Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
KN 172
SAT 030332
Batch No.: Kn-Gi-8251/104

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, open
Vehicle:
other: ethanol:water, 7:3 (v/v)
Concentration / amount:
1 % , 2.5 %, 5 %
Challenge
Concentration / amount:
1 % , 2.5 %, 5 %
No. of animals per dose:
4
Details on study design:
concentration were selected due to pretest (up to 25 %) results, highest technically achievable concentration without severe toxicity
Positive control substance(s):
yes
Remarks:
Alpha-hexylcinnamalaldehyd

Study design: in vivo (LLNA)

Vehicle:
other: Ethanol:water, 7:3 (v/v)
Concentration:
1 %, 2.5 % and 5 % (w/v)
highest test concentration without severe toxicity
No. of animals per dose:
4
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
Positive control was found to be a skin sensitizer (EC3=9.4 %)

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: Inthis study STIMULATION INDICES of 2.2, 2.7 and 2.1 were determined with the test item at concentrations of 1 %, 2.5 % and 5 % (wlv) in ethanol/water, 7:3 (v/v).

Any other information on results incl. tables

VIABILITY / MORTALITY

All treated animals survived the scheduled study period with the exception of three animals

in Group 4 (5 %), in which two animals (No. 14 and 16) died one or two days after the first

application. One animal (No. 15) was euthanized on the second application day due to

severe sedation, hunched posture and slight ear swelling.

CLINICAL SIGNS

No test item-related clinical signs were observed in any animals of the control group or

Group 2 (1 %). On the third application day, a slight ear swelling was obse~eda t both

dosing sites in all mice of Group 3 (2.5 %), persisting for a total of three days. On the second

application day, a slight ear swelling was observed at both dosing sites in one remaining

animal in Group 4 (5 %), persisting for the remainder of the in-life phase of the study. In

addition, this animal showed slight to moderate sedation and slight hunched posture one day

after the first topical application, persisting for a total of four days or for the remainder of the

in-life phase of the study.

BODY WEIGHTS

The body weight of the animals, recorded prior to the lSaptp lication and prior to necropsy,

was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test item KN 172 was found to be a non-sensitizer when tested at up to concentration of
5 % (wlv) in ethanol:water, 7:3 (VIV).
KN 172 causes systemic toxicity at concentration of 5 % (wlv) in mice.


Executive summary:

In order to study a possible contact allergenic potential of KN 172, three groups each of four

female mice were treated daily with the test item at concentrations of 1 %, 2.5 % and 5 %

(wlv) in ethanokwater, 7:3 (V/V) by topical application to the dorsum of each ear lobe (left and

right) for three consecutive days. A control group of four mice was treated with the vehicle

(ethanol:water, 7:3 (V/V)) only. Five days after the first topical application the mice were

injected intravenously into a tail vein with radio-labelled thymidine (3~-methytlh ymidine).

Approximately five hours after intravenous injection, the mice were sacrificed, the draining

auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node

cells were prepared from pooled lymph nodes which were subsequently washed and

incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node

cells was determined by the incorporation of 3~-methytlhymidine measured in a ßscintillation

counter.

To avoid loss of the test item applied to the outer ear surface the test item was carefully

dosed. To avoid missing of the test item from the ears a hair dryer was used to immediately

dry the wet ears.

No test item-related clinical signs were observed in any animals of the control group or

Group 2 (1 %). On the third application day, a slight ear swelling was observed at both

dosing sites in all mice of Group 3 (2.5 %), persisting for a total of three days. On the second

application day, a slight ear swelling was observed at both dosing sites in one remaining

animal in Group 4 (5 %), persisting for the remainder of the in-life phase of the study. In

addition, this animal showed slight to moderate sedation and slight hunched posture one day

after the first topical application, persisting for a total of four days or for the remainder of the

in-life phase of the study.

All treated animals survived the scheduled study period with the exception of three animals

in Group 4 (5 %), in which two animals (No. 14 and 16) died one or two days after the first

application. One animal (No. 15) was euthanized on the second application day due to

severe sedation, hunched posture and slight ear swelling.

A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test

concentrations resulted in 3-fold or greater increase in incorporation of 3 ~comp~ared d ~

with concurrent controls, as indicated by the STIMULATION INDEX (S.I.).

Group 2 I 1 1 2.2 In order to study a possible contact allergenic potential of KN 172, three groups each of four

female mice were treated daily with the test item at concentrations of 1 %, 2.5 % and 5 %

(wlv) in ethanokwater, 7:3 (VIV) by topical application to the dorsum of each ear lobe (left and

right) for three consecutive days. A control group of four mice was treated with the vehicle

(ethanol:water, 7:3 (VIV)) only. Five days after the first topical application the mice were

injected intravenously into a tail vein with radio-labelled thymidine (3~-methytlh ymidine).

Approximately five hours after intravenous injection, the mice were sacrificed, the draining

auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node

cells were prepared from pooled lymph nodes which were subsequently washed and

incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node

cells was determined by the incorporation of 3~-methytlh ymidine measured in a ßscintillation

counter.

To avoid loss of the test item applied to the outer ear surface the test item was carefully

dosed. To avoid missing of the test item from the ears a hair dryer was used to immediately

dry the wet ears.

No test item-related clinical signs were observed in any animals of the control group or

Group 2 (1 %). On the third application day, a slight ear swelling was observed at both

dosing sites in all mice of Group 3 (2.5 %), persisting for a total of three days. On the second

application day, a slight ear swelling was observed at both dosing sites in one remaining

animal in Group 4 (5 %), persisting for the remainder of the in-life phase of the study. In

addition, this animal showed slight to moderate sedation and slight hunched posture one day

after the first topical application, persisting for a total of four days or for the remainder of the

in-life phase of the study.

All treated animals survived the scheduled study period with the exception of three animals

in Group 4 (5 %), in which two animals (No. 14 and 16) died one or two days after the first

application. One animal (No. 15) was euthanized on the second application day due to

severe sedation, hunched posture and slight ear swelling.

A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test

concentrations resulted in 3-fold or greater increase in incorporation of 3 ~comp~ared d ~

with concurrent controls, as indicated by the STIMULATION INDEX (S.I.).

Group 2 I 1 1 2.2 In order to study a possible contact allergenic potential of KN 172, three groups each of four

female mice were treated daily with the test item at concentrations of 1 %, 2.5 % and 5 %

(wlv) in ethanokwater, 7:3 (VIV) by topical application to the dorsum of each ear lobe (left and

right) for three consecutive days. A control group of four mice was treated with the vehicle

(ethanol:water, 7:3 (VIV)) only. Five days after the first topical application the mice were

injected intravenously into a tail vein with radio-labelled thymidine (3~-methytlh ymidine).

Approximately five hours after intravenous injection, the mice were sacrificed, the draining

auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node

cells were prepared from pooled lymph nodes which were subsequently washed and

incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node

cells was determined by the incorporation of 3~-methytlh ymidine measured in a ßscintillation

counter.

To avoid loss of the test item applied to the outer ear surface the test item was carefully

dosed. To avoid missing of the test item from the ears a hair dryer was used to immediately

dry the wet ears.

No test item-related clinical signs were observed in any animals of the control group or

Group 2 (1 %). On the third application day, a slight ear swelling was observed at both

dosing sites in all mice of Group 3 (2.5 %), persisting for a total of three days. On the second

application day, a slight ear swelling was observed at both dosing sites in one remaining

animal in Group 4 (5 %), persisting for the remainder of the in-life phase of the study. In

addition, this animal showed slight to moderate sedation and slight hunched posture one day

after the first topical application, persisting for a total of four days or for the remainder of the

in-life phase of the study.

All treated animals survived the scheduled study period with the exception of three animals

in Group 4 (5 %), in which two animals (No. 14 and 16) died one or two days after the first

application. One animal (No. 15) was euthanized on the second application day due to

severe sedation, hunched posture and slight ear swelling.

A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test

concentrations resulted in 3-fold or greater increase in incorporation of 3 ~comp~ared d ~

with concurrent controls, as indicated by the STIMULATION INDEX (S.I.).

No dose-response relation was observed.

Calculation of the EC 3 value was not done because no test

concentrations produced a STIMULATION INDEX (S.I.) of 3 or

higher.

In order to study a possible contact allergenic potential of KN 172, three groups each of four

female mice were treated daily with the test item at concentrations of 1 %, 2.5 % and 5 %

(wlv) in ethanokwater, 7:3 (VIV) by topical application to the dorsum of each ear lobe (left and

right) for three consecutive days. A control group of four mice was treated with the vehicle

(ethanol:water, 7:3 (VIV)) only. Five days after the first topical application the mice were

injected intravenously into a tail vein with radio-labelled thymidine (3~-methytlh ymidine).

Approximately five hours after intravenous injection, the mice were sacrificed, the draining

auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node

cells were prepared from pooled lymph nodes which were subsequently washed and

incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node

cells was determined by the incorporation of 3~-methytlh ymidine measured in a ßscintillation

counter.

To avoid loss of the test item applied to the outer ear surface the test item was carefully

dosed. To avoid missing of the test item from the ears a hair dryer was used to immediately

dry the wet ears.

No test item-related clinical signs were observed in any animals of the control group or

Group 2 (1 %). On the third application day, a slight ear swelling was observed at both

dosing sites in all mice of Group 3 (2.5 %), persisting for a total of three days. On the second

application day, a slight ear swelling was observed at both dosing sites in one remaining

animal in Group 4 (5 %), persisting for the remainder of the in-life phase of the study. In

addition, this animal showed slight to moderate sedation and slight hunched posture one day

after the first topical application, persisting for a total of four days or for the remainder of the

in-life phase of the study.

All treated animals survived the scheduled study period with the exception of three animals

in Group 4 (5 %), in which two animals (No. 14 and 16) died one or two days after the first

application. One animal (No. 15) was euthanized on the second application day due to

severe sedation, hunched posture and slight ear swelling.

A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test

concentrations resulted in 3-fold or greater increase in incorporation of 3 ~comp~ared d ~

with concurrent controls, as indicated by the STIMULATION INDEX (S.I.).

Test item concentration S.I.

% (w/v)

Group 2 1 2.2

Group 3 2.5 2.7

.Groupe4 5 2.1

No dose-response relation was observed.

Calculation of the EC 3 value was not done because no test

concentrations produced a STIMULATION INDEX (S.I.) of 3 or

higher.