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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation
Remarks:
in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-07-17 to 2002-09-03
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD guideline-conform study, performed under GLP
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaCruBR
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, UK.
- Age at study initiation: 7 - 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: in groups of four in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum): Free access to food (Certified Rat and Mouse Diet (Code 5LF2) supplied by IPS Product Supplies Limited, Wellingborough, Northants, UK)
- Water (e.g. ad libitum): Free access to mains tap water
- Acclimation period: >= 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 2002-07-05 to 2002-07-09
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0.5, 5 and 50% w/v
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
No range finding test was performed.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Kimber I, Hilton J and Weisenberger C (1989) "The Murine Local Lymph Node Assay for Identification of Contact Allergens : A Preliminary Evaluation of in situ Measurements of Lymphocyte Proliferation", Contact Dermatitis 21,215-220 and Basketter D A and Scholes E W (1992) Comparison of the Local Lymph Node Assay with the Guinea Pig Maximisation Test for the Detection of a Range of Contact Allergens, Food and Chemical Toxicology 30,65-69.
- Criteria used to consider a positive response: The test substance will be regarded as a sensitiser if at least one concentration of the test material
results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as "not a moderate to strong sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:
For the purpose of the study the test material was freshly prepared in acetone/olive oil 4: 1. The mice were treated by daily application of 25 µL of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 0, 1,2). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
Five days following the first topical application of the test material (Day 5) all mice were injected via the tail vein with 250 µL of phosphate buffered saline containing 3H-methyl thymidine ( 3HTdR: 80 µCi/mL , specific activity 2.0 Ci/mmol, Amersham Pharmacia Biotech UK Ltd) giving
a total of 20 µCi to each mouse.
Parameter:
SI
Remarks on result:
other: 0.5 % w/v test substance in acetone/olive oil 4:1: 28.3 5 % w/v test substance in acetone/olive oil 4:1: 84.7 50 % w/v test substance in acetone/olive oil 4:1: not applicable as all animals were dead on Day 3.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Control (actetone/olive oil 4:1): 3955.95 dpm 0.5 % w/v test substance in acetone/olive oil 4:1: 112010.5 dpm 5 % w/v test substance in acetone/olive oil 4:1: 335033.2 dpm 50 % w/v test substance in acetone/olive oil 4:1: not applicable as all animals were dead on Day 3.

Clinical Observations and Mortality Data

Two animals treated with 50% w/v were found dead three days after application of the test material. The two remaining animals treated with 50% w/v were killed in extremis three days after application of the test material. Signs of systemic toxicity noted in animals treated with 50% w/v were hunched posture, lethargy, pilo-erection, decreased respiratory rate, laboured respiration, hypothermia and splayed gait. There were no deaths noted in animals treated with 0.5% or 5% w/v. Signs of systemic toxicity noted in animals treated with 5% w/v were hunched posture, pilo-erection and lethargy. No signs of systemic toxicity were noted in the test animals treated with 0.5% w/v or control animals during the study.

Bodyweights

All animals treated with 5% w/v showed bodyweight loss. Bodyweight changes of the test animals treated with 0.5% w/v between Day 0 and Day 5 were comparable to those observed in the control group animals.

Tables

- Table 1: Dpm, Dpm/Node and Test/Control Ratio

Concentration (% w/v) in Acetone/Olive Oil 4:1

Dpm

Dpm/Node a

Test/Control Ratio b

Result

Acetone/Olive Oil 4:1

3955.95

494.5

N/A

N/A

0.5

112010.5

14001.3

28.3

Positive

5

335033.2

41879.2

84.7

Positive

50

-

-

-

-

a = Dpm/node obtained by dividing the Dpm value by 8 (total number of lymph nodes)

b = Test/Control ratio of 3.0 or greater indicates a positive result

N/A = Not applicable

- = All animals dead on Day 3

- Table 2: Individual Clinical Observations and Mortality Data

Concentration (% w/v) in Acetone/Olive Oil 4:1

Animal Number

Days

0

1

2

3

4

5

Acetone/Olive Oil 4:1

1-1

0

0

0

0

0

0

1-2

0

0

0

0

0

0

1-3

0

0

0

0

0

0

1-4

0

0

0

0

0

0

0.5

2-1

0

0

0

0

0

0

2-2

0

0

0

0

0

0

2-3

0

0

0

0

0

0

2-4

0

0

0

0

0

0

5

3-1

0

0

0

H

HPL

HPL

3-2

0

0

0

H

HPL

HPL

3-3

0

0

0

H

HPL

HPL

3-4

0

0

0

H

HPL

HPL

50

4-1

0

0

HL

HLPHoRd RIWsX*

 

 

4-2

0

0

HL

X

 

 

4-3

0

0

HI,

HLPHoRd
R1WsX*

 

 

4-4

0

0

HL

X

 

 

0 = No signs of systemic toxicity, H = Hunched posture, L = Lethargy, P = Pilo-erection, Ho = Hypothermia, Ws = Splayed gait, Rd = Decreased respiratory rate, Rl = Laboured respiration,X = Animal dead, X* = animal killed in extremis

- Table 3: Individual Bodyweights and Bodyweight Changes

Concentration (% w/v) in Acetone/Olive Oil 4:1

Animal Number

Bodyweight (g)

Changes

Day 0

Day 5

Acetone/Olive Oil 4:1

1-I

19

19

0

1-2

18

18

0

1-3

18

18

0

1-4

21

21

0

0.5

2-1

20

20

0

2-2

18

18

 

2-3

19

20

1

2-4

19

19

0

5

3-1

18

17

-1

3-2

19

18

-1

3-3

20

17

-3

3-4

21

18

-3

50

4-1

18

-

-

4-2

18

-

4-3

19

-

-

4-4

19

-

-

- = Animal dead
Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test material was considered to be a sensitiser under the conditions of the test.
Executive summary:

A study was performed to assess the potential of the test material to produce delayed type hypersensitivity in the mouse. The study is indicated for use as a first stage in the assessment of skin sensitisation potential (OECD Guidelines for the Testing of Chemicals, No

406 1992 "Skin Sensitisation").

Three groups, each of four animals, were treated with 50 µL of the test material (25 µL per ear) as a solution in acetone/olive oil 4:1 at concentrations of 0.5, 5 and 50% w/v. A further group of four animals was treated with acetone/olive oil 4:l alone.

The test/control ratio for each concentration was found to be 28.3 (0.5 % w/v in acetone/olive oil 4:1) and 84.7 (5 % w/v in acetone/olive oil 4:1), respectively. Two animals treated with 50 % w/v were dead on Day 3 and the remaining 2 animals were killed in extremis three days after application of the test material. Therefore, no test/control ratio could be determined for the highest dose group.

The test material was considered to be a sensitiser under the conditions of the test.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

A reliable study is available (Sanders 2002/KS K1/skin sensitisation) conducted generally compliant to OECD TG 429 and GLP (CBA/Ca mice, 0.5, 5 and 50 % (w/v) in acetone/olive oil (4:1 v/v)).

The SI values calculated for the substance concentrations 0.5, 5.0 and 50 % (w/v) were 28.3, 84.7 and “not determinable” respectively. In the high dose groups all animals died or were killed in extremis after the second exposure. The EC3 value could not be calculated, since all S.I. values are above 3. Based on these results according to the recommendations made in the test guidelines, Gaskamine 240 would be regarded as skin sensitizer. As there is a dose response and the S.I: is > 3 at a concentration of 0.5 % (w/v) the substance is deemed a strong sensitiser (category 1A) according to CLP.


Migrated from Short description of key information:
Strong sensitiser (category 1A) according to CLP

Justification for selection of skin sensitisation endpoint:
OECD guideline-conform study, performed under GLP.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:
Migrated from Short description of key information:
No data available

Justification for classification or non-classification

Based on the above stated assessment of the skin sensitisation potential (S.I: is >3 at a concentration of 0.5 % (w/v)) the submission item needs to be classified as skin sensitiser.

Regulation (EC) No 1272/2008 (CLP): Skin sensitising category 1A, H317 “May cause an allergic skin reaction”.

Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC): Xi; R43 “May cause sensitisation by skin contact”.

For classification of the test item as to its respiratory sensitisation properties the respective data are lacking.