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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:
2018-01-25 to 2018-02-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The substance is not considered to fall under the applicability domain of the in vitro tests for skin sensitization (OECD test guidelines 442D, 442C and 442E). Based on the low water solubility, the test substance cannot be tested up to and including the highest concentration required in the test guidelines. Based on fast hydrolysis the substance is considered to be unstable under the test conditions described in the test guidelines and due to the formation of titanium hydroxide precipitate will be formed. Furthermore, the test guideline OECD 442C is not applicable for the testing of metal compounds, since they are known to react with proteins with mechanisms other than covalent binding.
Thus, the in vitro tests 442D, 442C and 442E are considered not reliable to test the potential for skin sensitization of the substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

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

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 23kg
- Housing: the animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

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

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
50%, 25% and 10% v/v in dimethylformamide
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
- Irritation: none
- Systemic toxicity: none
- Ear thickness measurements: yes

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
The test item will 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 control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitizer".

TREATMENT PREPARATION AND ADMINISTRATION:
- Groups of four mice per concantration 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 tip of the pipette. A further group of four mice received the vehicle alone in the same manner.
- Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 0.25 mL (250 µL) of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 µCi to each mouse.
- Clinical Observations: twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6.
- Body Weights: recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).
- Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.
- Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells 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 labeled with the study number and dose concentration. The lymph node cell 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 pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 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 ¿-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 (Beckman Instruments Inc, Fullerton, CA, USA).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
a-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
3.03
Test group / Remarks:
50% (v/v) test item in dimethyl formamide
Remarks on result:
other: Stimulation Index of 3.0 or greater indicates a positive result
Parameter:
SI
Value:
2.58
Test group / Remarks:
25% (v/v) in dimethyl formamide
Remarks on result:
other: Stimulation Index of 3.0 or greater indicates a positive result
Parameter:
SI
Value:
2
Test group / Remarks:
10% (v/v) in dimethyl formamide
Remarks on result:
other: Stimulation Index of 3.0 or greater indicates a positive result
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
- disintegrations per minute/node: 965.70 (Vehicle), 1930.51 (10 (% v/v)), 2491.01 (25 (% v/v)), 2923.08 (50 (% v/v))

DETAILS ON STIMULATION INDEX CALCULATION
- The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).


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

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

Any other information on results incl. tables

Table 1: Main Test - Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration
(% v/v) in dimethyl formamide

dpm

dpm/Node (a)

Stimulation Index (b)

Result

Vehicle

7725.62

965.7

na

na

10

15444.11

1930.51

2

Negative

25

19928.11

2491.01

2.58

Negative

50

23384.6

2923.08

3.03

Positive

dpm = Disintegrations per minute

a = Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

b = Stimulation Index of 3.0 or greater indicates a positive result

na = Not applicable

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test item was considered to be a sensitizer under the conditions of the test.
The test item was classified as a contact sensitizer (Category 1B) according to the Globally Harmonized System of Classification and Labelling of Chemicals.