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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
Reliability:
1 (reliable without restriction)

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
The relative humidity in the animal room was between approximately a minimum of 27 - 64% instead of 45 – 65% for several hours on five not subsequent days. This deviation to the study plan, however, does not affect the validity of the study.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid: viscous

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
The animals were distributed into the test groups at random. All animals belonging to the same experimental group were kept in one cage. In the main experiment, the animals were identified by tail tags. In the pre-experiment, animals were identified by cage number. At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: group
Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top
Bedding: granulated soft wood bedding
Feed: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
Water: tap water, ad libitum
Environment: temperature 22 + 2°C relative humidity approx. 45-65% (except for few hours on five not subsequent days, see deviation section) artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
methyl ethyl ketone
Concentration:
0, 0.5, 1 and 2.5%
No. of animals per dose:
4
Positive control substance(s):
other: no positive control
Statistics:
The mean values and standard deviations were calculated in the body weight tables. However, both biological and statistical significance were considered together.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
0% (control group)
Key result
Parameter:
SI
Value:
1.36
Test group / Remarks:
0.5%
Key result
Parameter:
SI
Value:
4.72
Test group / Remarks:
1%
Key result
Parameter:
SI
Value:
2.43
Test group / Remarks:
2.5%
Key result
Parameter:
EC3
Remarks on result:
not determinable
Remarks:
The EC3 value could not be calculated, since the S.I. of the mid dose is above the threshold value of 3 and above the value of the high dose.
Cellular proliferation data / Observations:
Viability / Mortality
No deaths occurred during the study period.

Clinical Signs
No signs of systemic toxicity were observed during the study period. On day 4 to 6, the animals treated with a test item concentration of 1 and 2.5% showed an erythema of the ear skin (Score 1). Animals treated with 0.5% test item concentration did not show any signs of local skin irritation.

Body Weights
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age. The individual body weight values are included in Annex 2.

Any other information on results incl. tables

In order to study a possible skin sensitising potential of VP 112/18, three groups each of four female mice were treated once daily with the test item at concentrations of 0.5, 1 and 2.5% (w/w) in MEK by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be achieved whilst avoiding excessive local skin irritation as confirmed by three pre-experiments. A control group of four mice was treated with the vehicle (MEK) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). 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 3H-methyl thymidine measured in a β-scintillation counter. All treated animals survived the scheduled study period and no signs of systemic toxicity were observed. On days 4 to 6, the animals treated with a test item concentration of 1 and 2.5% showed an erythema of the ear skin (Score 1). Animals treated with 0.5% test item concentration did not show any signs of local skin irritation. A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices (S.I.) of 1.36, 4.72 and 2.43 were determined with the test item at concentrations of 0.5, 1 and 2.5% in MEK, respectively. A conventional dose response showing increased sensitizing effects with increasing VP 112/18 concentrations was not observed. The Stimulation Index obtained in the mid dose group was above the threshold index of 3. However, the Stimulation Index obtained for the high dose group was below that of the mid dose group and below the threshold index of 3. An EC3 value could therefore not be calculated. Based on the observed irritation, it cannot be ruled out (although signs of irritation were not excessive) that the irritant potential of the test item might have contributed to the increase in S.I. values observed in the mid and high dose group.

Applicant's summary and conclusion

Conclusions:
The results obtained with the test item VP 112/18 were found to be equivocal under the test conditions of this study.
Executive summary:

In the study the test item VP 112/18 formulated in MEK was assessed for its possible skin sensitising potential. For this purpose a local lymph node assay was performed using test item concentrations of 0.5, 1 and 2.5%. The highest concentration tested was the highest concentration that could be achieved whilst avoiding excessive local skin irritation as confirmed by three pre-experiments. The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. On days 4 to 6, the animals treated with a test item concentration of 1 and 2.5% showed an erythema of the ear skin (Score 1). Animals treated with 0.5% test item concentration did not show any signs of local skin irritation. In this study Stimulation Indices (S.I.) of 1.36, 4.72 and 2.43 were determined with the test item at concentrations of 0.5, 1 and 2.5% in MEK, respectively. A conventional dose response showing increased sensitizing effects with increasing VP 112/18 concentrations was not observed. The Stimulation Index obtained in the mid dose group was above the threshold index of 3. However, the Stimulation Index obtained for the high dose group was below that of the mid dose group and below the threshold index of 3. An EC3 value could therefore not be calculated. Based on the observed irritation, it cannot be ruled out (although signs of irritation were not excessive) that the irritant potential of the test item might have

contributed to the increase in S.I. values observed in the mid and high dose group. Therefore, the results obtained with the test item VP 112/18 were found to be equivocal.