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Description of key information

A Local Lymph Node Assay (LLNA) was conducted in order to determine the skin sensitising potential of VCA. The LLNA study was conducted according to the OECD Guideline 429 and was GLP compliant. Female CBA/Ca mice were used as the test species and were administered 25 µL (per ear) of undiluted test item or the test item in a concentration of 50% or 25% v/v in acetone/olive oil 4:1. There were no deaths and no signs of systemic toxicity were noted in the test or control animals during the test. Body weight change of the test animals was comparable to that observed in the corresponding control group. The concentration of test item expected to cause a threefold increase in 3HTdR incorporation (EC3 value) was calculated to be 26%. The test item was considered to be a sensitiser under the conditions of the study and classified as Category 1B according to Regulation (EC) No 1272/2008.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 September 2016 - 8 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
In accordance with Annex VII of REACH, Column 2, an in vivo skin sensitisation study meeting the requirements set out in Article 13(3), first paragraph, and Article 13(4) was initiated before 11 October 2016 and is therefore considered as appropriate to address the standard information requirement for this endpoint.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 12 weeks old
- Weight at study initiation: 15 to 23 g
- Housing: suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum): free access to 2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK
- Water (e.g. ad libitum): free access to mains tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25 µL (per ear) of undiluted test item or the test item in a concentration of 50% or 25% v/v in acetone/olive oil 4:1.
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
A preliminary screening test was performed using 2 mice (1 mouse per test item concentration) using available information regarding the systemic toxicity/irritancy potential of the test item. The mice were treated by daily application of 25 µL of the undiluted test item or the test item at a concentration of 50% v/v in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days. The mice were 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 each 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 (Mitutoyo Corporation), 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 sensitisation.
- Irritation: No signs of visual local skin irritation
- Systemic toxicity: No signs of systemic toxicity
- Ear thickness measurements: No signs of irritation indicated by >= 25% increase in mean ear thickness

MAIN STUDY
- Test Item Administration: Groups of four mice were treated with the undiluted test item or the test item at concentrations of 50% or 25% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. 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. 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.
3H-Methyl Thymidine Administration: Five days after 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 uCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 uCi to each mouse.
- 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 ill health were recorded. Body weights were recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).
- Terminal Procedures: Five hours following 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. 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 labelled with the study number a 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 (approx 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). After approx. 18 hours incubation at approx. 4 °C the precipitates were recovered by centrifugation at 2100 rpm (approx 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 B-scintillation counting. The "Poly Q(TM)" vials containing the samples and scintillation fluid were placed in the sample charger of the scintillator and left to stand in darkness for approx 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 approx 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).
- Data Evaluation: 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 ration of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test item will be regarded as a sensitiser 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-sensitiser". The EC3 value was also calculated. The EC3 value is the concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation. The equation used for the calculation of EC3 is: EC3 = c + [[(3 - d) / (b - d)] x (a - c)]. The results were also interpreted according to Regulation (EC) No 1272/2008.
Positive control substance(s):
other: Hexylcinnamaldehyde, tech., 85%
Positive control results:
The positive control substance, Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser under the conditions of the test.
Parameter:
SI
Remarks:
Test substance concentration 25 %
Value:
ca. 2.89
Parameter:
SI
Remarks:
Test substance concentration 50 %
Value:
ca. 4.83
Parameter:
SI
Remarks:
Test substance concentration 100 %
Value:
ca. 6.68
Key result
Parameter:
EC3
Value:
ca. 26
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Test substance concentration 25% v/v in acetone/olive oil 4:1 gave a Stimulation Index of 2.89 and a Negative result.
Test substance concentrations of 50% and 100% v/v in acetone/olive oil 4:1 gave Stimulation Indexes of 4.83 and 6.68 respectively, and therefore Positive results.

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

EC3 CALCULATION
EC3 = c + [[(3 - d) / (b - d)] x (a - c)]
a = 50
b = 4.83
c = 25
d = 2.89
EC3 = 25 + [[(3 - 2.89) / (4.83 - 2.89) x (50 - 25)] = 26

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 group animals over the same period.

See 'attached background material' for full results tables.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test item was considered to be a sensitiser under the conditions of the study and classified as Category 1B according to Regulation (EC) No 1272/2008.
Executive summary:

A Local Lymph Node Assay (LLNA) was conducted in order to determine the skin sensitising potential of VCA. The LLNA study was conducted according to the OECD Guideline 429 and was GLP compliant. Female CBA/Ca mice were used as the test species and were administered 25 µL (per ear) of undiluted test item or the test item in a concentration of 50% or 25% v/v in acetone/olive oil 4:1. There were no deaths and no signs of systemic toxicity were noted in the test or control animals during the test. Body weight change of the test animals was comparable to that observed in the corresponding control group. The concentration of test item expected to cause a threefold increase in 3HTdR incorporation (EC3 value) was calculated to be 26%. The test item was considered to be a sensitiser under the conditions of the study and classified as Category 1B according to Regulation (EC) No 1272/2008.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

A Local Lymph Node Assay (LLNA) was conducted in order to determine the skin sensitising potential of VCA. The LLNA study was conducted according to the OECD Guideline 429 and EU Method B.42, and was GLP compliant. The concentration of test item expected to cause a threefold increase in 3HTdR incorporation (EC3 value) was calculated to be 26%. The test item was considered to be a skin sensitiser under the conditions of the study and classified as Category 1B according to Regulation (EC) No 1272/2008.