[No public or meaningful name is available]

Endpoint summary

Description of key information

The test item produced an EC3 value of 37 % (the concentration of test item expected to cause a 3-fold increase in 3HTdR incorporation) and was considered to be a sensitiser under the conditions of the test. The positive control α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 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 (OECD 429 and EU Method B.42).

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 January 2017 to 02 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHSD

Sex:

female

Details on test animals and environmental conditions:

ANIMAL INFORMATION
- Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands.
- On receipt the animals were randomly allocated to cages.
- The animals were nulliparous and non-pregnant.
- After an acclimatisation period of at least 5 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 8 to 12 weeks old.

ANIMAL CARE AND HUSBANDRY
- Animals were 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 Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.
- Temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70%, respectively.
- The rate of air exchange was at least fifteen changes per hour.
- Lighting was controlled by a time switch to give 12 hours continuous light and 12 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.

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

1 %, 10 % and 100 % test item

No. of animals per dose:

Five

Details on study design:

TEST ITEM PREPARATION AND ANALYSIS
- The test item was used undiluted and freshly prepared as a solution in acetone/olive oil 4: 1. This vehicle was chosen as it produced the most suitable formulation at the required concentration.
- The vehicle determination record is included as Annex 3 (attached).
- Test item was formulated within 2 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 was reflected in the GLP compliance statement.

REFERENCE ITEM PREPARATION
- The positive control item was freshly prepared as a 25% v/v dilution in acetone/olive oil 4: 1.

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. Any clinical signs of toxicity, if present, were also recorded. The body weight of the 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- 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 time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of ≥ 25 % was considered to indicate excessive irritation and limited biological relevance to the sensitisation endpoint.

MAIN TEST
- Groups of five mice were treated with undiluted test item or test item at concentrations of 10 % or 1 % 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, a-Hexylcinnamaldehyde, tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4: 1 was applied to the dorsal surface of each ear.
- Local skin irritation was scored daily. The thickness of each ear was measured and recorded pre- and post dose on Day 1, post-dose on Days 2 and 3 and on Days 4, 5 and 6.

3H-METHYL THYMIDINE ADMINISTRATION
- Five days following the first topical application of the test item, vehicle control item 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 f3-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).

DATA EVALUATION
- The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio 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 calculation of EC3 was EC3 = c + [[(3-d)/(b-d)] x (a-c)] where a = lowest concentration giving stimulation index > 3; b = actual stimulation index caused by 'a’; c = highest concentration failing to produce a stimulation index of 3; d = actual stimulation index caused by 'c'.

MAJOR COMPUTERISED SYSTEMS
- Delta Controls - ORCA view
- R version 3.0.2 (2013-09-25) - "Frisbee Sailing"

Positive control substance(s):

other: α-Hexylcinnamaldehyde tech, 85 % (Sigma Aldrich; batch MKBT2800V; purity 97.3 %; expiry date 30 August 2017; room temperature storage conditions)

Statistics:

STATISTICAL ANALYSIS
- 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 were met that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOV A) 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) were presented as *** (p < 0.001); ** (p < 0.0); * (p < 0.05); not significant (p ≥ 0.05).

Positive control results:

- The Stimulation Index for the positive control item was 8.33 (25 % v/v in acetone/olive oil 4:1; positive).

Parameter:

SI

Remarks:

test item

Value:

1.23

Test group / Remarks:

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

Remarks on result:

other: negative

Parameter:

SI

Remarks:

test item

Value:

2.8

Test group / Remarks:

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

Remarks on result:

other: negative

Key result

Parameter:

SI

Remarks:

test item

Value:

3.47

Test group / Remarks:

100 %

Remarks on result:

other: positive

Cellular proliferation data / Observations:

PRELIMINARY SCREENING TEST
- Clinical observations, body weight and mortality data are given in Appendix 1 (attached).
- Local skin irritation is given in Appendix 2 (attached).
- The ear thickness measurements and mean ear thickness changes are given in Appendix 3 (attached).
- 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 test item at concentrations of 10 % and 1 % v/v in acetone/olive oil 4: 1 were selected for the main test.

ESTIMATION OF PROLIFERATIVE RESPONSE OF LYMPH NODE CELLS IN MAIN TEST
- The radioactive disintegrations per minute per lymph node and the stimulation index are given in Appendix 4 (attached).
Stimulation Index values, expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group, were determined as 1.23 (1 % v/v in acetone/olive oil 4:1; negative); 2.80 (10 % v/v in acetone/olive oil 4:1; negative); 3.47 (100 %; positive).

CLINICAL OBSERVATIONS AND MORTALITY DATA
- Individual clinical observations and mortality data for test and control animals are given in Appendix 5 (attached).
- Local skin irritation is shown in Appendix 6 (attached).
- The ear thickness measurements and mean ear thickness changes are given in Appendix 7 (attached).
- There were no unplanned animal deaths during the study.
- No signs of systemic toxicity were noted in the test or control animals during the test.

BODY WEIGHT
- Individual body weights and body weight change for test and control animals are given in Appendix 8 (attached).
- 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.

CALCULATION OF EC3 VALUE
- The equation EC3 = c + [[(3-d)/(b-d)] x (a-c)] was used where a = 100; b = 3.47; c = 10; d = 2.80.
- The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 37 % (EC3 = 10 + [[(3-2.80)/(3.47-2.80)] x (100-10)] = 37).

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The test item produced an EC3 value of 37 % (the concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation) and was considered to be a sensitiser under the conditions of the test. The positive control α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 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:

GUIDELINE

An investigation was performed to assess the skin sensitisation potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The study was conducted in compliance with OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 22 July 2010) and Method B.42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008.

 

METHODS

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 undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 10 % or 1 % 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 sensitiser, a-Hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4: 1.

 

RESULTS

Stimulation Index values, expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group, were determined as 1.23 (1 % v/v in acetone/olive oil 4:1; negative); 2.80 (10 % v/v in acetone/olive oil 4:1; negative); 3.47 (100 %; positive). The Stimulation Index for the positive control item was 8.33 (25 % v/v in acetone/olive oil 4:1; positive). The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 37 %.

 

CONCLUSION

The test item produced an EC3 value of 37 % (the concentration of test item expected to cause a 3-fold increase in 3HTdR incorporation) and was considered to be a sensitiser under the conditions of the test. The positive control α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 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 conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

GUIDELINE

An investigation was performed to assess the skin sensitisation potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The study was conducted in compliance with OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 22 July 2010) and Method B.42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008.

 

METHODS

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 undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 10 % or 1 % 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 sensitiser, a-Hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4: 1.

 

RESULTS

Stimulation Index values, expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group, were determined as 1.23 (1 % v/v in acetone/olive oil 4:1; negative); 2.80 (10 % v/v in acetone/olive oil 4:1; negative); 3.47 (100 %; positive). The Stimulation Index for the positive control item was 8.33 (25 % v/v in acetone/olive oil 4:1; positive). The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 37 %.

 

CONCLUSION

The test item produced an EC3 value of 37 % (the concentration of test item expected to cause a 3-fold increase in 3HTdR incorporation) and was considered to be a sensitiser under the conditions of the test. The positive control α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 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.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

An EC3 value of 37 % was determined during investigation of skin sensitisation in the key local lymph node assay. In accordance with Guidance on the Application of the CLP Criteria (Version 4.1; June 2015), classification as a skin sensitiser (sub-category 1B) is appropriate.