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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
data is from experimental reports

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Principles of method if other than guideline:
The aim of the study was to assess the sensitizing capacity or allergenic potential of the test chemical by challenge (topical) application after the induction period of intradermal injection and epidermal (topical) application in guinea pigs.
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol
EC Number:
266-100-3
EC Name:
4-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol
Cas Number:
66068-84-6
Molecular formula:
C16-H28-O
IUPAC Name:
4-{5,5,6-trimethylbicyclo[2.2.1]heptan-2-yl}cyclohexan-1-ol
Test material form:
liquid
Details on test material:
Name of the test chemical: 3-(2,3,3-trimethyl-6-bicyclo[2.2.1]heptanyl)cyclohexan-1-ol
Common Name: Iso camphyl cyclohexanol
Molecular Formula: C16H28O
Molecular Weight: 236.396 g/mol
SMILES Notation: OC1CCC([C@@H]2[C@@H]3C[C@@H](C(C)(C)[C@@H]3C)C2)CC1
InChI: 1S/C16H28O/c1-10-14-8-12(16(10,2)3)9-15(14)11-4-6-13(17)7-5-11/h10-15,17H,4-9H2,1-3H3
Substance Type: Organic
Physical State: Liquid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CADILA PHARMACEUTICALS, GUJARAT, INDIA
- Microbiological status of animals, when known: Healthy young adult animals were used for study.
- Age at study initiation: Approximately 10- 12 Weeks (At the time of intradermal injection)
- Weight at study initiation: Minimum: 280 g; Maximum: 408 g (At the time of intradermal injection)
- Housing: Animals were housed in groups of 5 animals per cage in stainless steel cages.
- Bedding Material : Corn cob supplied by Sparconn Life Science, Bangalore
- Diet (e.g. ad libitum): All animals were provided conventional laboratory guinea pig diet (Nutrivet Life Sciences, Pune, India) ad libitum
- Water (e.g. ad libitum): Aqua guard filtered tap water was provided ad libitum via drinking bottles with 0.1 % Vitamin C (Ascorbic acid).
- Acclimation period: Animals were acclimatized to the test conditions for a period of 6 days for dose range finding and 11 days for main study prior to the test
- Indication of any skin lesions:
- Randomization: The animals were randomized for main study by validated VSS software.
- Animal Identification: During acclimatization, the animals were marked with numbers starting from 101 onwards, on inner side of ears by using permanent marker. After randomization all animals were marked by paw micro tattooing and cage cards. Following allocation to the study, each animal were assigned an individual cage card labelled with at least study no., study type, test system, sex, dose, experiment start date and experiment completion date.
Room Sanitation: The experimental room floor and work tops were swept and mopped with disinfectant solution every day.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Minimum: 18.70 °C; Maximum: 22.80 °C
- Humidity (%): Minimum: 50.70%; Maximum: 67.80%
- Air changes (per hr): More than 12 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light:12 hours dark
- IN-LIFE DATES: From: April 06, 2015 To: May 19, 2015

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
propylene glycol
Concentration / amount:
0.1 ml.
a) a (1:1) mixture (v/v) Fruend’s Complete Adjuvant (FCA) + distilled water.
b) 5.0 % (v/v) concentration ratio of test item in propylene glycol.
c) 5.0 % (v/v) concentration of test item formulated in a 1:1 mixture of FCA + distilled water.
Day(s)/duration:
24 hours
Adequacy of induction:
other: The concentration of the test item for each induction exposure was the highest to cause mild-to-moderate skin irritation
Route:
epicutaneous, semiocclusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.2 ml test item (as such) was applied to the right flank with filter paper
Day(s)/duration:
48 hours
Adequacy of induction:
other: The concentration of the test item for each induction exposure was the highest to cause mild-to-moderate skin irritation
Challenge
No.:
#1
Route:
epicutaneous, semiocclusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.2 ml test item (as such) on left flank and 0.2 ml of acetone with the filter paper was applied on right flank of each animal
Day(s)/duration:
24 hours
Adequacy of challenge:
other: The concentration of the test item used for the challenge exposure was the highest non-irritating dose.
No. of animals per dose:
18 (Range finding - 03 and Main Study -15)
Group 1: Control (G1) / 5 animals.
Group 2: Treatment (G2) /10 animals.
Details on study design:
RANGE FINDING TESTS:
Dose Range Finding Study
In order to check the animal’s tolerability to the test concentrations application, dose range finding study was performed.
One animal was used to determine the test concentration for intradermal induction and two animals for topical induction as well as challenge application. Pair of four intradermal injections with different concentration of test item to the guinea pig was made, i.e. two injections (100 µl) per concentration on each side of the dorsum. Before administration, 5.0, 2.5, 1.0 and 0.5% (v/v) test item concentrations were prepared with propylene glycol. After 24 and 48 hours post injection, sites were observed for erythema and oedema formation as per Draize Method.
At 5.0% (v/v) concentration, well defined erythema and slight oedema (edges of area well defined by definite raising) was observed at 24 and 48 hours post intradermal injection. At 2.5% (v/v) concentration, well defined erythema and very slight oedema (barely perceptible) was observed at 24 and 48 hours post intradermal injection. At 1.0% (v/v) concentration, very slight erythema (barely perceptible) and very slight oedema (barely perceptible) was observed at 24 and 48 hours post intradermal injection. At 0.5% (v/v) concentration, very slight erythema (barely perceptible) and very slight oedema (barely perceptible) was observed at 24 and 48 hours post intradermal injection.
As 5.0% (v/v) concentration was highest dose to cause mild to moderate erythematous reactions. Therefore, the suitable concentration for intradermal induction was determined as 5.0% (v/v) concentration of test item in propylene glycol.
To determine the topical and challenge concentration, the test was carried out with two guinea pigs with different concentrations of test item i.e. 25, 50, 75% (v/v) and 100% test item. After clipping the hair at the flank region of the animals, a filter paper (2 x 4 cm)2 was loaded with test item moistened with 0.2 ml of vehicle (80% ethanol). The filter paper was fixed by a semi occlusive dressing for a period of 24 hours. After bandage removal, the application sites were observed for erythema and oedema formation as per draize method at 24 and 48 hours.
During observations at 24 and 48 hours after topical application, no erythema and no oedema were observed. Therefore, the suitable concentration for topical application (dermal induction) and challenge exposure was selected 100% test item since that was the highest non-irritating dose.


MAIN STUDY
A. INDUCTION EXPOSURE
1. Intradermal
- No. of exposures: single
- Exposure period: 24 hours
- Test groups: 10 animals
- Control group: 5 animals
- Site: an area of approximately (4 x 6 cm)2 on the shoulder areas of each guinea pig was clipped.
- Frequency of applications: single
- Duration: 24 hours
- Concentrations:
test group =The volume of each injection was 0.1 ml.
a) a (1:1) mixture (v/v) Fruend’s Complete Adjuvant (FCA) + distilled water.
b) 5.0 % (v/v) concentration ratio of test item in propylene glycol.
c) 5.0 % (v/v) concentration of test item formulated in a 1:1 mixture of FCA + distilled water.
Control group = The volume of each injection was 0.1 ml.
a) a (1:1) mixture (v/v) Fruend’s Complete Adjuvant (FCA) + distilled water
b) Propylene glycol
c) 1:1 mixture of Injection a) and Injection b)

2. Epicutaneous, semi-occlusive
- No. of exposures: single
- Exposure period: 48 hours
- Test groups: 10 animals
- Control group: 5 animals
- Site: right flank
- Frequency of applications: single
- Duration: 48 hours
- Concentrations: test group = 0.2 ml test item (as such)
Control group = 0.2 ml of 80% ethanol was applied

B. CHALLENGE EXPOSURE
- No. of exposures: single
- Day(s) of challenge: day 21
- Exposure period: 24 hours
- Test groups: 10 animals
- Control group: 5 animals
- Site: right flank
- Concentrations: 0.2 ml test item (as such) on left flank and 0.2 ml of acetone with the filter paper was applied on right flank of each animal
- Evaluation (hr after challenge): At approximately 24 and 48 hours after patch removal of challenge application, the skin reaction for erythema / oedema was observed and recorded as per Magnusson & Kligman grading scale for the evaluation of challenge patch test reactions.

OTHER: Induction:
After 24 hours post intradermal injections, skin reaction of animals from both control and treatment groups were observed and recorded as per Draize method for the evaluation of challenge patch test reactions.
Topical Application
Day 6: Control and Treatment Groups
Approximately 24 hours before the topical application, the test area was painted with 0.5 ml of 10% Sodium Lauryl Sulphate in Vaseline on right flank, in order to create a local skin irritation.
Challenge controls:
no data available
Positive control substance(s):
not specified

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0.2 ml undiluted test chemical
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no dermal reactions or signs of toxicity were observed
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
72
Group:
test chemical
Dose level:
0.2 ml of undiluted test chemical
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no dermal reactions or signs of toxicity were observed
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
72
Group:
other: control group
Dose level:
0.2 ml of acetone
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no dermal reactions or signs of toxicity were observed
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

Table 1:Skin Response After the Challenge Application

 

Group:G1 (Control)                                                                                                          Sex:Male

Animal
No.

Reaction readings*
after removal of bandage

24 hours

48 hours

1

0

0

2

0

0

3

0

0

4

0

0

5

0

0

 

 

 

Group:G2 (Treatment)                                                                                                     Sex:Male

Animal
No.

Reaction readings*
after removal of bandage

24 hours

48 hours

6

0

0

7

0

0

8

0

0

9

0

0

10

0

0

11

0

0

12

0

0

13

0

0

14

0

0

15

0

0

Key:*= as per Magnusson & Kligman grading scale.

Table 2:Clinical Signs and Mortality

Sex:Male

Animal No.

Group

Experimental day 0 to 24

Clinical Signs

Mortality

1

G1 (Control)

Normal

No mortality/ morbidity

2

Normal

No mortality/ morbidity

3

Normal

No mortality/ morbidity

4

Normal

No mortality/ morbidity

5

Normal

No mortality/ morbidity

6

G2 (Treatment)

Normal

No mortality/ morbidity

7

Normal

No mortality/ morbidity

8

Normal

No mortality/ morbidity

9

Normal

No mortality/ morbidity

10

Normal

No mortality/ morbidity

11

Normal

No mortality/ morbidity

12

Normal

No mortality/ morbidity

13

Normal

No mortality/ morbidity

14

Normal

No mortality/ morbidity

15

Normal

No mortality/ morbidity


Table 3:Range Finding Study Results

 

Injection:Intradermal                           Sex:Male                                  Vehicle:Propylene glycol

Animal No.

Body weight (g)

Injection

Test item Concentration

(v/v %)

Observations after Intradermal injection

24 hours

48 hours

E

O

E

O

1

382

1

5.0

2

2

2

2

2

2.5

2

1

2

1

3

1.0

1

1

1

1

4

0.5

1

1

1

1

 

Application:Topical Application            Sex:Male                                     Vehicle:80% ethanol                                                                                                                                                                                 

Animal No.

Body weight (g)

Site

Test Item Concentration

(%)

Observations (After patch removal)

24 hours

48 hours

E

O

E

O

2

292

Left

100

0

0

0

0

Right

75

0

0

0

0

3

336

Left

50

0

0

0

0

Right

25

0

0

0

0

Keys:E = Erythema, O = Oedema


Table 4:Dermal Reaction

Main study – Induction (Intradermal)

Group:G1 (Control)                                                                                                         Sex:Male

Skin reaction after the intradermal injections of vehicle during induction period

Animal
No.

Reaction readings*
after Intradermal injection

At 24 hours (Day 1)

Erythema

Oedema

1

0

0

2

0

0

3

0

0

4

0

0

5

0

0

 

Group:G2 (Treatment)                                                                                                     Sex: Male

Skin reaction after the intradermal injections oftest itemduring induction period

Animal
No.

Reaction readings*
after Intradermal injection

At 24 hours (Day 1)

Erythema

Oedema

6

2

2

7

2

2

8

2

1

9

2

2

10

2

2

11

2

2

12

2

1

13

2

2

14

2

1

15

2

2

Key:*= as per Draize Method.

 


Table 4 (Continued)

Dermal Reaction

Induction (Topical Application)

Group:G1 (Control)                                                                                                          Sex:Male

Skin response after the topical application of vehicle during induction period

Animal
No.

Reaction readings*
after removal of bandage

At 24 hours (Day 10) – Right Flank

Erythema

Oedema

1

0

0

2

0

0

3

0

0

4

0

0

5

0

0

 

Group:G2 (Treatment)                                                                                                     Sex: Male

Skin response after the topical application oftest itemduring induction period

Animal
No.

Reaction readings*
after removal of bandage

At 24 hours (Day 10) – Right Flank

Erythema

Oedema

6

2

1

7

2

1

8

2

1

9

2

1

10

2

1

11

2

2

12

2

1

13

2

1

14

2

1

15

2

1

Key:*= as per as per Draize Method.


Table5:Body Weight (g)

Group:G1 (Control)                                                                                                          Sex:Male

Day

Animal No.

1

2

3

4

5

0

408

356

352

324

280

24

486

444

412

396

340

 

 

Group:G2 (Treatment)                                                                                                   Sex:Male

Day

Animal No.

6

7

8

9

10

11

12

13

14

15

0

406

360

354

318

296

370

366

356

308

282

24

494

424

436

388

340

412

454

416

376

368

 

Body weights – Summary(g)

 

Group

Prior to Dose

At termination

G1

(Control)

Mean

344.00

415.60

SD

46.90

54.49

N

5

5

G2

(Treatment)

Mean

341.60

410.80

SD

38.82

44.97

N

10

10

Key: N = Number of animals, SD = Standard Deviation.

 

Reliability Study Summary

 

Reliability check- Skin Sensitization Maximization Study of ‘Benzocaine (Ethyl 4-aminobenzoate)’ in Guinea pigwas conducted atsa-FORD (Sanctuary for Research and Development), Maharashtra, India. This study (sa-FORD Study No. 15_00_022) was performed as per OECD No. 406, OCSPP870.2600 (March 2003)and EC-B.6.

Study Period

Study Initiation Date                   : March 26, 2015

Experiment Start Date               : April 01, 2015

Experiment Completion Date    :May 01, 2015

Study Completion Date             :May 11, 2015

Main test was performed in 10 treated and 5 controls Dunkin Hartley male guinea pigs, hairs of all animals of both the groups were clipped off, 24 hours prior to induction (intradermal and topical) exposure; as well as challenge exposure of test item.

Based on results of the dose range finding study of Study No. 14_00_018; 5.0% w/v concentration of test item in propylene glycol for intradermal induction (day 0) and 100 mg (as such) of test item moistened with 0.2 ml of 80 % ethanol for topical (dermal) application on day 7 and 100 mg test item moistened with 0.2 ml of acetone for challenge application on day 21 were selected.

During intradermal induction exposure (day 0), animals of treatment group (G2) were injected with three pairs of intradermal injections [Injection a)Freund's Complete Adjuvant + distilled water(1:1),Injectionb)5% test item in propylene glycolandInjectionc)5% test item formulated in a 1:1 mixture (v/v) of FCA/ Distilled water] on either side of midline on shoulder region. Animals of control group also received three pairs of intradermal injections [Injection a) Freund’s Complete Adjuvant (FCA) + Distilled water (1:1), Injection b) propylene glycol and Injection c) 1:1 mixture of injection a) and Injection b) (v/v) on either side of midline on shoulder region.

10% sodium Lauryl Sulphate in Vaseline was applied to all animals of both the groups on day 6 on right flank to create local irritation.

Dermal induction was carried out on day 7. In animals of treatment group a filter paper (approximate size2 x 4 cm2) fully loaded with 100 mg (as such) of test item moistened with 0.2 ml of 80% ethanol was applied over the right flank, covered with secured non irritant adhesive tape for 48 hours. Animals of control group were treated in similar manner with a filter paper loaded with 80% ethanol for 48 hours. On day 10 (24 hours post patch removal) skin re­action for ery­thema and oedema of this dermal induction application was observed as perdraize method.

Challenge exposure was carried out on day 21. Patches of filter papers fully loaded with 100 mg (as such) of test item moistened with 0.2 ml of acetone was applied to left flank of animals and 0.2 ml of acetone to right flank of all animals of both treatment and control groups, respectively. Patches were held in contact for 24 hours by semi occlusive dressing. All animals were observed for allergic reaction (for ery­thema and oedema) as per theMagnusson & Kligman grading scale for the evaluation of challenge patch test reactions at approximately 24 and 48 hours after patch removal of challenge application.

The animals were observed daily for clinical signs and mortality/morbidity. Body weight was recorded prior to application and at the end of experiment.

No mortality was observed during study period. No test item related clinical signs and change in body weight observed.

Well defined erythema(09/10), very slight erythema(barely perceptible)(01/10) and slight oedema (01/10) and very slight oedema (barely perceptible)(09/10) were observed at 24 hour on day 1.Well defined erythema(05/10), very slight erythema (05/10) and very slight oedema (10/10) were observed at 24 hours after patch removal observed on day 10.

Discrete or patchy erythema was observed (06/10) and novisible change (04/10)was observed at 24 hour whereas discrete or patchy erythema (05/10) and novisible change (05/10)at 48 hour post patch removal of challenge application.

Results

Skin Reactions after Patch removal of Challenge Application (Male)

 

 

At 24 hours

 

At 48 hours

 

positive / total

 

positive / total

 

% positive of total

 

% positive of total

Control Group

 

 

 

 

 

100 mg (as such) Test item(left flank)

 

0 / 5

 

0 / 5

 

 

0

 

0

 

Acetone

 

0 / 5

 

0 / 5

 

(right flank)

 

0

 

0

 

Treatment Group

 

 

 

 

 

100 mg (as such) Test item(left flank)

 

6 / 10

 

5 / 10

 

 

60%

 

50%

 

Acetone

 

0 / 10

 

0 / 10

 

(right flank)

 

0

 

0

 

 

Under the conditions of thisstudy, 06/10 Guinea Pigs (60.0%) – at 24 hour and 05/10 Guinea Pigs (50.0%) – at 48 hour were found positive for sensitizer or allergic potential of Benzocaine (Ethyl 4-aminobenzoate) (CAS No.: 94-09-7). Hence, based on the 24 and 48 hours observation,guinea pigs were found positivefor sensitizer or allergic potential of Benzocaine (Ethyl 4-aminobenzoate) (CAS No.: 94-09-7).

 

The Globally Harmonized System of

Classification and Labelling of Chemicals (GHS)     :          “Sensitizer”

EU Classification, Labelling and Packaging of

Substances and Mixtures                                                     :         Category 1 (Positive)



Applicant's summary and conclusion

Interpretation of results:
other: not sensitizing
Conclusions:
Animals of the control (G1) and treatment (G2) group showed no allergic reaction to the challenge exposure during the 24 hours (day 23) and 48 hours (day 24) observations, after patch removal of challenge application. Under the conditions of this study and based on above results, it was concluded that, no sensitization or allergic potential for the test chemical was observed in male guinea pigs and classified as "Not Sensitizing"
Executive summary:

The aim of the study was to assess the sensitizing capacity or allergenic potential of the test chemical by challenge (topical) application after the induction period of intradermal injection and epidermal (topical) application in guinea pigs. The study was performed according to OECD 406 Guidelines.

Test was performed in 10 treatment and 5 control Dunkin Hartley male guinea pigs. Hairs of all animals of both the groups were clipped off, approximately 24 hours prior to induction (intradermal and topical) exposure; as well as challenge exposure of test item.

Based on results of the dose range finding study; 5.0% (v/v) concentration of test item using propylene glycol for intradermal induction (day 0),100% test itemfor topical (dermal) application on day 7 and for challenge application on day 21 were selected.

During intradermal induction exposure (day 0), animals of treatment group (G2) were injected with three pairs of intradermal injections [Injection a)Freund's Complete Adjuvant +distilled water (1:1),Injectionb)5.0% (v/v) test item in propylene glycolandInjectionc)5.0% test item formulated in a 1:1 mixture (v/v) of FCA + distilled water] on either side of midline on shoulder region. Animals of control group also received three pairs of intradermal injections [Injection a)Freund'sComplete Adjuvant (FCA) + distilled water (1:1), Injection b) propylene glycol and Injection c) 1:1 mixture of injection a) and Injection b) (v/v)] on either side of midline on shoulder region.

0.5 ml of 10% Sodium Lauryl Sulphate in Vaseline was applied to both the groups on day 6 on right flank, to create local skin irritation. Dermal induction was carried out on dayanimals of treatment group using a filter paperfully loaded with 0.2 mltest item (as such) for treated animals and 0.2 ml of 80% ethanol in control animals was applied over the right flank and secured with non irritant adhesive tape for 48 hours. On day 10 (24 hours post patch removal) skin re­action for ery­thema and oedema of this dermal induction application was observed as perDraize Method.

Challenge exposure was carried out on day 21. Patches of filter papersloaded with 0.2 mltest item (as such) was applied to left flank of animals and 0.2 ml of acetone to right flank of all animals of both treatment and control group animals. Patches were held in contact for 24 hours by semi occlusive dressing. All animals were observed for allergic reaction (for ery­thema and oedema) as per theMagnusson & Kligman grading scale for the evaluation of challenge patch test reactions at approximately 24 and 48 hours after patch removal of challenge application. No mortality was observed during study period. No test item related clinical signs and change in body weight observed. Increase in body weights were observed in all the experimental animals.

On day 1 post intradermal injections, all the animals of control group revealed no oedema and no erythema, whereas well defined erythema was observed in 10/10and very slight oedema (barely perceptible) was observedin 03/10 whereas slight oedema (edges of area well defined by definite raising)was observed in 07/10 animalsof treatment group.

On day 10 post topical application, all the animals of control group revealed no oedema and no erythema, whereas well defined erythema was observed in 10/10 and very slight oedema (barely perceptible) in 09/10 whereasslight oedema (edges of area well defined by definite raising)in 01/10 animals of treatment group.

Animals of the control (G1) and treatment (G2) group showed no allergic reaction to the challenge exposure during the 24 hours (day 23) and 48 hours (day 24) observations, after patch removal of challenge application. Under the conditions of this study and based on above results, it was concluded that, no sensitization or allergic potential for the test chemical was observed in male guinea pigs and classified as "Not Sensitizing"

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