Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12-20 February 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Modern guideline conform study performed in GLP certified laboratory

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Batch number: RW 2013-10-22
Colour: Orange
Storage conditions: Room temperature
Expiring date: 22. 10. 2018

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Species and strain: CBA/Ca Ola Hsd mice
Source: TOXI-COOP ZRT.
H-1103, Budapest, Cserkesz u. 90.
Hygienic level at arrival: SPF

Hygienic level during the study: Good conventional
Justification of strain: On the basis of comparative investigations in other laboratories, mice of the CBA/Ca strain were found to exhibit a more marked response than other strains. Females are used because the existing database is predominantly based on females.
Number of animals: 28 animals/main test (4 animals/treatment group)
Sex: Female, nulliparous, non-pregnant
Age of animals: Young adult mice;
10-12 weeks old (at start of the main test)
Body weight range
at starting: 18.2-22.5 g The weight variation in animals involved in the study did not exceed  20 % of the mean weight.
Acclimatization time: 7 days

Husbandry

Animal health: Only healthy animals were used
Housing during
acclimatization period: Grouped caging in small groups
Housing during the test: Grouped caging (4 animals/cage)
Cage type: Type II. Polypropylene / polycarbonate
Bedding: Laboratory bedding
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 – 70 %
Housing/Enrichment: Mice were group-housed to allow social interaction, and with deep wood sawdust bedding, to allow digging and other
normal rodent activities.

Food and Water Supply

Animals received ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, and tap water from municipal supply, as for human consumption, from a bottle ad libitum. For contents of standard diet for rats and mice and actual batch number of the diet see Appendix III.

Bedding

Lignocel Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (D-73494 Rosenberg (Germany) Holzmühle 1) was available to animals during the study.

Identification of Animals

The individual identification of the animals was performed by numbers on the tail. The cages were marked with identification cards, with information about study number, species, strain, sex, dose group and individual animal numbers.

Randomization

The animals were set in order of their body weight. The animals were randomly assigned to control and test groups using a randomization scheme. The randomization was checked by computer software according to the actual body weights verifying the homogeneity and deviations between the groups.

There were no deviations from these specifications during the experimental phase. The temperature and relative humidity were recorded during the acclimatization and experimental phases. Before housing the animals, the microbiological status of the room was checked.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Remarks:
Batch No.: 12673903, Expiry date: 05/2016, Supplier: Scharlau
Concentration:
Based on the preliminary test results the test item was examined in the main test at concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v) as formulations (apparently solutions) in N,N-Dimethylformamide (DMF).
No. of animals per dose:
4 animals/treatment group
Details on study design:
Preliminary Study
The maximum dose selection was performed according to the relevant guidelines and based on results of the formulation evaluation and the preliminary irritation / toxicity test. The preliminary irritation/toxicity screen was conducted in a similar experimental manner to the exposure phase of the main assay.
The test item was formulated in the selected vehicle (DMF) and tested at concentrations of 10 %, 5 % and 2.5 % (w/v). The formulations were adequately applicable on the ears of animals.
Three groups of 2 CBA/Ca mice were treated with the appropriate formulations once daily for 3 consecutive days. All animals were observed for any clinical signs of systemic toxicity or local irritation at the application site during the preliminary test. Body weights were recorded prior to the first treatment (on Day 1) and prior to termination (on Day 6). Irritation was monitored by measurement of ear thickness which was taken using digital micrometer on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6.
No mortality or any signs of systemic toxicity were observed during the preliminary test. No significant, treatment related effect on body weights was observed. No signs of significant irritation (indicated by an increased ear thickness of ≥ 25 % on any day of measurement) were observed in the treatment groups. Although erythema could not be reliably observed due to intensive orange colour of the test item an erythema score < 3 is considered.
Based on these results, the 10 % concentration was selected to be used as the maximum in the main test. The test item was tested also at three additional, lower concentrations (5 %, 2.5 % and 1 %) to evaluate dose-response relationship and ensure validity of the test in accordance with the relevant guidelines.

Main Study

Animals in the treatment groups were treated with the relevant vehicles (DMF or AOO), appropriate formulations of the test item or 25 % concentration of the positive control substance. The test item was administered at four different concentrations according to the results of the dose range finding test. For detailed data see the table below.

Test Concentrations in the Main Test

Groups Test item concentration (% w/v ) Positive control concentration (% w/v) No. of animals
1 Vehicle control for the PC: AOO - - 4
2 Positive control: HCA in AOO - 25 4
3 Yellow LF 6881 in DMF 10 - 4
4 Yellow LF 6881 in DMF 5 - 4
5 Yellow LF 6881 in DMF 2.5 - 4
6 Yellow LF 6881 in DMF 1 - 4
7 Vehicle control for the test item: DMF - - 4

PC = Positive control
AOO = Acetone:olive oil 4:1 (v/v) mixture
HCA = -Hexylcinnamaldehyde
DMF= N,N-Dimethylformamide
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The results were expressed as DPN (DPM divided by the number of pooled lymph nodes).
The stimulation index (SI = the DPN of a treated (positive control or test item) group divided by the DPN of the respective negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result. Based on the results EC3 value (dose calculated to induce a stimulation index of 3) of the test item was not calculated. Dose-response relationship was evaluated by linear regression. All calculations were made by Microsoft Excel Software.

Results and discussion

Positive control results:
The positive control item (25 % HCA in AOO) induced the appropriate (SI>3) stimulation over the control (SI value was 7.0), thus confirming the validity of the assay.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
No significantly increased lymphoproliferation (indicated by an SI  3) compared to the vehicle control was noted for Yellow LF 6881 at the tested concentrations. The stimulation index values were 1.1, 0.6, 0.5 and 0.9 at concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively. Dose response relationship was evaluated by linear regression using the SI values. No dose-related response was observed (p = 0.44, r = 0.56).
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
DPM, DPN and Stimulation Index Values for all Groups in the Main Test Test Group Measured Group* DPN Stimulation Name DPM/group DPM (DPM/Node) Index Values Vehicle control for the positive control (AOO): 8621 8599.5 1074.9 1.0 Positive control: 60045 60023.5 7502.9 7.0 25 % HCA in AOO Yellow LF 6881 6112 6090.5 761.3 1.1 10 % in DMF Yellow LF 6881 3621 3599.5 449.9 0.6 5 % in DMF Yellow LF 6881 2714 2692.5 336.6 0.5 2.5 % in DMF Yellow LF 6881 5171 5149.5 643.7 0.9 1 % in DMF Vehicle control for the test item (DMF): 5720 5698.5 712.3 1.0 HCA = Hexylcinnamaldehyde AOO = Acetone: Olive oil 4:1 (v/v) mixture DMF= N,N-Dimethylformamide *Group DPM = measured DPMgroup- average DPMbackground Average DPMbackground = 21.5

Any other information on results incl. tables

DPM, DPN and Stimulation Index Values for all Groups in the Main Test

Test Group

Measured

Group*

DPN

Stimulation

Name

DPM/group

DPM

(DPM/Node)

Index Values

Vehicle control for the positive control:

8621

8599.5

1074.9

1.0

AOO

 

 

 

 

Positive control:

60045

60023.5

7502.9

7.0

25 % HCA in AOO

 

 

 

 

Yellow LF 6881

6112

6090.5

761.3

1.1

10 % in DMF

 

 

 

 

Yellow LF 6881

3621

3599.5

449.9

0.6

5 % in DMF

 

 

 

 

Yellow LF 6881

2714

2692.5

336.6

0.5

2.5 % in DMF

 

 

 

 

Yellow LF 6881

5171

5149.5

643.7

0.9

1 % in DMF

 

 

 

 

Vehicle control for the test item:

5720

5698.5

712.3

1.0

DMF

 

 

 

 

 

HCA =a-Hexylcinnamaldehyde

AOO = Acetone: Olive oil 4:1 (v/v) mixture

DMF=N,N-Dimethylformamide

 

*Group DPM = measured DPMgroup- average DPMbackground

Average DPMbackground= 21.5

 

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In conclusion, under the conditions of the present Local Lymph Node Assay, Yellow LF 6881 tested at the maximum achievable concentration of 10 % (w/v) and at concentrations of 5 %, 2.5 % and 1 % (w/v) as formulations in an appropriate vehicle (DMF) was shown to have no skin sensitization potential.
Executive summary:

The maximum achievable Yellow LF 6881concentration was 10 % (w/v) in N,N-Dimethylformamide (DMF). Based on the preliminary test results the test item was examined in the LLNA as 10 %, 5%, 2.5 % or 1 % (w/v) formulations in DMF according to the relevant guidelines. In the main test 28 female CBA/Ca mice were allocated.

No mortality , no significant treatment related effect on body weights, any other signs of systemic toxicity or any sign of significant irritation or any other local effect were observed in any treatment group during the test. Visually larger lymph nodes than the control were observed in the positive control group only. Visual appearance of the lymph nodes was normal in the test item treated groups and in the negative control groups (both DMF and AOO). No significantly increased lymphoproliferation (indicated by an SI > 3) compared to the vehicle control was noted for Yellow LF 6881 at the tested concentrations. The stimulation index values were 1.1, 0.6, 0.5 and 0.9 at concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively. No dose-related response was observed (p = 0.44, r = 0.56).

The positive control item (25 % HCA in AOO) induced the appropriate (SI > 3) stimulation over the control (SI value was 7.0), thus confirming the validity of the assay.

According to evaluation criteria of the relevant guidelines the lack of a significantly increased lymphoproliferation (indicated by an SI ≥ 3) up to the maximum achievable concentration (based on solubility) of 10 % (w/v) and the lack of a dose-related response are considered evidence that Yellow LF 6881 has no skin sensitization potential.