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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)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2008
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
Details on test material:
- Physical state/Appearance: Beige powder
- Storage condition of test material: Room temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 15 to 23 g
- Housing: Suspended solid floor polypropylene cages furnished with softwood wood flakes. The animals were provided with environmental enrichment items (cardboard fun tunnels or suitable alternatives) which were considered not to contain any contaminant of a level that might have affected the integrity of the study.
- Diet: 2014C Teklad Global Rodent diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

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

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
PRE-SCREEN TEST
25% w/w

MAIN STUDY
5, 10 and 25% w/w
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TEST
One mouse was treated by daily application of 25 µL of test item solution to the dorsal surface of each ear for 3 consecutive days. The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The body weight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6. The thickness of each ear was measured using a Mitutoyo 547-300S gauge, pre-dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test item would be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to vehicle control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation would be classified as a ‘non-sensitizer’.

TREATMENT PREPARATION AND ADMINISTRATION
- The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3).
- The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the disposable pipette tip.
- A further group (vehicle control group) of four mice received the vehicle alone in the same manner.

³H-METHYL THYMIDINE ADMINISTRATION
- Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing ³H-methyl thymidine (³HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmoL) giving a total of 20 μCi to each mouse.

OBSERVATIONS
- Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
- Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

TERMINAL PROCEDURES
- Termination: Five hours following the administration of ³HTdR, all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group, the draining auricular lymph nodes were excised and processed. For each individual animal, 1 mL of PBS was added to the lymph nodes.
- Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labelled with the study number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells, and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5% trichloroacetic acid (TCA).
- Determination of ³HTdR Incorporation: After approximately 18 hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting. The Poly Q™ vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system.

DATA EVALUATION
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of ³HTdR incorporation into lymph node cells of test nodes relative to that recorded for the vehicle control nodes (Stimulation Index).
Positive control substance(s):
other: Phenylacetaldehyde
Statistics:
- Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate.
- Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data were first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data were both normally distributed and had homogeneity of variances were met, a parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non-parametric Kruskal-Wallis Rank Sum and Mann-Whitney U test procedures were used.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.35
Test group / Remarks:
5% test item
Parameter:
SI
Value:
1.55
Test group / Remarks:
10% test item
Parameter:
SI
Value:
1.62
Test group / Remarks:
25% test item
Cellular proliferation data / Observations:
PRE-SCREEN TEST
- Clinical observations: Off white coloured residual test item on the ears was noted on Days 2 and 3. No signs of systemic toxicity or visual local skin irritation were noted.
- Ear thickness: A greater than 25% (53.659%) increase in mean ear thickness was noted. As no other signs of irritation were noted the greater than 25% increase was considered to be due to residual test item on the ears rather than excessive irritation. Based on this the test item concencentrations of the main study were selected.

CELLULAR PROLIFERATION DATA
The results of the statistical analysis of the data indicated there was no significant difference between the control group and the test groups. (See Table in ‘any other information on results incl. tables)

DETAILS ON STIMULATION INDEX CALCULATION
The mean Stimulation Index is expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group

CLINICAL OBSERVATIONS
There were no deaths. No signs of systemic toxicity were noted in the test or vehicle control animals during the test.

BODY WEIGHTS
Body weight change of the test animals between Day 1 and Day 6 were comparable to that observed in the corresponding vehicle control group animals over the same period.

Any other information on results incl. tables

Table: Disintegrations per Minute and Stimulation Indices

Concentration

(% w/w) in propylene glycol

Mean dpm/Animal (Standard Deviation)

Stimulation Index

Result

Vehicle

2198.54 (±1030.98)

na

na

5

2976.82 (±584.65)

1.35

Negative

10

3416.71 (±2003.39)

1.55

Negative

25

3564.86 (±764.13)

1.62

Negative

The results of the statistical analysis of the data indicated there was no significant difference between the control group and the test groups

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this LLNA test according to OECD 429, the test item was not found to be sensitizing to the skin.
Executive summary:

A GLP compliant study according to OECD 429 was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25% w/w (the highest concentration that produced a suitable formulation), this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay (LLNA). Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of the test item as a suspension in propylene glycol at concentrations of 25%, 10% or 5% w/w. A further group of four animals was treated with propylene glycol alone.

No mortality or signs of systemic toxicity was observed in the test item treated animals during the study. No treatment related effects were observed on body weight. The Stimulation Index (SI) expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows: 5% w/w, SI 1.35 (negative); 10% w/w, SI 1.55 (negative); 25% w/w, SI 1.62 (negative). The results of the statistical analysis of the data indicated there was no significant difference between the control group and the test groups. In conclusion, under the conditions of the present assay, the substance tested in a suitable vehicle was shown not to have skin sensitisation potential in the Local Lymph Node Assay.