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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:
2016
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch 605803
- Expiration date of the lot/batch: 23 October 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Soluble at 600 mg/mL and stable for 20 hours with 1%mol decrease in DMSO

FORM AS APPLIED IN THE TEST (if different from that of starting material): Test material prepared in DMSO (w/w)

OTHER SPECIFICS: The test item preparations (w/w) were prepared within 4 hours prior to each dosing. No adjustment was made for specific gravity of the vehicle. Homogeneity was assessed by visual inspection of the solutions. Correction of the purity/composition of the test item is not applicable, since the test method requires a logical concentration range rather than specific dose levels to be dosed.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known:
- Age at study initiation: Approximately 10 weeks old
- Weight at study initiation: Average 20.79 g (18.7-23.3 g)
- Housing: Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions.
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): At least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12:12 light:dark cycle
- IN-LIFE DATES: From: 13 May 2016 To: 01 June 2016

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
0, 5, 10, and 25%
No. of animals per dose:
5 females/dose
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Soluble at 600 mg/mL in DMSO
- Irritation: Very slight erythema, scaliness (all animals) and/or scabs (2 animals) were noted for all animals between Days 2 and 6
- Systemic toxicity: Hunched posture and piloerection were noted for both animals treated at 50% on Day 3.
- Ear thickness measurements:Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values
- Erythema scores: Animal #1, #2, #3, and #4 erythema scores of 1 from day 2 through 5 at the left and right sites. No erythema observed on day 6.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Mouse Local Lymph Node Assay (LLNA) (OECD 429)
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
Induction (Days 1, 2, and 3): Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle DMSO).

Excision of the Nodes (Day 6): The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item. Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US). After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

Tissue Processing for Radioactivity (Day 6): Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter: 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

Radioactivity Measurements (Day 7): Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

OBSERVATIONS
- Mortality/Viability: twice daily
- Body weights: On day 1 (pre-dose) and day 6
- Clinical signs: Once daily on days 1-6 (on days 1-3 between 3 and 4 hours after dosing)
- Necropsy: No necropsy for gross macroscopic examination was performed according to study plan.
- Irritation: Once daily on days 1-6 (on days 1-3 within 1 hour after dosing) according to the following numerical scoring system. In addition, a description of all other (local) effects was recorded:

Irritation Grading for Erythema and Eschar formation:
No erythema ..............................................................................…………………………........... 0
Very slight erythema (barely perceptible) ....................................................………........…..… 1
Well-defined erythema ...................................................................……………………….......... 2
Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth) … 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema .......... 4
Positive control substance(s):
other: No concurrent positive control group was included in the study for both scientific and animal welfare reasons.
Statistics:
The EC3 value (the estimated test item concentration that will give a SI =3) was determined, using linear interpolation (Basketter DA, Lea LJ, Dickens A, Briggs, D, Pate I, Dearman RJ and Kimber I. A comparison of statistical approaches to the derivation of EC3 values from local lymph node assay dose responses. J Appl Toxicol 1999;19:261-266)

Results and discussion

Positive control results:
For both scientific and animal welfare reasons, no concurrent positive control group was included in the study.
An extensive data base is available with reliability checks performed at half year intervals during at least the past 9 years showing reproducible and consistent positive results.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1
Variability:
+/- 0.2
Test group / Remarks:
0% (control)
Key result
Parameter:
SI
Value:
1.5
Variability:
+/- 0.3
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
3.2
Variability:
+/- 0.6
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
4.2
Variability:
+/- 0.7
Test group / Remarks:
25%
Key result
Parameter:
EC3
Value:
9.4
Test group / Remarks:
Estimated test item concentration (%) that will give a SI = 3
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION: Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 1148, 2529 and 3330 DPM, respectively. The mean DPM/animal value for the vehicle control group was 787 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 1.5 +/- 0.3, 3.2 +/- 0.6 and 4.2 +/- 0.7, respectively.

EC3 CALCULATION: The EC3 value (the estimated test item concentration that will give a SI = 3) was determined using linear interpolation. The EC3 determined was 9.4%

CLINICAL OBSERVATIONS: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.

IRRITATION: The slight to well-defined erythema, scaliness or scabs of the ears as shown by the animals was considered not to have a toxicologically significant effect on the activity of the nodes.

BODY WEIGHTS: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

MACROSCOPIC EXAMINATION: All auricular lymph nodes of the control animals and animals treated at 5% and of most animal treated at 10% were considered normal in size. Most nodes of the animals treated at 25% were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Based on the results of the study, MTDID 51670 is a GHS Category 1B skin sensitizer.
Executive summary:

The dermal sensitization potential of the test article, MTDID 15670, was evaluated in a mouse local lymph node assay (LLNA) using female CBA/J mice (5/dose). The study design was based on OECD 429 (2010) and was performed in accordance with GLP. The test material was formulated in DMSO within 4 hours prior to each dosing. The test material in formulation (25µL/ear) was applied topically at 5, 10, and 25% to the dorsal surface of both ears once daily on day 1, 2, and 3; the top dose was determined in a preliminary study. Control animals were treated in the same way as the experimental animals, except that the vehicle was administered without the test material. On day 6, each animal was injected via the tail vein with 0.25 mL sterile PBS containing 20 µCi of 3H-methyl thymidine. Five hours after tail injection, all animals were killed via intraperitoneal injection of Euthasol. The draining (auricular) lymph node of each ear was excised and the relative size of the nodes (as compared to normal) was estimated by visual examination. Abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal. Disintegrations per minute (DPM) were determined for each animal and for each dose. A stimulation index (SI) was calculated for each group using individual SI values. If the results indicate a SI3, the test item may be regarded as a skin sensitizer. Slight to well-defined erythema, scaliness or scabs of the ears was observed in all animals and was considered to not to have toxicologically significant effect on lymph node activity. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. All auricular lymph nodes of the control animals and animals treated at 5% and of most animal treated at 10% were considered normal in size. Most nodes of the animals treated at 25% were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 1148, 2529 and 3330 DPM, respectively. The mean DPM/animal value for the vehicle control group was 787 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 1.5±0.3, 3.2±0.6 and 4.2±0.7, respectively. These results indicate that the test item elicits a SI ≥ 3. The data show a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 9.4% was calculated. Based on the results of the study, MTDID 51670 is a GHS Category 1B skin sensitizer.