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Administrative data

Description of key information

The substance Fret 10 -0367 was tested in LLNA study (OECD TG 429) at non-irritating concentrations of 50%, 25% and 10% v/v in acetone/olive oil 4:1. Stimulatin Indices for all the tested concentrations were below 3. The substance is considere a non-sensitizer under the testing conditions.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 26 September 2016 and 01 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
EC No. 440/2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd strain
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
- Microbiological status of animals, when known: not reported
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23g
- Housing: 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: >5 days
- Indication of any skin lesions: not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25”C
- Humidity (%): 30 - 70%
- Air changes (per hr): >15
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 26 September 2016 To: 01 NOvember 2016
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
undiluted (as supplied or 50%, 25% or 10% in acetone/olive oil 4:1
No. of animals per dose:
Preliminary Screening Test = 1 per dose

Main Test = 5 per dose
Details on study design:
Study Design

Preliminary Screening Test
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using two mice, one mouse per test item concentration. 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 (Days 1, 2, 3). 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 according to the scale included as Table 1. 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 sensitization.

Main Test
Test Item Administration
Groups of five mice were treated with the test item at concentrations of 50%, 25% or 10% 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 (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 five mice received the vehicle alone in the same manner.
The positive control animals were similarly treated to the test animals except that 25 µL of the positive control item, α Hexylcinnamaldehyde, at a concentration of 25% v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item, vehicle control or positive control item (Day 6) all mice were injected via the tail vein with 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.

Observations
Clinical 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 signs of ill health during the test were recorded.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes.

Preparation of Single Cell Suspension:
A single cell suspension of the lymph node cells for each individual animal 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 cells 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 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)
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non parametric Kruskal Wallis Rank Sum and Mann Whitney U test procedures were used.
Probability values (p) are presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P>0.05 (not significant)
Positive control results:
The positive control α Hexylcinnamaldehyde gave a Stimulation Index of greater than 3 (8.59) when tested at a concentration of 25% v/v in acetone/olive oil 4:1
Key result
Parameter:
SI
Value:
ca. 0.74
Test group / Remarks:
10% v/v in acetone/olive oil 4:1
Remarks on result:
other: Negative
Key result
Parameter:
SI
Value:
ca. 1.79
Test group / Remarks:
25% v/v in acetone/olive oil 4:1
Remarks on result:
other: Negative
Key result
Parameter:
SI
Value:
ca.
Test group / Remarks:
50% v/v in acetone/olive oil 4:1
Remarks on result:
other: Negative
Cellular proliferation data / Observations:
Preliminary Screening Test
No signs of systemic toxicity were noted.
Very slight erythema and a greater than 25% increase in mean ear thickness were noted in the animal treated with the undiluted test item.
No visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted in the animal treated with the test item at a concentration of 50% v/v in acetone/olive oil 4:1.
Based on this information the dose levels selected for the main test were 50%, 25% and 10% v/v in acetone/olive oil 4:1.

Main Test
Estimation of the Proliferative Response of Lymph Node Cells
The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are summarised in Table 2 (below):

Clinical Observations and Mortality Data
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Body Weight
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.

Table 2: Stimulation Indices

 Teatment Group  Concentration  Stimulation Index  Result
 Test Item

10% v/v in acetone/olive oil 4:1

 0.74  Negative
 

25% v/v in acetone/olive oil 4:1

 1.79

 Negative

 

50% v/v in acetone/olive oil 4:1

 2.09

 Negative

 Positive Control Item

25% v/v in acetone/olive oil 4:1

 8.59

 Positive

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be a non-sensitizer under the conditions of the test.
The test item does not meet the criteria for classification according to the Globally Harmonized Classification System and to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.
The positive control α Hexylcinnamaldehyde gave a Stimulation Index of greater than 3 (8.59) when tested at a concentration of 25% v/v in acetone/olive oil 4:1 thus demonstrating the sensitivity and reliability of the test system.
Executive summary:

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50% v/v, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay.  Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in acetone/olive oil 4:1 at concentrations of 50%, 25% or 10% v/v.  A further group of five animals was treated with acetone/olive oil 4:1 alone.  A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, α Hexylcinnamaldehyde, at a concentration of 25% v/v in acetone/olive oil 4:1.

Results

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

 Teatment Group  Concentration  Stimulation Index  Result
 Test Item

10% v/v in acetone/olive oil 4:1

 0.74  Negative
 

25% v/v in acetone/olive oil 4:1

 1.79

 Negative

 

50% v/v in acetone/olive oil 4:1

 2.09

 Negative

 Positive Control Item

25% v/v in acetone/olive oil 4:1

 8.59

 Positive

Conclusion

The test item was considered to be a non-sensitizer under the conditions of the test.

The test item does not meet the criteria for classification according to the Globally Harmonized Classification System and to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

The positive control α Hexylcinnamaldehyde gave a Stimulation Index of greater than 3 (8.59) when tested at a concentration of 25% v/v in acetone/olive oil 4:1 thus demonstrating the sensitivity and reliability of the test system.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Justification for classification or non-classification

In view of the absence of skin sensitisation in the LLNA study (OECD TG 429) the substance Fret 10-0367 does not need to be classified for skin sensitisationaccording to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and its amendments.