Cyclohexanemethanol, 4-(1-methylethyl)-

Endpoint summary

Description of key information

The results of a GLP in vitro skin irritation study using the EpiSkin model indicated that the substance has potential for skin irritation. Although other data, such as from a murine local lymph node assay, do not support irritation, the results of the in vitro assay are considered key to establishing a potential hazard.  

The substance was determined to not be an eye irritant in an in vivo eye irritation study conducted according to OECD test guidelines.

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 August 2014 - 18 August 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted according to OECD Testing Guideline and was compliant per GLPs.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

26 July 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

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:

Following the REACH strategy, the EPISKIN™ Reconstructed Human Epidermis Model method was used to assess skin irritation.

Vehicle:

unchanged (no vehicle)

Details on test system:

- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit, SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 14-EKIN-030
- Production date: not reported
- Shipping date: not reported
- Delivery date: 12 August 2014
- Expiry date: 18 August 2014
- Date of initiation of testing: 13 August 2014

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: not reported. 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++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.
- Observable damage in the tissue due to washing: none reported
- Modifications to validated SOP: none reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm (without a reference filter)
- Filter: not appicable
- Filter bandwidth: not applicable
- Linear OD range of spectrophotometer: no data reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: negative control OD values: 0.847, 0.771 and 0.933
- Barrier function: IC50 = 2.1 mg/ml ( ≥ 1.5 mg/ml)
- Morphology: Well-differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thinck stratum corneum
- Contamination: on the blood of the donor, absence of HIV1 and 2 antibodies, heptatitis C antobodies, hepatitis B antgen HB. On the epidermal cells absence of bacteria, fungus and mycoplasma
- Reproducibility: not reported

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: not applicable

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if relative mean tissue viability is ≤ 50% after 15 minutes of exposure.
- The test substance is considered to be non-irritating to skin if relative mean tissue viability is > 50% after 15 minutes of exposure.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

- The test item was used as supplied (undiluted).
- Amount(s) applied (volume or weight with unit): 10 μL (26.3 μL /cm2) of the test item was applied to the epidermis surface.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Lot/batch no 1528370

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Lot/batch no 1294323
- Concentration (if solution): Sodium Dodecyl Sulphate (SDS) at a 5% (w/v) aqueous solution

Duration of treatment / exposure:

The EpiSkin™ human epidermis skin constructs were treated with the undiluted test item for an exposure period of 15 minutes.

Duration of post-treatment incubation (if applicable):

At the end of the exposure period, tissues were rinsed and incubated at 37 °C, 5% CO2 in air for 42 h.

Number of replicates:

Triplicate tissues for test item, negative and positive controls

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minute exposure period and 42 h post-exposure incubation period

Value:

15.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive 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: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Direct MTT Reduction: The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Assessment of Color Interference with the MTT endpoint: The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

Table 7.3.1/1: Mean OD562Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD562of tissues	Mean OD562 of triplicate tissues	± SD of OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.847	0.850	0.081	99.6	100*	9.6
	0.771			90.7
	0.993			109.8
Positive Control Item	0.218	0.208	0.011	25.6	24.5	1.3
	0.196			23.1
	0.211			24.8
Test Item	0.125	0.130	0.008	14.7	15.3	1.0
	0.126			14.8
	0.139			16.4

Assay validity:

- The relative mean tissue viability for the positive control treated tissues was 24.5% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.3%. The positive control acceptance criterion was therefore satisfied.

- The mean optical density for the negative control treated tissues was 0.850 and the standard deviation value of the percentage viability was 9.6%. The negative control acceptance criterion was therefore satisfied.

- The standard deviation calculated from individual percentage tissue viabilities of the three identically test item treated tissues was 1.0%. Therefore, the test item acceptance criterion was satisfied.

Interpretation of results:

Category 2 (irritant)

Conclusions:

Under the experimental conditions of this study, the test item is classifided as irritant to the skin (Category 2) according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKINTM reconstructed human epidermis model. The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item. Triplicate tissues were treated with 10 μL (26.3 μL/cm2) of the undiluted test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 h. The cell viability was determined by mitochondrial dehydrogenase activity, assessed by the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5‑diphenyltetrazolium bromide) to a soluble, coloured, formazan product. The prediction model uses the percentage viability values (compared to negative control viability) to identify irritant and non-irritant substances.

This assay was valid with negative and positive controls showing results within the acceptable range.

The test substance with a mean tissue viability of 15.3 ± 1.0 %, was predicted as irritant to the skin. The quality criteria required for acceptance of results in the test were satisfied.

Under the experimental conditions of this study, the test item is classifided as irritant to the skin (Category 2) according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Summer 1978.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study appeared to follow OECD 405 with the following deviations: age of the animals not reported; eyes were not examined 24 hours pre-treatment; uncertain whether test material was placed in the conjunctival sac of the eye.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

age of the animals not reported; eyes were not examined 24 hours pre-treatment; uncertain whether test material was placed in the conjunctival sac of the eye.

Principles of method if other than guideline:

The Draize irritation procedure was applied.:
J.H. Draize, “Dermal toxicity”, in Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics, publ., The Association of Food and Drug Officials of the United States, 1959, p. 51.

GLP compliance:

no

Remarks:

Study pre-dates GLP

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

no data

Vehicle:

unchanged (no vehicle)

Controls:

other: The untreated left eye served as the control.

Amount / concentration applied:

Instillation of 0.1 gram of the test material.

Duration of treatment / exposure:

single application

Observation period (in vivo):

All eyes were examined 24, 48, and 72 hours and at four, six, seven, and eight days after dosing.

Number of animals or in vitro replicates:

6 per group

Details on study design:

no data

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.3

Max. score:

3

Reversibility:

fully reversible within: 96 hours

Irritation parameter:

conjunctivae score

Basis:

animal: #2 & #4

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

conjunctivae score

Basis:

animal: #3 & #6

Time point:

24/48/72 h

Score:

0.3

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0.6

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

animal: #1, #4 & #5

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal: #2, #3 & #6

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal: #1, #2, #3, #4, #5 & #6

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal: #1, #2, #3, #4, #5 & #6

Time point:

24/48/72 h

Score:

0

Max. score:

2

Other effects:

no data

Table 7.3.2/1: Irritant/corrosive response data each animal at each observation time up to removal from the test

 

Score at time point / Reversibility	Cornea	Iris (/2)	Conjunctivae
	Opacity (/4)		Redness (/3)	Chemosis (/4)	Discharge (/3)
24 h	0 / 0 / 0 / 0 / 0 / 0	0 / 0 / 0 / 0 / 0 / 0	2 / 2 / 1 / 2 / 1 / 1	1 / 0 / 0 / 1 / 1 / 0	2 / 0 / 1 / 1 / 2 / 0
48 h	0 / 0 / 0 / 0 / 0 / 0	0 / 0 / 0 / 0 / 0 / 0	1 / 1 / 0 / 1 / 1 / 0	0 / 0 / 0 / 0 / 0 / 0	1 / 0 / 0 / 0 / 1 / 0
72 h	0 / 0 / 0 / 0 / 0 / 0	0 / 0 / 0 / 0 / 0 / 0	1 / 0 / 0 / 0 / 0 / 0	0 / 0 / 0 / 0 / 0 / 0	0 / 0 / 0 / 0 / 0 / 0
96 / 120 / 144h	0 / 0 / 0 / 0 / 0 / 0	0 / 0 / 0 / 0 / 0 / 0	0 / 0 / 0 / 0 / 0 / 0	0 / 0 / 0 / 0 / 0 / 0	0 / 0 / 0 / 0 / 0 / 0
Average 24h, 48h, 72h	0.0 / 0.0 / 0.0 / 0.0 / 0.0 / 0.0	0.0 / 0.0 / 0.0 / 0.0 / 0.0 / 0.0	1.33 / 1.0 / 0.33 / 1.0 / 0.67 / 0.33	0.33 / 0.0 / 0.0 / 0.33 / 0.33 / 0.0	1.0 / 0.0 / 0.33 / 0.33 / 1.0 / 0.0
Reversibility*)	-	-	c.	c.	c.
Average time for reversion	-	-	72 h	48 h	72 h

*) Reversibility: c. = completely reversible; n.c. = not completely reversible; n. = not reversible

 E = scoring masked

Interpretation of results:

GHS criteria not met

Conclusions:

The test material caused slight eye irritation; however, these effects were not noted by 96 hours post-application. The mean scores of conjunctival redness and conjunctival oedema were below 2 in all animals following grading at 24, 48 and 72 hours after installation of the test material.

Executive summary:

The test material caused slight eye irritation; however, these effects subsided and were not noted in any animal by 96 hours post-application. The mean scores of conjunctival redness and conjunctival oedema were below 2 in all animals following grading at 24, 48 and 72 hours after installation of the test material. Based on the data, the substance was considered to be a slight primary irritant to the eye of the rabbit.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin/Eye Corrosion

The substance has not been tested for corrosivity, but it is not expected to be corrosive to skin or eyes based on its lack of chemical groups associated with oxidising properties. The substance has been shown to have potential skin irritation properties based on an in vitro skin irritation assay (EpiSkin), but application to the skin of the ears of mice in a Local Lymph Node Assay showed that only Grade 1 erythema (barely perceptible) was observed after 3 days of dermal application with the substance at 100% strength (see below). The substance was shown not to be an eye irritant. As such, given the weight of evidence, it would not appear that the substance is corrosive to skin or eyes.

 

Skin Irritation

The potential for the substance to cause skin irritation was assessed using the EpiSkin reconstructed human epidermis model in an in vitro assay. The results of the test conducted using triplicate cultures at a test dose of 26.3 μL /cm² showed relative mean viability of 15.3% following the 15-minute application to the reconstructed human epidermal tissue cultures. These results indicated the substance is an irritant.

 

Dermal irritation was observed in all animals in an acute dermal study in rabbits (AMR Biological Research Inc, 1979; see Section 7.2 and Section 7.2.3), and this would appear to be consistent with the in vitro data for the substance. However, it should be noted that the test substance was applied to the ventral surface of the animals on shaved and abraded skin (a deviation according to OECD Testing Guideline No. 402). Abrading the skin could have altered its permeability. It also was unclear in the study report whether residual test substance was removed (with water or an appropriate solvent) at the end of the 24-hour exposure period. Taking into consideration the study design deviations and the lack of a Control group, the dermal irritation findings in this study should be interpreted with care as any signs of irritation caused by the substance may have been exacerbated due to the abraded state of the skin, and thus these results alone do not provide conclusive evidence of a dermal irritation effect of the substance.

 

The potential for skin irritation was assessed as part of a murine Local Lymph Node assay (LLNA) (Harlan Laboratories Ltd., 2012) conducted according to OECD Guideline for the Testing of Chemicals No. 429 and in compliance with GLP (see IUCLID Section 7.4.1). Even when applied as an undiluted test material to the ears of mice for 3 days, there was only very slight erythema (barely perceptible) which had resolved by 1 day after the last of the 3 applications; no evidence of oedema was found, and it was concluded that there was no evidence of excessive skin irritation due to the test substance at any test concentration. No erythema or oedema was observed at the diluted test concentration of 25%. These data indicate that the substance may only be a ‘weak’ irritant when applied to intact skin at 100% strength, and would not be an irritant at lower concentrations such as 25%. 

 

In conclusion, the results of the in vitro EpiSkin study provide evidence of a potential for skin irritation by the substance. The severity of skin irritation that might be observed following application to intact skin in vivo cannot be estimated based on available data, given that the results of a dermal toxicity study in rabbits was not properly conducted according to the requirements for an in vivo skin irritation test and thus cannot be considered to contribute relevant information for the assessment of potential for skin irritation in vivo.  However, the results of the LLNA study in mice indicated that there was no evidence of excessive skin irritation following 3 days of repeated application using the substance both neat (100%) and diluted (50%), and no evidence of skin irritation at all at 25%.

 

Eye Irritation

The in vivo eye irritation study with the substance was conducted according to the OECD Testing guideline No. 405 (Acute Eye Irritation/Corrosion). While the study pre-dates GLP, it was considered well-designed and the results were well documented. The substance was instilled in the right eye of six (6) New Zealand albino rabbits in single doses of 0.1 g. Although redness and chemosis were observed in some animals, all effects subsided and were fully reversed in all animals by 96 hours. The authors considered the substance to be slightly irritating to the eye. However, based upon the CLP regulations, from the data provided in the study the substance is not classified as an eye irritant.

 

Respiratory Irritation

For respiratory irritation, human data are the most relevant to form the basis for assessment, as there are no validated testing guidelines in the EU or per OECD for in vitro  or in vivo evaluation of respiratory irritation (see ECHA 2014 guidance R.7a, Section R7.2.3.1 page 179 and Section R7.2.4.1, page 185). There are no human data such as indicated in Section R7.2.4.1 of the ECHA 2014 guidance that indicate respiratory reactions to the substance (e.g., from occupational exposure or consumer experience). In addition, it is justifiable to consider dermal and ocular irritation data when considering possible respiratory irritation (ECHA 2014 guidance, Section R7.2.1.2, page 167). While in vitro data have indicated an adverse skin irritation effect of the substance, in vivo application in an LLNA test (Harlan Laboratories Ltd., 2012) showed it is not severely irritating and, moreover, the substance is not an eye irritant. The substance is not corrosive and not severely irritating, which further supports a low respiratory irritation hazard (ECHA 2014 guidance, Section R7.2.1.1, note on page 166). Therefore, in summary, based on the available data the substance would not be expected to cause an adverse effect on the respiratory tract (not a respiratory tract irritant).

Justification for classification or non-classification

Based on the positive results of an in vitro skin irritation study using a validated test system, the substance meets the criteria for classification as a skin irritant (Category 2) according to Regulation (EC) No 1272/2008, Annex I section 3.2.

 

The mean 24, 48, 72 hour corneal opacity was 0, iritis was 0, conjunctival redness was <2, and conjunctival oedema was <2. As a result, the substance does not meet the criteria for classification for eye irritation according to Regulation (EC) No 1272/2008, Annex I section 3.3.

 

Based on the available data from skin and eye irritation, the substance is not corrosive or severely irritating to the lungs; as such, in the absence of human data to indicate otherwise, the substance is not of concern for respiratory tract irritation. As a result, the substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008, Annex I section 3.8.2.2.1