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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:
04 to 24 October 2013
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)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd) strain
Sex:
female
Details on test animals and environmental conditions:
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatization period of at least five days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were eight to twelve weeks old.The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study.The temperature and relative humidity were controlled to remain within target ranges of 19 to 25 degC and 30 to 70%, respectively. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.Justification: Mice are the preferred species of choice since quantitative methods have been developed for the measurement of skin sensitization responses in the mouse and are specified in the appropriate test guidelines.
Vehicle:
other: the test item was used undiluted and freshly prepared as a solution in butanone
Concentration:
Treated with the undiluted test item or the test item at concentrations of 25% or 10% v/v in butanone (50% was not suitable for dosing)
No. of animals per dose:
4
Details on study design:
Test Item Formulation and Experimental Preparation
For the purpose of the study, the test item was used undiluted and freshly prepared as a solution in butanone. This vehicle was chosen as it produced the most suitable formulation at the required concentration. The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Procedure
Preliminary Screening Test
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The 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 according to the scale included below. Any clinical signs of toxicity, if present, were also recorded. The body weight 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 four mice were treated with the undiluted test item or the test item at concentrations of 25% or 10% v/v in butanone (50% was not suitable for dosing). 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 micropipetteand 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 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: 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. 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 project 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 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 deg C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by 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 (Beckman Instruments Inc, Fullerton, CA, USA).Interpretation of ResultsThe 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).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
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).
Positive control results:
Introduction
A study was performed to assess the sensitivity of the strain of mouse used at these laboratories to a known sensitizer. The methodology for the LLNA is detailed in the OECD Guideline for the Testing of Chemicals No. 429 and Method B.42 of Commission Regulation (EC) No. 440/2008. The study described in this document is based on these test methods but has been refined in order to reduce the number of animals required. The reduced LLNA (rLLNA) has been endorsed by the non-Commission members of the European Centre for the Validation ofAlternative Methods (ECVAM) Scientific Advisory Committee (ESAC) at its 26th meeting held on 26 – 27 April 2007 at ECVAM, Ispra, Italy.
Test item:α-Hexylcinnamaldehyde, tech., 85%
Study number:41301405
Study dates:17 April 2013 to 23 April 2013
Methods
A group of five animals was treated with 50 µL (25 µL per ear) of α-Hexylcinnamaldehyde, tech., 85% as a solution in butanone at a concentration of 15% v/v. A further control group of five animals was treated with butanone alone.ResultsThe Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:
Positive
Conclusion
α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.
Key result
Parameter:
SI
Remarks on result:
other: The stimulation indices per dose level are listed below. The substance is a non sensitiser
Key result
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM values are listed below
Interpretation of results:
not sensitising
Remarks:
Migrated informationCriteria used for interpretation of results: EU
Conclusions:
The test item was considered to be a non-sensitizer under the conditions of the test.
Executive summary:

The test item was considered to be a non-sensitizer under the conditions of the test. The stimulation indices derived are as follows:

Concentration (% v/v) in butanone

Stimulation Index

Result

10

1.93

Negative

25

1.72

Negative

100

2.35

Negative

 

No skin irritation was noted during the study. 

No classification is applicable.

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

The test item was considered to be a non-sensitizer under the conditions of the test. The stimulation indices derived are as follows:

Concentration (% v/v) in butanone

Stimulation Index

Result

10

1.93

Negative

25

1.72

Negative

100

2.35

Negative

 

No skin irritation was noted during the study. 

No classification is applicable.


Short description of key information:
Skin sensitisation using the LLNA.

Justification for selection of skin sensitisation endpoint:
GLP study conducted in accordance with current OECD guidelines.

Respiratory sensitisation

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

White mineral oils do not have a history of being respiratory sensitisers. This effect is not applicable to this category of substances.


Short description of key information:
Brief assessment of potential respiratory sensitisation based on history of use.

Justification for selection of respiratory sensitisation endpoint:
Based on history of use of white mineral oils.

Justification for classification or non-classification

A Klimish one rated GLP study conducted in accordance with current OECD guidelines is available to assess skin sensitisation. The results are negative.

White mineral oils do not have a history of being respiratory sensitisers. This effect is not applicable to this category of substances.

No classification is applicable.