Registration Dossier

Administrative data

Description of key information

Not sensitising, OECD 406 (GPMT), BASF (1990)

Not sensitising, Open Epicutaneous Test, Klecak (1985)

Not sensitising, HMT or RIPT, Klecak (1985)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Principles of method if other than guideline:
Magnusson B and Kligman AM, 1969. The identification of contact allergens by animal assay. The guinea pig maximization test. J. Invest. Dermatol. 52: 268-276.
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study was conducted before the OECD TG on the LLNA was published.
Species:
guinea pig
Strain:
other: Pirbright White, Dunkin Hartley HOE DHPK [SPF-LAC] BO
Sex:
female
Details on test animals and environmental conditions:
- Source: Versuchstierzucht, Hagemann GmbH & Co . KG, D-4923 Extertal 1, FRG
- Rational for choice of animals: Because of the high sensitivity of guinea pigs to a sensitization of the skin which is supposed to be most like that of man. This animal species is the test object acknowledged worldwide for sensitization studies
- Weight at study initiation: 288-340g
- Randomization: According to Salfi, R: A Long Period Random Number Generator with Application to Permutation. Compstat 1974, pp. 28 - 35.
- Housing: 5/cage- Diet: 341 .4 mm-Kaninchen- Meerschweinchen-Haltungsdiät; ad libitum (Supplier : Firma Klingentalmühle AG , CH-4303 Kaiseraugst, Switzerland)
- Water: ad libitum (tap water; about 2 g of ascorbic acid per 10 L water was added to the drinking water twice a week. Quality of water and feed used was ascertained via analysis
- Acclimation: no data
- Clipping of the test animals: if required, about 3 to 5 hours before each reading and before each test substance application at the appropriate application sites

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal and epicutaneous
Vehicle:
olive oil
Concentration / amount:
Induction; Intradermal: 5%Induction; topical: 50%Challenge: 25%Rechallenge:25%
Route:
epicutaneous, occlusive
Vehicle:
olive oil
Concentration / amount:
Induction; Intradermal: 5%Induction; topical: 50%Challenge: 25%Rechallenge:25%
No. of animals per dose:
- Control I: 10
- Control II: 10
- Test group: 20
Details on study design:
PRETEST TESTS: (performed 4 wks before main study), Epidermal Application
- No. of animals: 4/concentration
- Site of application: flanks, respectively on the same areas
- Route of exposure: epicuteneous, patches of filter paper (2 x 2 cm) saturated with the test substance
- Type of coverage: occlusive
- Frequency/Duration: 2X for 24h within a period of 96h
- Evaluation (hr after patch removal): 24h and 48h, scoring of findings using Draize score system for scoring skin irritation
- Result: The minimum irritant concentration was found with a 50% test substance preparation in olive oil DAB 9 and the maximum non-irritant concentration with 25% test substance preparation in olive oil DAB 9 (48 h after the beginning of application).

MAIN STUDYA. INDUCTION EXPOSURE
- Intradermal Injection:
- Site: shoulder
- Concentration: 5% (0.1 mL/site)
- Vehicle: olive oil DAB 9
- Test group 1: Freund's complete adjuvant (FCA) 1:1 with physiological saline(0.9%).
- Test group 2: The test article in olive oil DAB 9.
- Test group 3: Freund's complete adjuvant 1:1 with physiological saline (0.9%) containing the test article- Control group 1: Freund's complete adjuvant (FCA) 1:1 with physiological saline(0.9%).
- Control group 2: Olive oil DAB 9.
- Control group 3: Freund's complete adjuvant 1:1 with physiological saline (0.9%)
- Frequency of applications: 1x- Evaluation (hr after injection): 24h, scoring of findings using Draize score system for scoring skin irritationTopical Induction Exposure:
- Time Schedule: about 1 week after intradermal injections
- Site: same as intradermal injection
- Concentration: 50% cyclohexanol in Olive oil DAB 9 (test groups), olive oil DAB 9 (control groups)
- Route of exposure: epicuteneous, patches of filter paper (2 x 4 cm) saturated with the test substance
- Type of coverage: occlusive
- Duration: 48h.
- Evaluation (hr after injection): 48h, scoring of findings using Draize score system for scoring skin irritation

B. CHALLENGE EXPOSURE
- 1st Challenge: Test groups and control group 1 treated identically in the following manner
- Day(s) of challenge: 2 weeks after topical induction exposure
- Route of exposure: epicutaneous, applied to a 2 x 2cm piece of filter paper
- Exposure period: 24 h- Site: intact clipped flank, right flank (test substance, 25% in vehicle), left flank (vehicle). Left flank of each control group 2 animal was treated with the vehicle. Right flank was not treated
- Vehicle: olive oil DAB 9
- Evaluation (hr after challenge): 24h, 48h and 72h, scoring of findings using Draize score system for scoring skin irritation

- 2nd Challenge- Test groups, control groups 1 and 2 treated identically in the following manner
- Start of challenge: 7days after 1st challenge
- Route of exposure: epicutaneous, applied to a 2 x 2cm piece of filter paper
- Exposure period: 24 h- Site: intact clipped flank, right flank (vehicle), left flank (test substance; 25% in vehicle)- Vehicle: olive oil DAB 9
- Evaluation (hr after challenge): 24h, 48h and 72h, scoring of findings using Draize score system for scoring skin irritation
Positive control substance(s):
no
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25% in olive oil DAB 9
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
slight erythema: 5/10 animals. no other findings
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25% in olive oil DAB 9
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no symptom
Remarks on result:
no indication of skin sensitisation
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
25% in olive oil DAB 9
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no symptom
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25% in olive oil DAB 9
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
slight erythema: 12/20 animals. no other findings
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25% in olive oil DAB 9
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no symptom
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
25% in olive oil DAB 9
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no symptom
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
25% in olive oil DAB 9
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
slight to moderate erythema: 14/20. no other findings
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
25% in olive oil DAB 9
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no symptom
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
72
Group:
negative control
Dose level:
25% in olive oil DAB 9
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no symptom
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
25% in olive oil DAB 9
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
slight to moderate erythema: 14/20. no other findings
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
25% in olive oil DAB 9
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no symptom
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
72
Group:
test group
Dose level:
25% in olive oil DAB 9
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no symptom
Remarks on result:
no indication of skin sensitisation

At intradermal induction, injection of 5% cyclohexanol in olive-oil DAB 9 caused distinct erythema and oedema in all 20 test animals at the 24h observation time point. Necrotic skin changes in addition to distinct oedema could be observed at the injection sites of 5% cyclohexanol in Freunds adjuvant / 0.9% aqueous NaCl-solution (1:1) in all 20 test animals. The control animals injected with olive oil DAB 9 (vehicle) showed only distinct erythema. After percutaneous induction with the minimal irritating concentration (50% cyclohexanol in olive-oil DAB 9), necrotic skin changes (caused by the intradermal induction) accompanied by distinct oedema were observed in all test animals at the 48h observation time point. All animals of control group 1 and 2 which received olive oil DAB 9 (vehicle) exhibited incrustation, partially open (caused by the intradermal induction) in addition to distinct erythema and oedema. After first challenge with the maximal non-irritating concentration (25% cyclohexanol in olive-oil DAB 9) as well as with olive oil DAB 9 (vehicle), slight to distinct erythema were recorded in the controls as well as the test group after 24 h. These skin irritation effects however, were no longer present at the 48 h and 72h observation time points. A similar trend was also noticed after the second challenge where slight to distinct erythema seen after 24h in test and control animals, rechallenged with 25% cyclohexanol in olive-oil DAB 9 and olive oil DAB 9 (vehicle), was no longer present at the 48 and 72h observation time points. Hence, no animal, neither in the control groups nor in the test group showed positive reactions after the first challenge (20 days after intradermal induction) or after the rechallenge (7 days after challenge).

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the study, the test material was found to not be sensitising.
Executive summary:

In a dermal sensitization GLP study with cyclohexanol (98.8%) in olive oil, adult female Pirbright White, Dunkin Hartley HOE DHPK [SPF-LAC] BO guinea pigs were tested using the method of Magnusson and Kligman (Guinea Pig Maximization Test (GPMT)) according to the OECD TG 406. Two control groups were employed in the main study, each comprising of 10 animals. 20 guinea pigs were used in the test group. At intradermal induction, injection of 5% cyclohexanol in olive-oil DAB 9 caused distinct erythema and oedema in all 20 test animals at the 24h observation time point. Necrotic skin changes in addition to distinct oedema could be observed at the injection sites of 5% cyclohexanol in Freunds adjuvant / 0.9% aqueous NaCl-solution (1:1) in all 20 test animals. The control animals injected with olive oil DAB 9 (vehicle) showed only distinct erythema. After percutaneous induction with the minimal irritating concentration (50% cyclohexanol in olive-oil DAB 9), necrotic skin changes (caused by the intradermal induction) accompanied by distinct edema were observed in all test animals at the 48h observation time point. All animals of control group 1 and 2 which received olive oil DAB 9 (vehicle) exhibited incrustation, partially open (caused by the intradermal induction) in addition to distinct erythema and oedema. After first challenge with the maximal non-irritating concentration (25% cyclohexanol in olive-oil DAB 9) as well as with olive oil DAB 9 (vehicle), slight to distinct erythema were recorded in the controls as well as the test group after 24 h. These skin irritation effects however, were no longer present at the 48 h and 72h observation time points. A similar trend was also noticed after the second challenge where slight to distinct erythema seen after 24h in test and control animals, rechallenged with 25% cyclohexanol in olive-oil DAB 9 and olive oil DAB 9 (vehicle), was no longer present at the 48 and 72h observation time points. Hence, no animal, neither in the control groups nor in the test group showed positive reactions after the first challenge (20 days after intradermal induction) or after the rechallenge (7 days after challenge).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In a dermal sensitisation GLP study with cyclohexanol (98.8%) in olive oil (BASF 1990), adult female Pirbright White, Dunkin Hartley HOE DHPK [SPF-LAC] BO guinea pigs were tested using the method of Magnusson and Kligman (Guinea Pig Maximization Test - GPMT) according to the OECD TG 406. Two control groups were employed in the main study, each comprising of 10 animals. 20 guinea pigs were used in the test group. At intradermal induction, injection of 5% cyclohexanol in olive-oil DAB 9 caused distinct erythema and oedema in all 20 test animals at the 24h observation time point. Necrotic skin changes in addition to distinct oedema could be observed at the injection sites of 5% cyclohexanol in Freund's adjuvant / 0.9% aqueous NaCl-solution (1:1) in all 20 test animals. The control animals injected with olive oil DAB 9 (vehicle) showed only distinct erythema. After percutaneous induction with the minimal irritating concentration (50% cyclohexanol in olive-oil DAB 9), necrotic skin changes (caused by the intradermal induction) accompanied by distinct oedema were observed in all test animals at the 48h observation time point. All animals of control group 1 and 2 which received olive oil DAB 9 (vehicle) exhibited incrustation, partially open (caused by the intradermal induction) in addition to distinct erythema and oedema. After first challenge with the maximal non-irritating concentration (25% cyclohexanol in olive-oil DAB 9) as well as with olive oil DAB 9 (vehicle), slight to distinct erythema were recorded in the controls as well as the test group after 24 hours. These skin irritation effects however, were no longer present at the 48h and 72h observation time points. A similar trend was also noticed after the second challenge where slight to distinct erythema seen after 24h in test and control animals, rechallenged with 25% cyclohexanol in olive-oil DAB 9 and olive oil DAB 9 (vehicle), were no longer present at the 48h and 72h observation time points. Hence, no animal, neither in the control groups nor in the test group showed positive reactions after the first challenge (20 days after intradermal induction) or after the rechallenge (7 days after challenge). This study is considered to be relevant, adequate, and reliable for the purposes of classification and risk assessment.

Cyclohexanol was included in a summary report of 268 dermal sensitisation studies (Klecak 1985) conducted on guinea pigs using the open epicutaneous test (OET). The threshold irritating concentration of the test material was estimated from reactions read at 24 hours after application of the test substance. For the induction phase, 0.1 mL of the test material was applied to the clipped flank of the guinea pigs. The applications were prepared daily for three weeks or done five times weekly for four weeks. The application sites remained uncovered. Controls were untreated or treated only with the vehicle. For the challenge phase, an aliquot of the test material was applied to skin areas measuring 2 cm2 on the test and control guinea pigs and reactions read after 24, 48 and / or 72 hours. The material was considered sensitising if at least 1 of 6 animals showed a positive result with a non-irritating concentration in the challenge. The results were not reported for the irritation pre-screen. It was not specified if the concentration reported in the results was the minimal irritating concentration or the lower primary non-irritating concentration. 4% cyclohexanol was reported to produce no contact sensitization in guinea pigs. This study confirms the results from BASF (1990), and is considered to be supporting information.

Cyclohexanol was included in a summary report of 268 dermal sensitisation studies (Klecak 1985) conducted on humans using the HMT or HRIPT. Cyclohexanol was reported as not being a human skin sensitiser under the conditions of the tests. This study confirms the results from BASF (1990), and is considered to be supporting information.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results of the key study (BASF 1990), the substance is not classified for skin sensitisation according to the criteria set out in Regulation (EC) No. 1272/2008.