Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Remarks:
This in vivo study was performed before entry into force of EU Regulation 2016/1688
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-04-28 to 2014-06-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant 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:
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
Details on test material:
- Name of test material (as cited in study report): Tris(2-hydroxyethyl)methylammonium methyl sulphate- Substance type: organic- Physical state: yellowish liquid- Expiration date of the lot/batch: July 2015- Storage condition of test material: At room temperature, avoid temp. <5 and >30°C- Analytical purity: 98.9 %- Lot/batch No.: Lot. 29734916K0

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst, The Netherlands- Age at study initiation: 9 - 10 weeks- Housing: groupwise in Makrolon Type III cages- Diet: ad libitum (2018C Teklad Global 18% protein rodent diet)- Water: ad libitum (tap water)- Acclimation period: at least five daysENVIRONMENTAL CONDITIONS- Temperature (°C): 22 ± 2- Humidity (%): 45 - 65- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
25, 50 and 100 %
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used, was 100 % of the undiluted test item. - Irritation: To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals treated with 50 % and 100 % of the test substance once daily on three consecutive days. On day 1 to 3, the animal treated with 50% test item concentration showed an erythema of the ear skin (score 1). The animal treated with 100 % test item concentration showed an erythema of the ear skin (score 2 on day 1 and score 1 between days 2 to 6). No relevant increases in ear thickness or ear weight were observed. Thus, the test item in the main study was assayed at 25, 50 and 100 %. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.MAIN STUDYTopical applicationEach test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25 and 50% (w/w) in PG, as well as with the undiluted test item (100 %). The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (∅ ∼ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).Administration and determination of incorporated 3H-methyl-thymidineFive days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 20.1 μCi of 3H-methyl thymidine (equivalent to 80.3 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.Approximately five hours after treatment with 3HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.Determination of lymph node weight, cell count and ear weightsAfter excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance. Furthermore, the lymph node cell count was determined for each animal. For this, the volume of the cell suspensions was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined using a cell counter (CAS®1, Schärfe System). The values obtained were taken down manually. After the lymph nodes had been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.Interpretation of raw dataThe proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/animal values were determined, mean scintillation-background DPM was subtracted from test and control raw data.A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:• First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.• Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.Furthermore, an index was calculated for the lymph node weight and –cell count as well as for the ear weight by dividing the mean values of the test item treated groups by the mean of the vehicle treated group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response (see Ref. a) and the cut-off value for the ear weight index regarding a positive response (ear irritation) was reported to be 1.1 (see Ref. b). However, these cut-off values mentioned in the respective papers have been determined using a different strain of mice and can thus not be implicitly adopted.(a) Ehling G., Hecht, M., Heusener J., Gamer A.O., van Loveren H., Maurer Th., Riecke K., Ullmann L., Ulrich P., Vandebriel R., Vohr H.-W. (2005): An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay 2nd round. Toxicology, 212, 69–79.(b) Ulrich P., Streich J., Suter W. (2001): Intralaboratory validation of alternative endpoints in the murine local lymph node assay for the identification of contact allergic potential: primary ear skin irritation and ear-draining lymph node hyperplasia induced by topical chemicals. Archives of Toxicology, 74, 733-744.ObservationsIn addition to the sensitising reactions the following observations and data were recorded during the test and observation period:Mortality/viability, body weights, ear thickness, ear weights, lymph node weights, lymph node cell counts and clinical signs (local and systemic)
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation).A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as a statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05).The Dean-Dixon-Test and the Grubb’s test were used for detection of possible outliers (performed with Microsoft Excel 2007). Two outlier animals (animal 4 and 17) were detected in the Dean-Dixon-Test but and in the Grubb’s test, but were not excluded from any subsequent calculations. However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:
The stimulation index of alpha-hexylcinnamaldehyde at concentrations of 10 and 50 % were 1.76 and 6.79, respectively. Compared to the historical control values the S.I. of 6.79 was well within the range of the available data.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 25, 50 and 100 % were 1.4, 1.6 and 2.31, respectively. An EC3 value could not be calculated, since all S.I.s are below the threshold value of 3.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100 % were 1155.2, 1325.4 and 1912.2 DPM, respectively. The mean DPM/animal value for the vehicle control group was 826.6 DPM.

Any other information on results incl. tables

Additional observations

No mortality occurred and no signs of systemic toxicity were observed. 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 measured lymph node weights and -cell counts of all animals treated were recorded after sacrifice. A statistically significant or biologically relevant increase in lymph node weights or –cell counts was not observed in any of the test item treated groups in comparison to the vehicle control group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response. The indices determined for the lymph node cell count did not reached or exceed this threshold.

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A biologically relevant or statistically significant increase in ear weights was not observed. Furthermore, the cut-off value (1.1) of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice was not reached or exceeded in any of the treated groups.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information