Reaction mass of bis(2-methylpropyl) (1R,2R,3R,6S)-3,6-dimethylcyclohexane-1,2-dicarboxylate and bis(2-methylpropyl) (1S,2S,3R,6S)-3,6-dimethylcyclohexane-1,2-dicarboxylate

Endpoint summary

Description of key information

Skin irritation, In-Vitro (EPISKIN human epidermis skin construct) - Not irritating

Eye irritation, In-Vitro (Bovine Corneal Opacity and Permeability Assay) - 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:

13 March 2012 - 28 May 2012

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:

other: Standard Operating Procedure, In Vitro Skin Irritation Test: Human Epidermis Model (L’Oreal 2009)

Deviations:

not specified

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: H1L002
- Expiration date of the lot/batch: 30 September 2013

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark

- Purity: 99.0%
- Molecular weight: 312.45
- Chemical formula: C18H32O4

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
Episkin model, Batch number 12-EKIN-021, Expiry date 28 May 2012

After incubation of at least 24 hours in maintenance medium, triplicate tissues were dosed for 15±0.5 minutes with the test substance, negative or positive control at room temperature.
A maximum of four samples were applied in a block with a minimum of 1 minute intervals between each application of substance. On application of 10µL, the positive control was spread over the tissue for approximately 30 seconds and then respread with a curved flat spatula after 7 minutes application time.

After 15±0.5 minutes, each tissue was rinsed with 25 mL sterile Dulbeccos PhosphateBuffered Saline (DPBS) to remove residual test substance. Inserts were then blotted on absorbent paper to remove remaining DPBS. Each insert was then transferred to a well containing 2 ml maintenance medium and incubated for 42±1 hour at 37±2°C in a humidified atmosphere of 5% CO2 in air.

After 42±1 hour, each insert was transferred to a well containing 2 mL of 0.3 mg/mL MTT and incubated for 3 hours ± 5 minutes at 37±2°C in a humidified atmosphere of 5% CO2 in air.

At the end of 3 hours ± 5 minutes, the triplicate inserts were blotted on absorbent paper. The epidermis was removed from the insert using a biopsy punch, the epidermis separated from the collagen matrix using forceps and both parts placed in a micro tube. When all tissues had been punched, the tissues were vortexed with 500 μL of acidic isopropanol (0.04 N HCl final concentration).

The tissues were extracted by storing at 2-8ºC, protected from light, for 48 to70 hours.

After formazan extraction, duplicate 200 µL aliquots of the extractant from each micro tube were pipetted into the wells of flat-bottomed 96-well plates. The extractant was mixed by vortexing prior to taking the aliquots. The absorbance was read at 540 nm, with six wells containing acidified isopropanol (0.04 N HCl final concentration) as a blank.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
Applied as such (no vehicle)

NEGATIVE CONTROL
Dulbecco's Phosphate Buffered Saline (DBPS) with magnesium and calcium

POSITIVE CONTROL
5% Sodium Dodecyl Sulphate in distilled water

10 µL of each of the above solutions were applied to tissues

Duration of treatment / exposure:

15±0.5 minutes

Duration of post-treatment incubation (if applicable):

42±1 hours

Number of replicates:

Triplicate tissues

Irritation / corrosion parameter:

% tissue viability

Value:

140.7

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The test substance, SIBE 138, was diluted to 10% v/v with distilled water to obtain an aqueous solution for pH measurement. The pH of the test substance, measured using pH indicator paper, was approximately 7.0.
The mean absorbance of the triplicate negative control values was 0.723 which was between the minimum and maximum values of 0.6 and 1.5. The standard deviation (SD) of the percentage viability was 16 which was below the maximum value of 18.
The percentage mean viability of the positive control was 25.2 ± 5.5 of the negative control. These were below the maximum acceptance values of 40% viability and SD of 18.

Interpretation of results:

GHS criteria not met

Conclusions:

It was concluded that the test substance, SIBE 138, with a mean tissue viability of 140.7 ± 15.2%, was predicted as non-irritant to the skin.

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:

20 March 2012 - 22 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Characteristics of donor animals: Eyes were excised from animals aged approximately 24 months
- Storage, temperature and transport conditions of ocular tissue: Eyes were transported and maintained at ambient temperature in sufficient HBSS containing 1% (v/v) penicillin / streptomycin solution to fully cover the eyes in
- Time interval prior to initiating testing: The eyes were used within 4 hours of excision
- indication of any existing defects or lesions in ocular tissue samples: Eyes were examined for defects (opacity, scratches, cuts, pigmentation, etc.) prior to use and those exhibiting defects were discarded
- Indication of any antibiotics used: Excised eyes were maintaned and transported in HBSS containing 1% (v/v) penicillin / streptomycin solution

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration: Undiluted

Duration of treatment / exposure:

10 minutes ± 30 seconds

Duration of post- treatment incubation (in vitro):

Two hours ± 10 minutes

Number of animals or in vitro replicates:

Three / triplicate

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At least three times or until the wash medium was clear with no discolouration (four times in the case of SIBE138)

Irritation parameter:

in vitro irritation score

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Value:

0.333

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

The test substance, SIBE 138, was diluted to 10% w/v with 0.9% saline to obtain an aqueous solution for pH measurement. The pH of the test substance, measured using pH indicator paper, was approximately 7.0.

Sample Opacity ± SD = 0.333 ± 0.000

Permeability ± SD = 0.013 ± 0.008

In vitro irritancy Score ± SD = 0.5 ± 0.1

In vitro classification = Non-Corrosive/Non-Severe irritant

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance, SIBE 138, elicited an In Vitro Irritancy Score of 0.5 ± 0.1 and was predicted to be a non-corrosive / non-severe eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In-vitro studies performed in compliance with GLP and according to relevant OECD and EC test guidelines are available to assess the irritation potential of SIBE138 to the skin (Huntingdon Life Sciences study LBM0038, 2013) and the eyes (Huntingdon Life Sciences study LBM0032, 2013). Each of the available studies concluded a clear negative result. On the basis of the available data it is concluded that a classification for skin or eye irritation is not required according to Annex I of the CLP Regulation (Regulation (EC) 1272/2008, including Amendments to the 4th May 2017).