Registration Dossier

Administrative data

Description of key information

Skin irritation/corrosion: not irritating (OECD 439, GLP, K, rel. 1).

Eye irritation: not irritating (OECD 405, K, rel.2)

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:
From 2012-09-04 to 2012-10-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 439 and in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2008
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Monitoring Programme (inspected on 10 July 2012)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Several in vivo studies were presumably conducted on the substance. However discordant results were reported, bringing doubts on the identity, purity and composition of the tested substances. As it was not possible to obtain certificates of analysis of the tested batches, it was deemed necessary to conduct a new in vitro study on the substance.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit
- Supplier: SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 12-EKIN-037
- Production date: not reported
- Shipping date: not reported
- Delivery date: 09 October 2012
- Date of initiation of testing: 09 October 2012 (Pre-incubation); 10 October 2012 (main test)
- Expiry date: 15 October 2012

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature, 15 minutes
- Temperature of post-treatment incubation (if applicable): 37°C, 42 hours

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 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.
- Observable damage in the tissue due to washing: none observed
- Modifications to validated SOP: none

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: 540 nm
- Filter: without a reference filter
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 22.2 ± 0.3 (CV = 1.2 %) (≥ 19.5)
- Barrier function: IC50 = 2.1 mg/ml (IC50 ≥ 1.5 mg/ml)
- Morphology: Well-differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
- Contamination:
On blood : absence of HIV1 and 2 antibodies (Architect Abbott); absence of hepatitis C antibodies (Architect Abbott); absence of hepatitis B antigen HBs (Architect Abbott).
On epidermal cells: absence of bacteria, fungus and mycoplasma.
- Reproducibility: All values for the control groups were within the ranges obtained by the testing laboratory in the preceding 120 studies. This was taken to show the correct functioning of the test system.

NUMBER OF REPLICATE TISSUES: Triplicate tissues for test item, negative and positive controls

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not applicable, the test item did not directly reduce MTT.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- Relative mean tissue viability is ≤50%: Irritant
- Relative mean tissue viability is >50%: Non-Irritant (CLP)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL
- Concentration (if solution): as supplied.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): as supplied

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5% (w/v)
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Vehicle:
unchanged (no vehicle)
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
62.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
5.8%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the mean OD540 for the negative control treated tissues was 0.740 and the standard deviation value of the viability was 6.62%. The negative control
acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: Yes; the relative mean tissue viability for the positive control treated tissues was 5.8% relative to the negative control treated tissues and the standard devi
ation value of the viability was 0.7%. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 6.9%. The test acceptance criterion was therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline: All values for the control groups were within the ranges obtained by the testing laboratory in the preceding 120 studies.

Table 7.3.1/1: Mean OD540 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

OD540 of tissues

Mean OD540 of triplicate tissues

± SD of OD540

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.684

0.740

0.049

92.4

100*

6.6

0.762

103.0

0.773

104.5

Positive Control Item

0.044

0.043

0.005

5.9

5.8

0.7

0.038

5.1

0.048

6.5

Test Item

0.437

0.463

0.059

59.1

62.6

6.9

0.431

58.2

0.522

70.5

SD=   Standard deviation

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

Interpretation of results:
study cannot be used for classification
Conclusions:
The test material is not classified as irritating to skin in this in vitro assay according to the criteria of the Regulation (EC) No (1272/2008 (CLP).
Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT (3‑[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

This test was designed to be compatible with the OECD Guideline No. 439 and was performed in compliance with GLP.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre‑labelled 96‑well plate. The optical density was measured at 540 nm.

The relative mean viability of the test item treated tissues was 62.6 ± 6.9 %, after the 15‑minute exposure period.

The quality criteria required for acceptance of results in the test were satisfied.

The test material was considered to be non-irritant according to the criteria of the Regulation (EC) No 1272/2008 (CLP).

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
From 1982-01-14 to 1982-01-26
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Comparable to guideline study with acceptable restrictions: the study is non-GLP.
Qualifier:
according to guideline
Guideline:
other: USA IRLG Testing Standards and Guidelines Work Group (January 1981)
Deviations:
not specified
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: H. Fortkamp, 4540 Lengerich.
- Age at study initiation: 11-17 weeks
- Weight at study initiation: 2.20-2.63 kg bw
- Housing: individually, in steel greed floor cages over trays containing softwood sawdust bedding.
- Diet: ad libitum (Ssniff K Alleindiät für Kanichen, Ssniff Versuchstier-GmbH, 4770 Soest/Westfalen). Not contaminated.
- Water: main water ad libitum. Not contaminated.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Controls:
other: right eye served as control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
Duration of treatment / exposure:
Eye were not rinsed after instillation
Observation period (in vivo):
Examination 1, 24, 48, 72 hours, and then 4 and 7 days after instillation
Number of animals or in vitro replicates:
6 females
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): no done

SCORING SYSTEM: Draize scale

TOOL USED TO ASSESS SCORE: no data

Other: initial pain reaction caused by the test article was made using a 6 point scale.
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
6 animals
Time point:
24/48/72 h
Score:
0.04
Max. score:
4
Reversibility:
fully reversible within: 48 hrs
Irritation parameter:
iris score
Basis:
mean
Remarks:
6 animals
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
6 animals
Time point:
24/48/72 h
Score:
0.17
Max. score:
3
Reversibility:
fully reversible within: 48 hrs
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
6 animals
Time point:
24/48/72 h
Score:
0.04
Max. score:
4
Reversibility:
fully reversible within: 48 hrs
Irritation parameter:
other: discharge
Basis:
mean
Remarks:
6 animals
Time point:
24/48/72 h
Score:
0
Max. score:
3
Irritant / corrosive response data:
Practically no initial pain was seen in any animal on instillation. One hour after dosing moderate chemosis (grade 2) associated with moderate reddening (grade 2) were observed in 2/6 animals. The remaining 4 animals all showed slight chemosis (grade 1) and slight reddening (grade 1). The day after dosing one animal still showed slight chemosis associated with moderate reddening an on this occasion diffuse areas of corneal opacity and a slight ocular discharge were also noted. One further animal showed slight reddening only whilst the eyes of the remaining 4 animals appeared normal. Two days after treatment the eyes of all animals appeared normal.
Other effects:
None

Table 7.3.2/1: Mean irritant/corrosive response data for all animals at each observation time up to removal from the test

 

Score at time point / Reversibility

Cornea

Iris

(/2)

Conjunctivae

Opacity

(/4)

Area

(/4)

Redness

(/3)

Chemosis

(/4)

Discharge

(/3)

1 h

0.00

0.00

0.00

1.33

1.33

0.00

24 h

0.13

0.25

0.00

0.50

0.13

0.00

48 h

0.00

0.00

0.00

0.00

0.00

0.00

72 h

0.00

0.00

0.00

0.00

0.00

0.00

Average 24h, 48h, 72h

0.04

0.08

0.00

0.17

0.04

0.00

Reversibility*)

c.

c.

-

c.

c.

-

Average time (unit) for reversion

48h

48h

-

48 h

48 h

-

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

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, the test material is not classified as irritating to eyes according to the criteria of the Regulation (EC) No 1272/2008 (CLP) and of the GHS
Executive summary:

In an eye irritation study performed similarly to the OECD guideline No. 405, 0.1 mL of undiluted test material was instilled into the left eye of 6 female New Zealand White Rabbit. The eyes were not rinsed after the instillation of the test item. The right eye of each rabbit served as control. Animals were observed at 1, 24, 48 and 72 hours, and the 4 and 7 days after dosing. The reactions in the conjunctiva (redness, chemosis and discharge), the iris and the cornea (opacity and area involved) were scored according to the Draize scale.

Practically no initial pain was seen in any animal on instillation. One hour after dosing moderate chemosis (grade 2) associated with moderate reddening (grade 2) were observed in 2/6 animals. The remaining 4 animals all showed slight chemosis (grade 1) and slight reddening (grade 1). The day after dosing one animal still showed slight chemosis associated with moderate reddening an on this occasion diffuse areas of corneal opacity and a slight ocular discharge were also noted. One further animal showed slight reddening only whilst the eyes of the remaining 4 animals appeared normal. 48 hours after treatment the eyes of all animals appeared normal. 

 

The calculated mean score for each individual lesion for all 6 animals within 3 scoring times (24, 48 and 72 hrs) were as follows: 0.17 for redness, 0.04 for chemosis; 0.08 for cornea opacity; 0.04 for area of cornea involved and 0.00 for discharge, iris and lesions.

 

Under the test conditions, the test material is not classified as irritating to eyes according to the criteria of the Regulation (EC) No 1272/2008 (CLP) and of the GHS.

This study is considered as acceptable and satisfies the requirement for eye irritation endpoint.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

Several in vivo studies were presumably conducted on 4-tert-butylcyclohexyl acetate. However discordant results were reported, bringing doubts on the identity, purity and composition of the tested substances. As it was not possible to obtain certificates of analysis of the tested batches, it was deemed necessary to conduct a new in vitro study on the substance (Warren, 2012).

The purpose of this test was to evaluate the skin irritation potential 4 -tert-butylcylohexyl acetate using the EPISKIN reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. This test was designed to be compatible with the OECD Guideline No. 439 and was performed in compliance with GLP. The quality criteria required for acceptance of results in the test were satisfied. The relative mean viability of the test item treated tissues was 64.7 ± 7.6 %, after the 15‑minute exposure period. With a tissue viability > 50%, 4 -tert-butylcyclohexyl acetate was considered to be non-irritant to skin. The EPISKIN in vitro model is a stand-alone replacement test for in vivo skin irritation testing and allows identifying non-classified chemicals. This study was selected as the key study.

Based on the in vitro results, the sample tested in the study of Hazleton (1982, rel.2) is considered representative of 4 -tert-butylcyclohexyl acetate.

Eye irritation:

A key study was identified (Hazleton, 1982, rel. 2). In this eye irritation study performed similarly to the OECD guideline No. 405, 0.1 mL of undiluted 4 -tert-butylcyclohexyl acetate was instilled into the left eye of 6 rabbits. The eyes were not rinsed after the instillation of the test item. The right eye of each rabbit served as control. Practically no initial pain was seen in any animal on instillation. One hour after dosing moderate chemosis (grade 2) associated with moderate reddening (grade 2) were observed in 2/6 animals. The remaining 4 animals all showed slight chemosis (grade 1) and slight reddening (grade 1). The day after dosing one animal still showed slight chemosis associated with moderate reddening an on this occasion diffuse areas of corneal opacity and a slight ocular discharge were also noted. One further animal showed slight reddening only whilst the eyes of the remaining 4 animals appeared normal. 48 hours after treatment the eyes of all animals appeared normal. The calculated mean score for each individual lesion for all 6 animals within 3 scoring times (24, 48 and 72 hrs) were as follows: 0.17 for redness, 0.04 for chemosis; 0.08 for cornea opacity; 0.04 for area of cornea involved and 0.00 for discharge, iris and lesions. Under the test conditions, 4 -tert-butylcyclohexyl acetate is not classified as irritating to eyes.

The study of BASF (1970, rel.4), although poorly detailed, was considered as supporting since only slight redness was observed in rabbits 1 and 24 hours after instillation, no effects being apparent 8 days after instillation.

In the study of Leberco (1964, Rel.3), the test material was not clearly identified. It was assumed to be tested at 0.625 % but scores leading to classification were obtained. Since there was no indication of the vehicle(s) used and since the sample of Hazleton is considered representative of the substance, this study was disregarded.

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP).

Self classification:

Based on the available information no additional self-classification is proposed regarding both skin and eye irritation according to the CLP and to the GHS.

No data was available regarding respiratory irritation, however the substance not being classified for skin and eye irritation, no classification is expected for respiratory irritation.