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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
20 Feb - 12 Apr 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study. Refer to endpoint summary Sensitisation and IUCLID Section 13 for reporting and justification of the analogue approach.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPP 81-6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
Buehler test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): cyclohexane
- Molecular formula: C6H12
- Molecular weight: 84.16
- Smiles notation: C1CCCCC1
- InChl: 1S/C6H12/c1-2-4-6-5-3-1/h1-6H2
- Substance type: pure substance
- Physical state: clear liquid
- Analytical purity: 99.98%
- Purity test date: 1996-11-01

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Davidson Mill Breeding Labs, Jamesburg, USA
- Weight at study initiation: 314 – 319 g (main study); 307-379 g (screening study)
- Housing: animals were individually housed in stainless steel cages with elevated wire mesh flooring
- Diet: Purina Guinea Pig Diet #5025, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-27
- Humidity (%): 20-70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 12 Mar 1996 To: 12 Apr 1996

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: induction: 95% ethanol; challenge: acetone
Concentration / amount:
Induction: 10%
Challenge: 10%
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: induction: 95% ethanol; challenge: acetone
Concentration / amount:
Induction: 10%
Challenge: 10%
No. of animals per dose:
range-finding test: 20 (in test groups)
main study: 10 (controls), 10 (challenge controls), 20 (in test groups: 9 males and 11 females), 10 (positive controls: 3 males and 7 females), 10 (naive positive controls)
Details on study design:
RANGE FINDING TESTS: three groups of 4 animals (2 males and 2 females) were exposed to 5, 10, 15, 20 and 25% of test substance in 95% ethanol by using the patching technique described in the induction stage. Necrosis was observed after exposure to 25, 20 and 15% concentration of the test substance in 95% ethanol. At 5 and 10% concentration in 95% ethanol, very faint redness was noted in 1/4 animals. Thus, the 10% concentration was chosen for the induction phase. Two groups of 4 animals (2 males and 2 females) were exposed to 5, 10, 15, 20% of the test substance in acetone in order to select appropriate concentrations for the challenge exposure. After treatment with 20% test substance in acetone, very faint redness was observed in 1/4 animals. All other concentrations did not cause any irritations. Based on these results, 10% test substance was chosen for the challenge exposure.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6 h
- Test groups: test substance in 95% ethanol
- Control group: 95% ethanol
- Site: left flank
- Frequency of applications: every 7 days
- Duration: 0-21
- Concentrations: 10%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 28
- Exposure period: 6 h
- Test groups: test substance in acetone
- Control group: test substance in acetone and acetone
- Site: right flank (test substance) and left flank (vehicle)
- Concentrations: 10%
- Evaluation (hr after challenge): 24 and 48 h after patch removal

Challenge controls:
An additional challenge control group (5 males and 5 females) was used and treated with 10% of the test substance in acetone according to the conditions described for the challenge exposure.
Positive control substance(s):
yes
Remarks:
induction: 0.1% 1-chloro-2,4-dinitrobenzene (DNCB) in 50% ethanol/physiological saline; challenge: 0.07% DNCB in acetone

Study design: in vivo (LLNA)

Statistics:
The individual body weight gain was statistically evaluated by analysis of variance (ANOVA) and Neuman Keuls test. Statistical significance was indicated at p < 0.05.

Results and discussion

Positive control results:
0.1% of the positive control test substance DNCB in 50% ethanol/physiological saline induced no or moderate redness in the induction phase of the study. After challenge exposure with 0.07% DCNB in acetone, positive skin sensitisation reactions were seen in 8/10 animals (80%), thus meeting the reliability criteria for the non-adjuvant skin sensitisation test (≥ 15%).

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
induction: 0%; challenge: 10%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: induction: 0%; challenge: 10%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
induction: 10%; challenge: 10%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
redness was observed in 1 animal
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: induction: 10%; challenge: 10%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: redness was observed in 1 animal.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
induction: 0.1%; challenge: 0.07%
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
faint to moderate redness
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: induction: 0.1%; challenge: 0.07%. No with. + reactions: 8.0. Total no. in groups: 10.0. Clinical observations: faint to moderate redness.
Reading:
1st reading
Hours after challenge:
24
Group:
other: challenge control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group:
Reading:
1st reading
Hours after challenge:
24
Group:
other: naive positive control
Dose level:
0.07%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group:
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
induction: 0%; challenge: 10%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: induction: 0%; challenge: 10%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
induction: 10%; challenge: 10%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: induction: 10%; challenge: 10%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
induction: 0.1%; challenge: 0.07%
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
faint to moderate redness
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: induction: 0.1%; challenge: 0.07%. No with. + reactions: 8.0. Total no. in groups: 10.0. Clinical observations: faint to moderate redness.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: challenge control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group:
Reading:
2nd reading
Hours after challenge:
48
Group:
other: naive positive control
Dose level:
0.07%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group:

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The skin sensitisation potential of cyclohexane was investigated in a GLP-compliant study using a modified Buehler test method in accordance with OECD guideline 406.
9 male and 11 female guinea pigs were induced with 10% cyclohexane in ethanol and challenged with 10% cyclohexane in acetone. The test included concurrent negative (vehicle) and positive controls (1-chloro-2,4-dinitrobenzene, 0.1% in 50% ethanol: 0.9% saline at induction and 0.07% in acetone at challenge).
Induction treatment resulted in no redness (14/20 animals) to very faint redness on some tested animals (6/20 animals). Very faint redness was seen 24 h after challenge in 1/20 animals of the test group and no reactions were observed in any animal at 48 h post-challenge.
No skin reactions were observed in negative control animals. The incidence of sensitisation among the positive control animals was 8/10.
The incidence of sensitisation in cyclohexane-induced and -challenged animals was 0/20.

The study results do not fulfil the classification criteria for skin sensitisation according to Regulation (EC) No 1272/2008 and Directive 67&548/EEC.

CLP: not classified
DSD: not classified