SkinEthic Skin Irritation Test-42bis

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The human skin RHE model has been validated for this type of study.

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 µL

NEGATIVE CONTROL
- PBS buffer
- Amount(s) applied (volume or weight): 16 µL

POSITIVE CONTROL
- Sodium dodecyl sulfate
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5%

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

3 membranes for each group (test item, negative control and positive control)

Species:

other: Reconstructed human epidermis model

Details on test animals or test system and environmental conditions:

Reconstructed human epidermis model (RHE)
Supplier: SkinEthic
Test System: Human keratinocytes on a polycarbonate filter inserted in a sealed 24-well plate
Tissue Batch: 13 022A 0308
The tissue batch used fully met the quality and validity criteria as described in OECD TG 439

Type of coverage:

open

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

16 µL

Duration of treatment / exposure:

42 minutes (according to the suppliers protocol)

Number of animals:

The test item, the positive and the negative control were tested in batch-triplicates, i.e. 9 tissues were used for this study

Details on study design:

The study was performed according to OECD TG 439, Council Regulation (EC) No. 761/2009, Method B.46 and the SOP SkinEthic Skin Irritation Test-42bis (2009)

Irritation / corrosion parameter:

% tissue viability

Value:

90.33

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: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (mean OD = 2.183 at 570 nm)
- Acceptance criteria met for positive control: Yes (mean relative viability = 1.00%)
- Acceptance criteria met for variability between replicate measurements: yes (standard deviation values < 18%)

Irritant / corrosive response data:

not applicable

Other effects:

not applicable

This in vitro study was performed to assess the irritating potential of the test item by means of the Human Skin Model Test. The test item was applied topically to a human reconstructed skin model followed by a cell viability test. Cell viability was measured by dehydrogenase conversion of MTT into a blue formazan salt, which was quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict skin irritation. Triplicates of the human skin model RHE^TMwere treated with 16 µL of the test item, the negative (PBS buffer) or the positive control (5% aqueous dodecyl sulphate solution) for 42 minutes, respectively.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the treatment interval thus ensuring the validity of the test system. After treatment with the negative control the absorbance values reached the required acceptability criterion of an optical density (OD) >/=1.2 and </=2.5 for the treatment interval thus showing the quality of the tissues.

The mean relative tissue viability after treatment with the test item was 90.33% and, thus, higher than 50.0%. Therefore, the test item is considered to possess no irritating potential to the skin.

Under the experimental conditions reported, the test item is not irritating to the skin.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

Under the experimental conditions reported, the test item is not irritating to the skin.

Executive summary:

see above

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:

2014-12-10 to 2015-02-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

yes

Remarks:

Histopathology included

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

yes

Remarks:

histopathology included

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: DRZ22129-058
- Expiration date of the lot/batch: 2015-06-04
- Purity test date: 2014-06-04

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: controlled room temperature (15-25°C), below 70% RH
- Stability under test conditions: yes

Species:

other: Isolated chicken eyes

Strain:

other: COBB 500

Details on test animals or tissues and environmental conditions:

Source of chicken eyes: Chicken eyes were isolated from chicken heads, which were obtained from a slaughter house
Strain of chicken: COBB 500

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

30 µL of the test item was applied onto the entire surface of the cornea

Duration of treatment / exposure:

The eyes were rinsed with 20 mL physiological saline after an exposure period of 10 seconds from the end of application.

Number of animals or in vitro replicates:

Number of test material treated eyes: 3
Number of positive control treated eyes: 3
Number of negative control eye: 1

Details on study design:

The eye irritating potential of the test item was investigated in isolated chicken's eyes. The test was performed according to OECD TG No. 438.

Irritation parameter:

percent corneal swelling

Remarks:

at up to 75 min [%]

Value:

3.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Remarks:

at up to 240 min [%]

Value:

6.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Value:

1.67

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No.: 438 (26th July 2013). After the zero reference measurements, the eye was held in horizontal position and 30 µL of the liquid test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with saline. The positive control eyes were treated with 30 µL of 5% (w/v) Benzalkonium chloride solution. The negative control eye was treated with 30 µL of physiological saline (Salsol solution, NaCl 0.9% w/v). Based on this in vitro eye irritation in the isolated chicken eyes test, the test item is not classified as a severe irritant and not classified as non-irritant. To further establish the classification, histopathological observations were made on two sections of each of the 3 corneas treated with test item (6 sections). Microscopic evaluation showed very slight erosion of the corneal epithelium (6/6). No stromal and endothelial changes were observed as well as no effects on integrity of basement, Bowman’s and Descemet’s membranes. Thus, the test item was categorized as Category 2 (sub-category 2B). Taking a weight of evidence approach, the overall results were categorized as Category 2 (sub-category 2B).

Interpretation of results:

Category 2B (mildly irritating to eyes) based on GHS criteria

Remarks:

Migrated information

Conclusions:

Based on th eresults of this test, the test item should be classified as irritating to the eyes and categorized as Category 2 (sub-category 2B).

Executive summary:

see above

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance must not be classified as a skin irritant.

Based on the results of two in vitro eye irritation tests (BCOP, ICE), the substance should be classified as irritating to the eyes (Category 2).