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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17-04-2000 to 17-07-2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
Method employed in this study for the detection of delayed contact hypersensitivity was the guinea-pig maximization test described by B. Magnusson and A.M. Kligman - "The identification of contact allergens by animal assay, the guinea pig maximisation method"; Invest. Dermatol. 1969. 52, 268-276
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147 (2000)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: February 2000 ; signature: April 2000
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Scientific justification and information as to the availability of the study is attached by the applicant in 'attached background material'.

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
451-230-7
EC Name:
-
Cas Number:
55739-89-4
Molecular formula:
C10H18O
IUPAC Name:
2-ethyl-4,4-dimethylcyclohexan-1-one
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: room temperature, under nitrogen in the dark
- Other: colourless

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier
- Age at study initiation: Young adult (approximately 8 - 12 weeks old).
- Weight at study initiation: 351 – 447 g
- Housing: Individually or in pairs in solid-floor polypropylene cages furnished with woodflakes
- Diet (e.g. ad libitum): Complete maintenance diet for guinea pigs (details in the full study report).
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions; identical to the test

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23°C
- Humidity (%): 30 – 70%
- Air changes (per hr): at least 15 per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

IN-LIFE DATES: From: To: 17-04-2000 to 03-06-2000

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
arachis oil
Concentration / amount:
- Intradermal: 5% test item in Arachis Oil BP
Day(s)/duration:
Day 0
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Concentration / amount:
- Topical: 100% test material (undiluted)
Day(s)/duration:
Day 7
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Remarks:
Arachis Oil BP for the intradermal injections and in Arachis Oil BP for the topical applications (as applicable)
Concentration / amount:
- Challenge: 75% in Arachis Oil BP and 50% in Arachis Oil BP
Day(s)/duration:
Day 21
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Test group: 20 ; Control group: 10
Details on study design:
RANGE FINDING TESTS:
A preliminary irritation study was conducted in order to select test item concentrations to be used in the main study. Intradermal induction: two concentrations were investigated: 1% and 5% v/v in Arachis Oil BP. Two males were used each receiving four 0.1 mL injections of only one concentration of test item. The degree of erythema at the injection sites was assessed approximately 24, 48 and 72 hours and 7 days after injection. Topical Induction: Two males (intradermally injected with Freund's Complete
Adjuvant nine days earlier) were treated with the undiluted test Item and three preparations of the test material (75%, 50% and 25% v/v in arachis oil BP). Applications were made to the clipped flanks under occlusive dressings for an exposure period of 48 hours. The degree of erythema and oedema was evaluated approximately 1, 24 and 48 hours after dressing removal. Topical Induction: The undiluted test material and three preparations of the test item (75%, 50% and 25% v/v in arachis oil BP) were applied to the clipped flanks of two guinea pigs under occlusive dressings for an exposure period of 24 hours. These guinea pigs did not form part of the main study but had been treated identically to the control animals of the main study, up to Day 14. The degree of erythema and oedema was evaluated approximately 1, 24 and 48 hours after
dressing removal. The highest non-irritant concentration of the test item and one lower concentration were selected for the topical challenge stage of the main study.

Final concentrations for definitive testing based on preliminary irritation study:
- Intradermal: 5% test item in Arachis Oil BP
- Topical: 100% test item (undiluted)
- Challenge: 75% and 50% in Arachis Oil BP
% are percent v/v of test item in vehicle. Vehicle: Arachis Oil BP (intradermal induction and topical, as applicable), was chosen on the basis of the most suitable formulation at the required concentrations and maximising the solubility of the test item.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal induction; 1 epidermal induction (topical booster)
- Exposure period: Day 0 intradermal induction and Day 7 topical induction (topical booster)
- Test groups: duplicate injections as follows: 2 ID: Freund's Complete Adjuvant diluted at 50% in water; 2 ID: test item at 5% in arachis oil ; 2 ID: a test item at 5% in arachis oil in equal volumes v/v of FCA at 50% and water at 50%
- Control group: Vehicle and FCA only.
- Site: intradermal induction – three pairs of injections in clipped scapular region;
- Frequency of applications:
- Duration: 0-7 days. On day 7, shorn topical application of 100% test item. On day 7 : 48 hours for epidermal induction. The dressing was removed after 48 hours exposure
- Concentrations: Intradermal induction: A) A 1:1 w/w mixture of Freunds' Complete Adjuvant with water for injection; B) The test item at 5% concentration ; C) A 1:1 w/w mixture of the test item, at 5% and Freunds' Complete Adjuvant plus water (50:5)0. Topical induction: 100% test item concentration using occlusive dressing.
The control group were treated as described for the experimental group except that, instead of the test item, the vehicle was administered.

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 24 hours (topical challenge)
- Exposure period: Day 21 the dressing was removed after 24 hours exposure. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.
- Test groups: 1; test item 75% in Arachis Oil BP vehicle (and 50%) within the same test group
- Control group: 1; vehicle only
- Site: One flank (clipped)
- Concentrations: 75% and 50% in Arachis Oil BP using occlusive dressing.
- Evaluation (hr after challenge): 24 and 48 hours after dressing removal (at Day 23 and 24).
The control group were treated as described for the experimental group except that, instead of the test substance, the vehicle was administered.

OTHER: Mortality, toxicity and body weights along with irritation were examined as part of the study.
Challenge controls:
(Naive) negative control groups consisting of 10 males were exposed to the vehicle in the induction and challenge, consistent the main study with the difference that instead of test item only the vehicle was administered during induction.
Positive control substance(s):
yes
Remarks:
2-Mercaptobenzothiazole (5 and 10% ; no concurrent)

Results and discussion

Positive control results:
A reliability check was performed (six tests are listed with at least 6, 12 and 18 months conduct prior to the study and presented in the full study report) to check the sensitivity of the test system and the reliability of the experimental techniques used. The study used the same conditions as the main study using 2-Mercaptobenzothiazole (at 5 and 10 concentrations %v/v) as positive control.
The skin reactions observed in seven experimental animals in response to the > 20 % test substance concentration in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results lead to a sensitisation rate of 70 - 100% to the 10 %w/w concentration.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
75%
No. with + reactions:
0
Total no. in group:
19
Clinical observations:
Total no. in groups: 20.0 ; Number 5 terminated due to ill health on day 9 ; Clinical observations: None reported; maximum score = 0 (no visible change).
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
75%
No. with + reactions:
0
Total no. in group:
19
Clinical observations:
Total no. in groups: 20.0 ; Number 5 terminated due to ill health on day 9 ; Clinical observations: None reported; maximum score = 0 (no visible change).
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
Total no. in groups: 100.0 ; Number 22 found dead on day 9 ; Clinical observations: None reported; maximum score = 0 (no visible change).
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
Total no. in groups: 100.0 ; Number 22 found dead on day 9 ; Clinical observations: None reported; maximum score = 0 (no visible change).
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
No. with + reactions:
7
Total no. in group:
15
Clinical observations:
Non-concurrent Positive Control Groups: Total no. in groups: 15 ; minimum score = 70% and maximum score = 100% (details in the full study report)
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Table 1: Preliminary irritation study

Skin reactions after intradermal injection

Identification

 

Concentration

(% v/v)

Erythema grading

Systemic Toxicity

A (male)

24 hours

1

2

None

 

48 hours

 

2

None

 

72 hours

 

2

None

 

7 days

 

1-2

None

 

 

 

 

 

B (male)

24 hours

5

2

None

 

48 hours

 

2

None

 

72 hours

 

2

None

 

7 days

 

1-2

None

 

 

 

 

 

The concentration selected for the intradermal injection stage was 5% v/v in Arachis Oil BP

 

Table 2: Preliminary irritation study

Skin reactions after topical application

Identification

Concentration

(% v/v)

Skin Reactions (hours after removal of patches)

 

 

1

24

48

 

 

Er

Oe

Other

Er

Oe

Other

Er

Oe

Other

C (male)

100

2

0

-

1

0

-

0

0

-

 

75

2

0

-

1

0

-

0

0

-

 

50

2

0

-

0

0

-

0

0

-

 

25

1

0

-

0

0

-

0

0

-

 

 

 

 

 

 

 

 

 

 

 

D (male)

100

2

0

-

1

0

-

0

0

-

 

75

2

0

-

0

0

-

0

0

-

 

50

1

0

-

0

0

-

0

0

-

 

25

1

0

-

0

0

-

0

0

-

 

 

 

 

 

 

 

 

 

 

 

Where:

Er = erythema

Oe = oedema

- = no other reactions noted

The undiluted (100%) test item was selected for topical induction

 

Table 3: Preliminary irritation study

Topical application for challenge application

Identification

Concentration

(% v/v)

Skin Reactions (hours after removal of patches)

 

 

1

24

48

 

 

Er

Oe

Other

Er

Oe

Other

Er

Oe

Other

E (male)

100

2

0

-

1

0

-

0

0

-

 

75

1

0

-

0

0

-

0

0

-

 

50

1

0

-

0

0

-

0

0

-

 

25

1

0

-

0

0

-

0

0

-

 

 

 

 

 

 

 

 

 

 

 

F (male)

100

1

0

-

1

0

-

0

0

-

 

75

1

0

-

0

0

-

0

0

-

 

50

1

0

-

0

0

-

0

0

-

 

25

1

0

-

0

0

-

0

0

-

 

 

 

 

 

 

 

 

 

 

 

The concentration of test item for topical challenge was 75% and 50% v/v in Arachis Oil BP

 

Table 4: Challenge applications

Identification

Skin Reactions (hours after removal of patches)

 

24

48

 

50%

 

 

75%

 

 

50%

75%

 

Er

Oe

Other

Er

Oe

Other

Er

Oe

Other

Er

Oe

Other

1

0

0

-

0

0

-

0

0

-

0

0

-

2

0

0

-

0

0

-

0

0

-

0

0

-

3

0

0

-

0

0

-

0

0

-

0

0

-

4

0

0

-

0

0

-

0

0

-

0

0

-

5

*

*

*

*

*

*

*

*

*

*

*

*

6

0

0

-

0

0

-

0

0

-

0

0

-

7

0

0

-

0

0

-

0

0

-

0

0

-

8

0

0

-

0

0

-

0

0

-

0

0

-

9

0

0

-

0

0

-

0

0

-

0

0

-

10

0

0

-

0

0

-

0

0

-

0

0

-

11

0

0

-

0

0

-

0

0

-

0

0

-

12

0

0

-

0

0

-

0

0

-

0

0

-

13

0

0

-

0

0

-

0

0

-

0

0

-

14

0

0

-

0

0

-

0

0

-

0

0

-

15

0

0

-

0

0

-

0

0

-

0

0

-

16

0

0

-

0

0

-

0

0

-

0

0

-

17

0

0

-

0

0

-

0

0

-

0

0

-

18

0

0

-

0

0

-

0

0

-

0

0

-

19

0

0

-

0

0

-

0

0

-

0

0

-

20

0

0

-

0

0

-

0

0

-

0

0

-

 

 

 

 

 

 

 

 

 

 

 

 

 

21 (control group)

0

0

-

0

0

-

0

0

-

0

0

-

22

#

#

#

#

#

#

#

#

#

#

#

#

23

0

0

-

0

0

-

0

0

-

0

0

-

24

0

0

-

0

0

-

0

0

-

0

0

-

25

0

0

-

0

0

-

0

0

-

0

0

-

26

0

0

-

0

0

-

0

0

-

0

0

-

27

0

0

-

0

0

-

0

0

-

0

0

-

28

0

0

-

0

0

-

0

0

-

0

0

-

29

0

0

-

0

0

-

0

0

-

0

0

-

30

0

0

-

0

0

-

0

0

-

0

0

-

 

 

 

 

 

 

 

 

 

 

 

 

 

Where:

* = no data ; terminated on day 9 due to ill health

# = no data ; mortality on day 9

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the test material is not considered to be a contact sensitizer.
Executive summary:

The study was performed according to a method equivalent to guideline OECD TG 406 and EU Method B.6 and consistent with Magnusson-Kligman Guinea Pig Maximisation test under GLP to assess the skin sensitisation potential of the test item. Concentrations selected for the main study were based on the results of a preliminary study. In the main study, after induction (intradermic injection at 5% v/v in Arachis Oil BP and topical application at 100%) and a ten day rest phase of twenty experimental animals were challenged with 75% and 50% v/v in Arachis Oil BP along with parallel negative control group of ten animals: challenged at 75% and 50% v/v in Arachis Oil BP. The treated group (challenge treatment doses of 75%v/v and/or 50%v/v in Arachis Oil BP), gave no skin reactions at 24 and 48-hour observation. No skin reactions were noted in the control groups. There was no test item treatment related mortality. All surviving animals gained bodyweight at the end of the study. Under the conditions of this study, the test item is not considered to be a contact skin sensitiser.