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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 July 2014 to 24 September 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
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)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Appearance/physical state: Clear colourless liquid
- Storage conditions: Room temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
ANIMALS AND ANIMAL HUSBANDRY
- 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 acclimatisation 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.
- 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 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.

Study design: in vivo (LLNA)

Vehicle:
other: cottonseed oil
Concentration:
- Preliminary test: 100 % test item
- Initial main test: 100 %, 10 %, 1 % and 0.1 % test item (cottonseed oil used as vehicle)
- Additional main test: 100 %, 0.4 % and 0.1 % test item (dried cottonseed oil used as vehicle)
No. of animals per dose:
Five mice per group
Details on study design:
TEST ITEM
- The test item was used undiluted and freshly prepared as a solution in cottonseed oil for the initial test and in dried cottonseed oil for the additional test. These vehicles were chosen at the request of the Sponsor.
- The vehicle determination record is included as Appendix 3 (attached).
- 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 was reflected in the GLP compliance statement.

POSITIVE CONTROL ITEM
- The positive control item was freshly prepared as a 50% v/v dilution in cottonseed oil for the initial test and in dried cottonseed oil for the additional test.

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 and 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 as Appendix 4 (attached).
- 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-dose and 1 hour post dose on Day 1, 1 hour 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 equal to or greater than 25% is considered to indicate excessive irritation and thereby be of limited biological relevance to the endpoint of sensitiSation.

INITIAL MAIN TEST
- Groups of five mice were treated with the undiluted test item or the test item at concentrations of 10%, 1% or 0.1% v/v in cottonseed oil.
- 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 and 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, tech., 85%, at a concentration of 50% v/v in cottonseed oil was applied to the dorsal surface of each ear.
- The thickness of each ear was measured and recorded as described in the preliminary screening test.

ADDITIONAL MAIN TEST
- At the request of the Sponsor, due to an inconclusive result in the initial main test and concerns over the reactivity of the test item with the moisture content of the original vehicle (cottonseed oil), an additional main test was performed using dried cottonseed oil as the vehicle.
- Groups of five mice were treated with the undiluted test item or the test item at concentrations of 0.4% or 0.1% v/v in dried cottonseed oil.
- 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 and 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 (vehicle control) received the vehicle alone in the same manner.
- Another group of five animals (blank control) was untreated.
- An additional group of positive control animals was treated as described in the initial test.
- The thickness of each ear was measured and recorded as described in the preliminary screening test.

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.
- The blank control group in the additional test was also injected PBS containing 3H-methyl thymidine.

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 labelled 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 eighteen hours incubation at approximately 4 °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 p-scintillation counting. The "Poly Q" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left 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 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 RESULTS
- 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 sensitizer 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-sensitizer."
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.
- Probability values (p) were presented as *** (P < 0.001); ** (P < 0.01); * (P < 0.05); not significant (P ≥ 0.05).

Results and discussion

Positive control results:
- The Stimulation Index for the positive control item in the initial main test was 4.25 (50 % v/v α-Hexylcinnamaldehyde, tech., 85 % in cottonseed oil).
- The Stimulation Index for the positive control item in the additional main test was 4.53 (50 % v/v α-Hexylcinnamaldehyde, tech., 85 % in dried cottonseed oil).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.05
Test group / Remarks:
0.1 % test item v/v in dried cottonseed oil
Remarks on result:
other: negative
Parameter:
SI
Value:
1.31
Test group / Remarks:
0.4 % test item v/v in dried cottonseed oil
Remarks on result:
other: negative
Parameter:
SI
Value:
3.66
Test group / Remarks:
100 % test item in dried cottonseed oil
Remarks on result:
other: positive
Key result
Parameter:
other: EC3 value (%)
Value:
76
Remarks on result:
other: extrapolated EC3 value
Remarks:
see explanatory document attached
Cellular proliferation data / Observations:
PRELIMINARY SCREENING TEST
- Clinical observations, body weight and mortality data are given in Table 1 (attached).
- Local skin irritation is given in Table 2 (attached).
- The ear thickness measurements and mean ear thickness changes are given in Table 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, and at the request of the Sponsor, the undiluted test item and the test item at concentrations of 10%, 1% and 0.1% v/v in cottonseed oil were selected for the initial main test.

INITIAL MAIN TEST – ESTIMATION OF THE PROLIFERATIVE RESPONSE OF LYMPH NODE CELLS
- The radioactive disintegrations per minute per animal and the stimulation index are given in Table 4 (attached).
- 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 shown in the table below.

INITIAL MAIN TEST – CLINICAL OBSERVATIONS AND MORTALITY DATA
- Individual clinical observations and mortality data for test and control animals are given in Table 5 (attached).
- Local skin irritation is given in Table 6 (attached).
- The ear thickness measurements and mean ear thickness changes are given in Table 7 (attached).
- There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.
- Very slight erythema was noted up to Day 4 on the ears of animals treated with the vehicle (cottonseed oil), the test item at concentrations of 10%, 1% and 0.1% v/v in cottonseed oil and the positive control item at a concentration of 50% v/v in cottonseed oil. No signs of local skin irritation were noted in animals treated with the undiluted test item.

INITIAL MAIN TEST – BODY WEIGHT
- Individual body weights and body weight change for test and control animals are given in Table 8 (attached).
- Body weight change of the test animals between Day 1 and Day 6 were comparable to that observed in the corresponding control group animals over the same period.

ADDITIONAL MAIN TEST – ESTIMATION OF THE PROLIFERATIVE RESPONSE OF LYMPH NODE CELLS
- The radioactive disintegrations per minute per animal and the stimulation index are given in
Table 9 (attached).
- 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 shown in the table below.

ADDITIONAL MAIN TEST – CLINICAL OBSERVATIONS AND MORTALITY DATA
- Individual clinical observations and mortality data for test and control animals are given in Table 10 (attached).
- Local skin irritation is given in Table 11 (attached).
- The ear thickness measurements and mean ear thickness changes are given in Table 12 (attached).
- There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.
- Very slight erythema was noted on Day 1 on the ears of animals treated with the test item at a concentration of 0.4% v/v in dried cottonseed oil and on Days 1 to 3 on the ears of animals treated with the positive control item at a concentration of 50% v/v in dried cottonseed oil. No signs of local skin irritation were noted in the untreated (blank) control animals, the vehicle (dried cottonseed oil) control animals or animals treated with the undiluted test item or the test item at a concentration of 0.1% v/v in dried cottonseed oil.

ADDITIONAL MAIN TEST – BODY WEIGHT
- Individual body weights and body weight change for test and control animals are given in Table 13 (attached).
- Body weight change of the test animals between Day 1 and Day 6 were comparable to that observed in the corresponding control group animals over the same period.

Any other information on results incl. tables

RESULTS OF INITIAL MAIN TEST

Treatment Group

Concentration

Stimulation Index

Result

Test Item

0.1 % v/v in cottonseed oil

3.21

Positive

Test Item

1 % v/v in cottonseed oil

2.94

Negative

Test Item

10 % v/v in cottonseed oil

1.45

Negative

Test Item

100 %

0.99

Negative

Positive Control Item

50 % v/v in cottonseed oil

4.25

Positive

RESULTS OF ADDITIONAL MAIN TEST

Treatment Group

Concentration

Stimulation Index

Result

Test Item

0.1 % v/v in dried cottonseed oil

1.05

Negative

Test Item

0.4 % v/v in dried cottonseed oil

1.31

Negative

Test Item

100 %

3.66+

Positive

Positive Control Item

50 % v/v in dried cottonseed oil

4.53

Positive

+ = The mean radioactive incorporation was divided by the mean radioactive incorporation of the blank (untreated) control group for the undiluted test item

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Remarks:
see ECHA Guidance on information requirements and chemical safety assessment Chapter R.8: Characterisation of dose [concentration]-response for human health (Version 2.1; November 2012)
Conclusions:
The test item was considered to be a sensitiser under the conditions of the test and an extrapolated EC3 value of 72% has been derived. The positive control, α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 50% v/v in cottonseed oil or dried cottonseed oil, thus demonstrating the sensitivity and reliability of the test system.
Executive summary:

GUIDELINE

A study 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 investigation was designed to be compatible with OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitization: Local Lymph Node Assay" (adopted 22 July 2010) and Method B42 Skin Sensitization (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. Four 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 cottonseed oil at concentrations of 10 %, 1 % or 0.1 % v/v. A further group of five animals was treated with cottonseed oil alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, a-Hexylcinnamaldehyde tech., 85 %, at a concentration of 50 % v/v in cottonseed oil.

 

At the request of the Sponsor, due to an inconclusive result in the initial main test and concerns over the reactivity of the test item with the moisture content of the original vehicle (cottonseed oil), an additional main test was performed using dried cottonseed oil as the vehicle. Three groups 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 dried cottonseed oil at concentrations of 0.4 % or 0.1 % v/v. A further group of five animals was treated with dried cottonseed oil alone. Another group of five animals was untreated (blank). An additional concurrent positive control test was also performed using a further group of five animals.

 

RESULTS

In the initial main test, the Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group was determined to be 3.21 positive (0.1 % v/v test item in cottonseed oil), 2.94 negative (1 % v/v test item in cottonseed oil), 1.45 negative (10 % v/v test item in cottonseed oil) and 0.99 negative (100 % v/v test item). The Stimulation Index for the positive control item in the initial main test was 4.25 (50 % v/v α-Hexylcinnamaldehyde, tech., 85 % in cottonseed oil).

 

In the additional main test, the Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group was determined to be 1.05 negative (0.1 % v/v test item in dried cottonseed oil), 1.31 negative (0.4 % v/v test item in dried cottonseed oil) and 3.66 positive (100 % test item). The mean radioactive incorporation was divided by the mean radioactive incorporation of the blank (untreated) control group for the undiluted test item. The Stimulation Index for the positive control item in the additional main test was 4.53 (50 % v/v α-Hexylcinnamaldehyde, tech., 85 % in dried cottonseed oil).

 

CONCLUSION

The test item was considered to be a sensitiser under the conditions of the test and an extrapolated EC3 value of 72 % has been derived. The positive control, α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 50% v/v in cottonseed oil or dried cottonseed oil, thus demonstrating the sensitivity and reliability of the test system.