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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01.10.2014 to 18.12.2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
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:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
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:
liquid

In vivo test system

Test animals

Species:
mouse
Strain:
other: HsdWin:NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS

- Source: Harlan Nederland, Kreuzelweg 53, 5960 AD Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks
- Weight at study initiation: 29- 36g
- Housing: During the adaptation period up to 8 mice were housed together in conventional
Makrolon® type III cages, cages were changed at least twice a week. While during the study
period the animals were single-housed in type II cages.
All the animals used in this study were kept in the same room. In case animals from other
toxicological studies were kept in the same room, adequate spatial separation and appropriate
organization of the working procedures ensured that animals could not be confused.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 6 days
- Indication of any skin lesions: no, only healthy animals showing no signs of disease were used in the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 40-70%
- Air changes (per hr): About 1 0 changes per hour
- Photoperiod (hrs dark / hrs light): 12 h/12 h, with artificial illumination
- IN-LIFE DATES: From: 13.10.2014 To: 16.10.14

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Test item: 0 % (vehicle control), 25 %, 50 %and 100 %.
The test item was formulated immediately before each administration in acetone/Olive oil 4:1 (A/00)
Group 1 - Vehicle (A/OO)
Group 2 - 25% DINCD (in A/OO)
Group 3 - 50% DINCD (in A/OO)
Group 4 - 100% DINCD



No. of animals per dose:
6 female per group (4 groups)
Details on study design:
MAIN STUDY
The methods used in this study are in principle specified in guidelines (OECD 406, 1992;
EPA guideline OPPTS 870.2600, Skin Sensitization, March 2003; updated
OECD TG 429, 2010). A further modification was done by including the measurement of the ear swelling after treatment leading to a much more simplified and reliable assay (Integrated Model for the Differentiation of Skin reactions
(IMDS, (1 )). By comparing the specific immune reaction induced by the test item in the
draining lymph nodes (LN cell counts I LN weights) with the immediate non-specific acute
skin reaction (ear swelling I ear weight) it is possible to discriminate the irritant potential from the sensitizing potential of the compound tested. International standards have been
successfully classified using this modification (2). Such modifications are also authorized in
the updated OECD guideline 429 (20 1 0).

TREATMENT PREPARATION AND ADMINISTRATION:
The test item in the formulation, the test item pure or the vehicle were applied epicutaneously onto the dorsal part of both ears of the animals. This treatment was repeated on three consecutive days ( d 1, d2 and d3).
The volume administered was 25 µl/ear.
Based on our experiences with this test system and the known properties of the test item the
following concentrations were used: 0 % (vehicle control), 25 %, 50 % and 100 %.
Statistics:
When it was statistically reasonable, the values from treated groups were compared with those
from the control group(s; vehicle) by a one-way analysis of variance (ANOVA) when the
variances are considered homogeneous according to a homogeneity testing like Cochran's
test. Alternatively, if the variances are considered to be heterogenous (p:::; 0.05), a
non-parametric Kruskal-Wallis test has been used (Kruskal-Wallis ANOV A) at significance
levels of 5 %. Two sided multiple test procedures were done according to Dunnett or
Bonferroni-Holrn, respectively. Outlying values in the LN weights were eliminated at a
probability level of99% by Nalimov's method. In addition, for the LLNA/IMDS the smallest
significant differences in the means were calculated by Scheffe's method, which can be used
for both equal and unequal sample sizes.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1
Variability:
+/- 16.22 (SD in %)
Test group / Remarks:
Vehicle
Key result
Parameter:
SI
Value:
1.12
Variability:
+/- 23.98 (SD in %)
Test group / Remarks:
25 %
Key result
Parameter:
SI
Value:
1.12
Variability:
+/- 30.36 (SD in %)
Test group / Remarks:
50 %
Key result
Parameter:
SI
Value:
1.22
Variability:
+/- 31.01 (SD in %)
Test group / Remarks:
100 %

Applicant's summary and conclusion

Conclusions:
In conclusion, these results show that the test item DINCD has no sensitizing potential in
mice after dermal application up to and including a concentration of 100 %. The increases in
ear swelling, however, point to a weak to moderate irritant potential of the test item DINCD.
Therefore, the concentration of 100 % turned out to be the NOEL for the parameters
investigated in this study with respect to skin sensitization.