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Description of key information

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:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP.
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)
Remarks:
inspected July 2012; signature: November 2012
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised animal supplier
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: females 15 - 23g
- Fasting period before study: overnight before dosing
- Housing: Animals were housed individually suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum): pelleted diet ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hour dark / 12 hours light
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Preliminary test: undiluted (100% v/v).
Main test: 25, 50 and 100% v/v
No. of animals per dose:
Preliminary test: 1
Main test: 5
Details on study design:
RANGE FINDING TESTS:
A single mouse was treated by daily application of 25 μl of the undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was 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 bodyweight was recorded on Day 1 (prior to dosing) and on Day 6. The thickness of each ear was measured using an Oditest micrometer, pre-dose and post dose on Day 1, post dose on Days 2 and 3 and on Days 4, 5 and 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between Days 1 to 3 and Days 1 to 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.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: 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".

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of five 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 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 thickness of each ear was measured using an Oditest micrometer, pre-dose and post dose on Day 1, post dose on Days 2 and 3 and on Days 4, 5 and 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between Days 1 to 3 and Days 1 to 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.

3H-Methyl Thymidine Administration:
Five days following 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:80μCi/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.
- Bodyweights: The bodyweight of each mouse was 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. 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 labelled with the project 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 ten minutes. The pellet was resuspended in 10 ml of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 ml of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation:
After approximately eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by β-scintillation counting. The "Poly QTM" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty 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 twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system.


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 by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett’s Multiple Comparison test was used and for non-homogenous datasets Dunnett’s T3 Multiple Comparison Method was used.
Positive control results:
In a separate 'positive control study' performed according to OECD 429 during October 2012-November 2012, the sensitivity of the strain of mouse used in this study was assessed using the known sensitiser, α-hexylcinnamaldehyde. The positive control was tested at concentrations of 25% v/v in the same vehicle. The highest concentration tested showed a Stimulation Index (SI) of 6.29 and met the criteria for a 'positive' result.
Parameter:
SI
Remarks on result:
other: See table below
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See table below

In the preliminary screening test, no signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. Based on this information the undiluted test item and the test item at concentrations of 50% and 25% v/v in acetone/olive oil 4:1 were selected for the main test.

 

In the main test, there were no deaths or signs of systemic toxicity, and body weights were unaffected. The radioactive disintegrations per minute (dpm) and stimulation index (SI) are given in the table, below.

 Conc. (% w/w)

 Dpm

 SI

 Result

 0

 2108.73

 N/A

 N/A

 25

 3152.14

 1.49

 Negative

 50

 4427.28**

 2.10

 Negative

 100

 4441.10**

 2.11

 Negative

An SI of less than 3 was recorded at each of the 3 concentrations of substance evaluated.

 

**Significantly different from control group p<0.01

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the test material is considered to be non-sensitising to skin.
Executive summary:

The study was performed to assess the skin sensitisation potential of the test material in the CBA/Ca (CBA/CaOlaHsd) mouse following topical application to the dorsal surface of the ear. The study was performed to GLP and the method followed OECD 429. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100%, 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 undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 50% or 25% v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone. In the main test, there were no deaths or signs of systemic toxicity, and body weights were unaffected. A stimulation index (SI) of less than 3 was noted at each of the three test material concentrations evaluated. Under the conditions of the study the test item does not meet the criteria for classification as a sensitiser.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

OECD 429 LLNA 2013 - The study was performed to assess the skin sensitisation potential of the test material in the CBA/Ca (CBA/CaOlaHsd) mouse following topical application to the dorsal surface of the ear. The study was performed to GLP and the method followed OECD 429. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100%, 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 undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 50% or 25% v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone. In the main test, there were no deaths or signs of systemic toxicity, and body weights were unaffected. A stimulation index (SI) of less than 3 was noted at each of the three test material concentrations evaluated. Under the conditions of the study the test item does not meet the criteria for classification as a sensitiser.

 

Human RIPT 1995 - A human repeat insult patch test was performed to assess the skin sensitisation potential of the test material in human volunteers . The test substance was evaluated neat to determine its ability to sensitize the skin of normal volunteer subjects using an occlusive repeated insult patch study. One hundred eleven subjects between the ages of 18 and 66 were enrolled and 105 subjects completed the study. Under the conditions employed in this study; there was no evidence of sensitization to the test substance.

 

Equivalent to OECD 406 1992 - The study was conducted to assess the potential of the test substance to cause delayed dermal hypersensitivity in female Dunkin-Hartley guinea pigs. The study was performed to GLP and the method followed is similar to the Buehler method. From the results of this study it is concluded that the test material under the conditions of this test, produced irritant responses at concentrations of 5% and greater. However, the increases in the incidence and severity of responses to 25% and 12.5% concentrations in the test group are considered evidence of delayed dermal hypersensitivity. The large number of positives observed in the control group, questions the integrity of this study and validity of the conclusion that the test substance is a skin sensitiser.


Migrated from Short description of key information:
Skin sensitisation: non-sensitising, female mice, OECD 429, Harlan Laboratories Ltd 2013

Justification for selection of skin sensitisation endpoint:
Three studies available, one GLP compliant Klimisch 1 LLNA study; one Human RIPT study where reliability cannot be assigned and one in vivo study according to GLP and following an equivalent methodology to the Buehler method where reliability cannot be assigned. Selected study is an in vivo study indicating negative reaction to dermal exposure; supported by Human data. The Buehler method equivalent in vivo study has been disregarded as unreliable due to potential false-positive results observed from control data.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The substance does not meet classification criteria under EU Directive 67/548/EEC for skin sensitisation

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for skin sensitisation

The weight of evidence indicates that the substance has a low frequency of occurrence in humans and/or low potency in animals and therefore it cannot be presumed to have the potential to produce sensitisation in humans via the dermal route. This is from a high reliability in vivo study supported by human data. The Buehler method equivalent in vivo study has been disregarded as unreliable due to potential false-positive results indicated in the controls possibly related to irritation response rather than delayed hypersensistivity.