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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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Benzyltris(dimethylaminato)phosphorus(1+) tetrafluoroborate(1-)
EC Number:
302-080-5
EC Name:
Benzyltris(dimethylaminato)phosphorus(1+) tetrafluoroborate(1-)
Cas Number:
94088-77-4
Molecular formula:
C13H25BF4N3P
IUPAC Name:
benzyltris(dimethylaminato)phosphorus(1+) tetrafluoroborate(1-)
Test material form:
solid: crystalline

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: AnLab Prague, Czech Republic

- Females (if applicable) nulliparous and non-pregnant: yes

- Microbiological status of animals, when known: no data

- Age at study initiation: 8-10 weeks

- Weight at study initiation:

Mouse no. Initial body weight
1 20.98
2 19.60
3 20.98
4 19.57
5 19.87
Mean 20.20
S.D. 0.721

Positive control
Mouse no. Initial body weight
1 19.69
2 18.97
3 17.47
4 21.06
5 20.69
Mean 19.58
S.D. 1.437

Test substance (100%)
Mouse no. Initial body weight
1 21.40
2 19.56
3 21.02
4 19.65
5 23.60
Mean 21.05
S.D. 1.644

Test substance (50%)
Mouse no. Initial body weight
1 21.90
2 18.85
3 20.22
4 19.70
5 20.46
Mean 20.23
S.D. 1.121

Test substance (25%)
Mouse no. Initial body weight
1 20.01
2 20.08
3 22.02
4 22.26
5 19.41
Mean 20.76
S.D. 1.293

- Housing: The animals were housed individually in polypropylene cages suspended on stainless steel racks, in a room equipped with central air-conditioning. The room temperature was within the range of 22 ± 2°C, relative humidity was within the range of 50 - 60 %. The light regimen was set to a 12-hour light / 12-hour dark cycle The sanitation was performed according to standard operation procedures.

- Diet (e.g. ad libitum): laboratory food ssniff (ssniff Spezialdiäten GmbH, Germany) ad libitum

- Water (e.g. ad libitum): tab water ad libitum

- Acclimation period: 6 days prior to the start of treatment

- Indication of any skin lesions: The health condition of animals was examined by a veterinarian before initiation of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): see housing
- Humidity (%): see housing
- Photoperiod (hrs dark / hrs light): see housing

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
The required amount of the test item (according to the concentration) was mixed in vehicle shortly before the administration (i.e. 100% concentration was obtained by mixing of 1g of test item with 1mL of vehicle). The dose preparation data are listed in the raw data. The preparations were made freshly before each dosing occasion. The concentrations of the test substance used for test were 100, 50 and 25%.
No. of animals per dose:
5 females – control (vehicle)
5 females – positive control
15 females – test item (5 per group)
4 females - pre-screen test plus spare animals
Details on study design:
PRE-SCREEN TESTS:
The pre-screen test was conducted under the same conditions as the main LLNA study, except there was no assessment of lymph node proliferation. The undiluted test item was tested.
All mice (2 mice/group) were observed daily for any clinical signs of toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to the termination (day 6). Both ears of each mouse were assessed for any sign of irritation. Erythema scores were given following Table 1. Ear thickness was measured by a calliper on Day 1 (pre-dose), Day 3 and Day 6. Excessive local skin irritation is indicated by an erythema score ≥ 3 and/or an increase in ear thickness of ≥25%.
Erythema Scores
Observation Score
No erythema 0

Very slight erythema (barely perceptible) 1

Well-defined erythema 2

Moderate to severe erythema 3

Severe erythema (beet
redness) to eschar formation
preventing grading of erythema 4

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
Name of test method: Local Lymph Node Assay according to OECD Guideline 429

Criteria used to consider a positive response:

Clinical observation score

Clinical symptoms, local irritation and systemic toxicity were observed and rated according to a 4 class system
(no effect, weak effect, moderate effect and strong effect). Results are expressed as the Stimulation Index (SI).
The SI was obtained by dividing the pooled radioactive incorporation for each treatment group by the radioactive
incorporation of the pooled vehicle control group; this yields a mean SI. A substance is regarded as sensitizer in
the LLNA test when SI≥3. EC3 value, which induce stimulation indices, is determined by linear interpolation of
points on dose-response curve, immediately above and below of SI value, according to the equation:

EC3=c+[(d-3)/(b-d)]x(a-c)

a – higher concentration, b – SI of higher concentration, c – lower concentration d – SI of lower concentration
If all points are below the stimulation index of three, no EC3 value can be stated.

TREATMENT PREPARATION AND ADMINISTRATION:

Day 1: Each animal was identified and the body weight was recorded. To the dorsum of each ear 25µL of the appropriate dilution of the test item, or the vehicle alone was applied.
Days 2 and 3: The application procedure carried out on day 1 was repeated.
Days 4 and 5: No treatment.
Day 6: The body weight of each animal were recorded. 250µL of phosphate-buffered saline (PBS) containing 2 µCi (7.4 x 104 Bq) of 125I-iododeoxyuridine and 10-5M fluorodeoxyuridine was injected into all test and control mice via the tail vein. Five hours later, the animals were sacrificed. The draining auricular lymph nodes from each ear were excised and pooled in PBS for each experimental group (pooled treatment group approach) and weighted.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
2.07
Test group / Remarks:
Test substance (25 %)
Key result
Parameter:
SI
Value:
2.45
Test group / Remarks:
Test substance (50%)
Key result
Parameter:
SI
Value:
2.65
Test group / Remarks:
Test substance (100%)
Key result
Parameter:
EC3
Remarks on result:
other: The EC3 value could not be calculated, as all measured points were below the stimulation index of three.
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
In comparison with the control group, an increase of the pooled lymph node weight was recorded at all used concentrations. The pooled lymph node weight of treated groups were 0.0408g for 25% concentration, 0.0435g for 50% concentration and 0.0468g for 100% concentration of tested item. The lymph node weight of control group and positive control group were 0.0357g and 0.0737g, respectively. The DPM values for the three treated groups were 972 (25%), 1151 (50%) and 1243 (100%), respectively.

DETAILS ON STIMULATION INDEX CALCULATION
The SI values for the three treated groups were 2.07 (25%), 2.45 (50%) and 2.65 (100%), respectively.

EC3 CALCULATION
The EC3 value could not be calculated, as all measured points were below the stimulation index of three.

CLINICAL OBSERVATIONS:
Animals were carefully observed for any clinical symptoms, either of local irritation at the application site or systemic toxicity. The daily clinical observation of the animals did not show visible clinical signs.

BODY WEIGHTS:
The animal body weights were measured prior to the first treatment and at the scheduled sacrifice. No marked changes of mean body weight were observed during the study.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The skin sensitizing potential of Tecnoflon BA 104 was assessed using the murine local lymph node assay. Based on the results of this study, Tecnoflon BA 104 is not considered a skin sensitizer under the condition of this study.
Executive summary:

The sensitization potential of Tecnoflon BA 104 was evaluated using the Local Lymph Node Assay (LLNA). The LLNA has been developed to determine the contact sensitization potential of chemicals. Based on the recommendations of the OECD Guideline, the test item was formulated in Dimethyl sulfoxide (DMSO). The positive control (a-Hexylcinnamaldehyde) (25%) was formulated in acetone:olive oil 4:1 (v/v).

ThePre-screen testwas performed using a dose of 100 % (w/v). Based on the observations recorded in the preliminary test, the concentration of 100 % was selected as top dose for the main test. Five female mice (CBA/Ca) were topically exposed (dorsum of both ears) to the test item at concentrations of 25%, 50% and 100%, to the positive control and to the vehicle only. Lymphocyte proliferation was measured using incorporation of radioactive 125I-iododeoxyuridine and 10 -5 M fluorodeoxyuridinein the draining lymph nodes. The radioactive incorporation was expressed as disintegrations per minute (DPM)/pooled treatment group and compared with DPM value from the vehicle control group and expressed as the Stimulation Index (SI).

After application of the test itemat three concentrations (25%, 50% and 100% w/v) the animals did not show visible clinical symptomsor local irritation or systemic toxicity. In this study Stimulation Indices (SI) of  2.07, 2.45 and 2.65 were determined with the test item at concentration of 25, 50, and 100% in dimethyl sulfoxide, respectively. The EC3 value could not be calculated, as all measured points were below the Stimulation Index of three.

The test item Tecnoflon BA 104isnot considered a skin sensitizer under the test condition of this study.

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