Registration Dossier

Administrative data

Description of key information

GLP compliant, Klimish Grade 1, OECD 429 local lymph node sensitisation assay.

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

The positive control alpha-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.

GLP compliant, Klimish Grade 1, OECD 406 Guinea Pigs-Modified Buehler Design test

Based on the results of this study, Test Substance is considered to be a contact sensitizer in guinea pigs. The criterion for sensitization (dermal scores ≥ 1 in at least 15% of the test animals) was met. The results of the HCA positive control study demonstrated that a valid test was performed and indicated that the test design would detect potential contact sensitizers.

A Buehler study was conducted because the material is surface active and may be outside applicability of LLNA test. A LLNA study was conducted after confirming this substance is sensitizing to determine potency. 

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:
26 February 2016 to 15 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines
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)
Specific details on test material used for the study:
- Physical state: Yellow semi-solid
- Analytical purity: 100 %
- Expiration date of the lot/batch 23 November 2017
- Storage condition of test material: Room temperature in the dark
Details on study design:
Not applicable
Details on study design:
Not applicable
Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: Animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: Food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Ltd., Oxon, UK), ad libitum
- Water: Mains tap water, ad libitum
- Acclimation period: at least 5 days
-Randomly allocated to cages.

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: at least 15 changes per hour
- Photoperiod: 12 h dark / 12 h light

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Preliminary test: 50% w/w in acetone/olive oil 4:1
Main test: 1.5, 15 and 50% w/w in acetone/olive oil 4:1
No. of animals per dose:
Preliminary screening test: 1 female/dose
Main test: 5 females/dose
Details on study design:
PRELIMINARY SCREENING TEST:
- The mouse was treated by daily application of 25 µL of test item at concentrations of 50% w/w in acetone/olive 4:1 to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3).

MAIN TEST
- Mice were treated by daily application of 25 µL of test item at concentrations of 1.5, 15 and 50% w/w in acetone/olive 4:1 to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3) using an automatic micropipette.
- A further group of 5 mice received the vehicle alone in the same manner.
- The positive control animals were similarly treated except that alpha-Hexylcinnamaldehyde, tech., 85% was used at a concentration of 25% v/v in acetone/olive oil 4:1.

3H-METHYL THYMIDINE ADMINISTRATION AND TERMINAL PROCEDURES
- Following first topical application of test item, vehicle control or positive control item, on 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.
- Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation.
- The draining auricular lymph nodes were excised and pooled for each individual animal of each group. For each group 1 mL of PBS was added to the pooled lymph nodes.
- A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze.
- Lymph node cells were rinsed with 4 mL PBS into a petri dish, and cells suspension was transferred to a centrifuge tube. Petri dish was washed with 5 mL PBS and cells added to the centrifuge tube.
- Lymph node cells were pelleted at 1400 pm for 10 mins and re-suspended in 10 mL PBS and re-pelleted.
- The pellet was re-suspended in 3 mL of 5 % (w/v) trichloroacetic acid (TCA) to precipitate radioactive material.
- After 18 hours incubation at 4 °C, precipitate were recovered by centrifugation at 2100 rpm for 10 mins and re-suspended in 1 mL TCA and transferred to 10 mL of scintillation fluid.
- 3HTdR incorporation was measured by β –scintillation counting.

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).
- Criteria used to consider a positive response: 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.”
- The EC3 value was also calculated (EC3 is the concentration of test item expected to cause a 3 fold in 3HTdR incorporation.
- The equation used for the calculation of EC3 is EC3= c + [[(3-d)/(b-d)] x (a-c)].
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 homogeinity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric once 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 test procedures were used.
Positive control results:
Stimulation index for α-hexylcinnamaldehyde at 25 % v/v in acetone/olive oil 4:1 was 5.30 and classified as a sensitizer.
Key result
Parameter:
EC3
Remarks:
(%)
Value:
9
Key result
Parameter:
SI
Value:
0.76
Test group / Remarks:
Stimulation index for 1.5% test item.
Remarks on result:
other: Negative
Key result
Parameter:
SI
Value:
4.81
Test group / Remarks:
Stimulation index for 15% test item.
Remarks on result:
other: Positive
Key result
Parameter:
SI
Value:
8.18
Test group / Remarks:
Stimulation index for 50% test item.
Remarks on result:
other:
Remarks:
positive
Cellular proliferation data / Observations:
PRELIMINARY SCREENING TEST
- Clinical observations, body weight and mortality data are given in Appendix 1 (attached) and 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 skin irritation or irritation were observed.
-Based on this information, dose levels of 1.5, 15 and 50% were selected for the main test.

MAIN TEST
- The radioactive disintegrations per minute per lymph node and the stimulation index are given in Appendix 4 (attached).
- The Stimulation Index for 1.5, 15 and 50% were 0.76, 4.81 and 8.18, respectively. Thus, the test item was found negative at 1.5% and positive at 15 in 50%.
- Individual clinical observations and mortality data for test and control animals are given in Appendix 5 (attached) and local irritation is given in Appendix 6 (attached).
- The ear thickness measurements and mean ear thickness are given in Appendix 7 (attached).
- No death occurred and no signs of systemic toxicity were observed during the test.
- Individual body weights and body weight change are given in Appendix 8 (attached).
- Body weight change of test animals between Day 1 and 6 was comparable to that observed in the corresponding control group animals.

The Stimulation Index for the test tiem was 0.76 (negative), 4.81 (positive), 8.18 (positive) at concentration of 1.5, 15 and 50% test item in acetone/olive oil 4:1, respectively.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test item was considered to be a sensitizer under the conditions of the test.
The positive control alpha-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:

INTRODUCTION

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.


METHOD

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50% w/w, this concentration was selected as the highest dose level to be 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 concentration of 1.5, 15 and 50% w/w. A further group of five animals was treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using alpha-hexyclinnamaldehyde tech., 85% at a concentration of 25% v/v in acetone/olive oil 4:1.


RESULTS

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

The Stimulation Index for the test tiem was 0.76 (negative), 4.81 (positive), 8.18 (positive) at concentration of 1.5, 15 and 50% test item in acetone/olive oil 4:1, respectively.

The Stimulation Index for the positive control item was 5.30 (positive) at a concentration of 25% positive control in acetone/olive oil 4:1.

The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 9%.

 

CONCLUSION

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

The positive control alpha-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:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
Guinea Pigs-Modified Buehler Design
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Mar-2016 - Dec-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
The design of this study was based on the study objective(s), the overall product development strategy for the test substance, and the following study design guidelines: OECD Guideline 406 and EPA Health Effects Test Guideline OPPTS 870.2600.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
Buehler test
Justification for non-LLNA method:
The study was conducted because the material is surface active and may outside applicability of LLNA test. A LLNA study was conducted after confirming this substance is sensitizing to determine potency.
Species:
guinea pig
Strain:
other: Hartley-derived albino guinea pigs
Sex:
male/female
Details on test animals and environmental conditions:
Animal Identification
Each animal was identified by a cage card and plastic ear tag.

Environmental Acclimation
The animals were acclimated to their designated housing for at least 7 days before the first day of dosing.

Selection, Assignment, and Disposition of Animals
The animals chosen for study were arbitrarily selected from healthy animals. All animals received a detailed pretest observation prior to dosing. Only healthy animals were chosen for study use.
The male range-finding animals were approximately 5 weeks of age on the day prior to dosing with body weights of 358 grams and 389 grams. The female range-finding animals were approximately 6 weeks of age on the day prior to dosing with body weights of 345 grams and 362 grams.
The male second range-finding animals were approximately 5 weeks of age on the day prior to dosing with body weights of 332 grams and 368 grams. The female second range-finding animals were approximately 6 weeks of age on the day prior to dosing with body weights of 329 grams and 374 grams.
The male main phase animals were approximately 6 weeks of age on the day prior to Induction 1 dosing with body weights ranging from 358 grams to 467 grams. The female main phase animals were approximately 6 weeks of age on the day prior to Induction 1 dosing with body weights ranging from 348 grams to 453 grams.
The disposition of all animals was documented in the Study Records.

Husbandry

Housing
The animals were pair housed (2 animals of the same sex and same dosing group together) throughout the study in polycarbonate cages containing direct bedding material equipped with an automatic watering valve. Housing and care were as specified in the USDA Animal Welfare Act (9 CFR, Parts 1, 2, and 3) and as described in the Guide for the Care and Use of Laboratory Animals from the National Research Council.1

Environmental Conditions
Temperatures of 70°F to 73°F (21°C to 23°C) with a relative humidity of 41% to 54% were maintained. A 12-hour light/12-hour dark cycle was maintained, except when interrupted for designated procedures. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
PMI Nutrition International Certified Guinea Pig Chow No. 5026 was provided ad libitum throughout the study, except during designated procedures. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the dietary analyses were provided by the supplier for each lot of diet and are on file at the Testing Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap water after treatment by reverse osmosis and ultraviolet irradiation was freely available to each animal via an automatic watering system, except during designated procedures. Water bottles and/or hydrogel supplemental water were provided when required. The water is analyzed semi-annually for microbial contamination and for total dissolved solids, hardness, and various environmental contaminants. Results of these analyses are maintained on file at the Testing Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.

Animal Enrichment
Beginning at receipt, guinea pigs were pair housed in solid bottom cages containing direct bedding material. In addition, a timothy hay cube was provided to each animal at least weekly.

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.
Route:
epicutaneous, occlusive
Vehicle:
other: Mineral oil
Concentration / amount:
On the day prior to the first induction dose administration (Day -1), all main phase animals were weighed and the hair was removed from the left side of the test and HCA test animals. On the day following clipping (Day 0), chambers were applied @ 100% (neat test substance)
Day(s)/duration:
The induction procedure was repeated on Day 7 and Day 14 so that a total of 3 consecutive induction exposures were made to the test animals.
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: Mineral oil
Remarks:
Challenge
Concentration / amount:
On the day prior to challenge dose administration, the test, HCA test, challenge control, and HCA challenge control animals were weighed and the hair was removed from the right side of the animals. On the day following clipping (Day 28), chambers were applied at 25 and 15 % in Mineral oil
Day(s)/duration:
day 28
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
other: Mineral oil
Remarks:
Rechallenge
Concentration / amount:
On the day prior to rechallenge dose administration, the test and rechallenge control animals were weighed and the hair was removed from the right side of the animals. On the day following clipping (Day 35), chambers were applied @ 1.5 % in mineral oil
Day(s)/duration:
Day 35
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Range finder
2 males 2 females per dose

Main study 10 males 10 femails
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test substance. This study was designed such that it did not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
Details on study design:
Test Substance Characterization
The Sponsor provided to the Testing Facility documentation of the identity, strength, purity, composition, and stability for the test substance. A Certificate of Analysis was provided to the Testing Facility and is presented in see attached Appendix 2.

Analysis of Test Substance
The stability of the bulk test substance was not determined during the course of this study. Information to support the stability of each lot of the bulk test substance was provided by the Sponsor.


Reserve Samples
A reserve sample was collected for each batch (lot) of test substance, control substance, and positive control substance components (HCA, ethanol, and acetone) and maintained under the appropriate storage conditions by the Testing Facility.

Test Substance Inventory and Disposition
Records of the receipt, distribution, storage, and disposition of the test substance (including empty containers) were maintained. With the exception of reserve samples, all unused Sponsor-supplied bulk test substance will be returned to the Sponsor (after issuance of the final
reports of all studies using this material). All empty containers were maintained for the duration of the study.

Dose Formulation and Analysis

Preparation of Test Substance
The test substance, Test Substance, was administered as received and/or diluted with the control substance on the day of dosing during the range-finding phases, induction, challenge, and rechallenge. Selected doses were achieved by adjustment of test substance concentration in the control substance. The test and control substances were warmed to approximately 40°C prior to dilution and/or dispensation. The test substance formulations were cooled to room temperature prior to administration. Details of the preparation and dispensing of the test substance have been retained in the Study Records.

HCA Preparation
HCA dosing formulations were prepared at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared, protected from light, and dispensed on the day of dosing. Details of the preparation and dispensing of the positive control substance have been retained in the Study Records.

Sample Collection and Analysis
No samples for analytical analysis were collected by the Testing Facility.

Test System

Receipt
On 29 Mar 2016, 44 male and 44 female Hartley-derived albino guinea pigs were received from Charles River Laboratories, Inc., Stone Ridge, NY. The animals were examined and weighed on the day following receipt.

Justification for Test System and Number of Animals
The Hartley-derived guinea pig was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.


The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test substance. This study was designed such that it did not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

Experimental Design – Range-Finding Phases

Text Table 1
Experimental Design for the First Range-Finding Phase


Site No. Test Material Dose Level Number of Animals
Males Females
1 Test Substance 100%a
2 Test Substance 75%b 2 2
3 Test Substance 50%b
4 Test Substance 25%b


a As received.
b The vehicle used was mineral oil.



Text Table 2
Experimental Design for the Second Range-Finding Phase

Site No. Test Material Dose Level Number of Animals
Males Females
1 Test Substance 20%a
2 Test Substance 12.5%a 2 2
3 Test Substance 5%a
4 Test Substance 1%a
a The vehicle used was mineral oil.

Justification of Route and Dose Levels
The dermal route of exposure was selected because this is the intended potential route of human exposure.
Four graded levels were utilized for this procedure. Optimally, the range-finding studies were expected to produce no systemic toxicity and a spectrum of dermal responses that include Grades 0, ±, 1, and 2 unless the test substance was not dermally irritating at 100%.

Administration of Test Materials
On the day prior to dosing, the guinea pigs selected for the topical range-finding studies were weighed and the hair removed from the right and left side of the animals with a small animal clipper. Care was taken to avoid abrading the skin during the clipping procedures.
On Day 0, 8 concentrations of the test substances were prepared and a 0.3 mL dose of the appropriate 4 different concentrations were applied to the clipped area of the first or second topical range-finding animals according to Text Table 1 or Text Table 2. The 25 mm Hill Top Chamber® backed by adhesive tape (occlusive patch) was applied to the clipped surface as quickly as possible. The trunk of the animal was wrapped with elastic wrap to prevent removal
of the chamber and the animal was returned to its cage.
Approximately 6 hours after chamber application, the binding materials were removed. The test sites were then wiped 2 times with gauze moistened in mineral oil, followed by dry gauze, and then wiped with gauze moistened in reverse osmosis deionized (RODI) water, followed by dry gauze, to remove test substance residue and the animals were returned to their cages.

In-life Procedures, Observations, and Measurements – Range-Finding Phases
The in-life procedures, observations, and measurements listed below were performed for all range-finding animals.

Mortality/Moribundity Checks
The animals were observed for general health/mortality and moribundity twice daily, once in the morning and afternoon, throughout the study.

Clinical Observations

Detailed Clinical Observations
The animals were removed from the cage and examined in detail before dosing on Day 0.


Dermal Observations
The test sites of each topical range-finding animal were graded for irritation at approximately
24 hours and 48 hours after chamber application using the Macroscopic Dermal Grading System in see attached Appendix 3 according to Buehler.2

Body Weights
Each topical range-finding animal was weighed on the day prior to dosing (Day -1).

Scheduled Euthanasia
Following the 48-hour scoring interval, all range-finding animals were euthanized by carbon dioxide inhalation and discarded.

Experimental Design – Main Phase

Text Table 3 Experimental Design for the Main Phase



Phase/Treatment Number of Animals
Group Induction 1 to 3 Challenge Rechallenge a Males Females
Test Test Substance (100%)b Test Substance (25% and 15%)b Test Substance (1.5%)b 10 10
Challenge Control - Test Substance (25% and 15%)b - 5 5
Rechallenge Control - - Test Substance (1.5%)b 5 5
HCA Test 5.0% HCAc 2.5% and 1.0% HCA d - 5 5
HCA Control - 2.5% and 1.0% HCAd - 5 5
- = not applicable.
a The second rechallenge group was maintained on study; however, the second rechallenge procedure was not required as the rechallenge results were definitive.
b The vehicle used was mineral oil.
c The vehicle used was ethanol.
d The vehicle used was acetone.

Justification of Route and Dose Levels
The dermal route of exposure was selected because this is a potential route of human exposure.
The dose concentration for the main induction phase was based upon the results of the range-finding portion of the study. A second range-finding phase was performed to aid in determining the appropriate dose concentration for the challenge phase. The test substance concentration used for challenge was expected to produce no systemic toxicity and dermal
responses generally consisting of Grades 0 to ±. A rechallenge was performed to assess a lower formulation concentration. The test substance concentration used for rechallenge was expected to produce no systemic toxicity and dermal responses consisting of Grades 0 to ±.


Administration of Test Materials
On the day prior to dosing, the guinea pigs selected for the main study had the hair removed with a small animal clipper. Care was taken to avoid abrading the skin during the clipping procedures.
On the following day, a 0.3 mL dose of the appropriate test or positive control substance was placed on a 25 mm Hilltop Chamber® backed by adhesive tape (occlusive patch). The chamber was then applied to the clipped surface of the appropriate animals as quickly as possible.
Following chamber application, the trunk of the animal was wrapped with elastic wrap to prevent removal of the chamber and the animal was returned to its cage.
Approximately 6 hours after chamber application, the binding materials were removed. The test sites were then wiped 2 times with gauze moistened in mineral oil, followed by dry gauze, followed by gauze moistened in RODI water, followed by dry gauze, to remove test substance residue, and the animals were returned to their cages.


Induction
On the day prior to the first induction dose administration (Day -1), all main phase animals were weighed and the hair was removed from the left side of the test and HCA test animals. On the day following clipping (Day 0), chambers were applied as indicated in Text Table 4.

Text Table 4 Induction Dosing


Group Test Substance Induction No. Dose Volume (mL) Concentration (%) Site No. of Animals
Males Females

Test Test Substance 1 0.3 100a 1 10 10
2 0.3 100a 1
3 0.3 100a 1

HCA Test HCA 1 0.3 5.0b 1 5 5
2 0.3 5.0b 1
3 0.3 5.0b 3c
a As received.
b The vehicle used was ethanol.
c Based on irritation, the test site was changed for Induction 3.
The induction procedure was repeated on Day 7 and Day 14 so that a total of 3 consecutive induction exposures were made to the test animals.


Challenge
On the day prior to challenge dose administration, the test, HCA test, challenge control, and HCA challenge control animals were weighed and the hair was removed from the right side of the animals. On the day following clipping (Day 28), chambers were applied as indicated in Text Table 5.



Text Table 5 Challenge Dosing


Group Test Substance Dose Volume (mL) Concentration (%) Site No. of Animals
Males Females
Test Test Substance 0.3 25a 2 10 10
Test Test Substance 0.3 15a 4 10 10
Challenge Control Test Substance 0.3 25a 2 5 5
Challenge Control Test Substance 0.3 15a 4 5 5
HCA Test HCA 0.3 2.5b 2 5 5
0.3 1.0b 4
HCA Control HCA 0.3 2.5b 2 5 5
0.3 1.0b 4
a The vehicle used was mineral oil.
b The vehicle used was acetone.

Rechallenge
On the day prior to rechallenge dose administration, the test and rechallenge control animals were weighed and the hair was removed from the right side of the animals. On the day following clipping (Day 35), chambers were applied as indicated in Text Table 6.

Text Table 6 Rechallenge Dosing


Group Test Substance Dose Volume (mL) Concentration (%) Site No. of Animals
Males Females
Test Test Substance 0.3 1.5a 6 10 10
Rechallenge Control Test Substance 0.3 1.5a 2 5 5
a The vehicle used was mineral oil.

In-life Procedures, Observations, and Measurements
The in-life procedures, observations, and measurements listed below were performed for main study animals.

Mortality/Moribundity Checks
The animals were observed for general health/mortality and moribundity twice daily, once in the morning and afternoon, throughout the study.

Clinical Observations

Detailed Clinical Observations
The animals were removed from the cage and examined in detail before dosing on Day 0.

Dermal Observations
The test sites of each main study animal were graded for irritation at approximately 24 hours and 48 hours after chamber application (induction) or 24 hours and 48 hours after chamber removal


(challenge and rechallenge) using the Macroscopic Dermal Grading System in see attached Appendix 3 according to Buehler.2

Body Weights
Each main study animal was weighed on the day prior to the first induction (Day -1) and on the day prior to challenge and rechallenge dosing for the appropriate test, challenge control, and rechallenge control animals.

Scheduled Euthanasia
Following the rechallenge phase 48-hour scoring interval, all main study animals were euthanized by carbon dioxide inhalation and discarded.

COMPUTERIZED SYSTEMS
Critical computerized systems used in the study are listed below. All computerized systems used in the conduct of this study have been validated; when a particular system has not satisfied all requirements, appropriate administrative and procedural controls were implemented to assure the quality and integrity of data.

Text Table 7
Critical Computerized Systems

System Name Version No. Description of Data Collected and/or Analyzed
Systems 600 Apogee Insight System 3.11 Temperature and humidity
Instem Life Science Systems, DISPENSE 8 Test material receipt, accountability, and formulation activity

STATISTICAL ANALYSIS
The sensitization potential of the test substance was based on the dermal responses observed on the test and control animals at challenge or rechallenge. Generally, dermal scores of ≥ 1 in the test animals with scores of 0 to ± noted in the controls were considered indicative of sensitization. Dermal scores of 1 in both the test and control animals were generally considered equivocal unless a higher dermal response (≥ grade 2) was noted in the test animals. Group mean dermal scores were calculated for challenge and rechallenge. A response of at least 15% in a nonadjuvant test was expected for a mild to moderate sensitizer in this study design.

RETENTION OF RECORDS, SAMPLES, AND SPECIMENS
All study-specific raw data, electronic data, documentation, protocol, retained samples and specimens, and final reports will be archived at the Testing Facility no later than the date of Final Report issuance, and then transferred to the archive at Charles River Laboratories, Inc., Horsham, PA. Five years after issue of the Audited Draft Report, the Sponsor will be contacted to determine the disposition of these materials.
Electronic data generated by the Testing Facility were archived as noted above, except that the data collected using Dispense 8 and reporting files stored on SDMS were archived at the Charles River Laboratories facility located in Wilmington, MA.
Challenge controls:
Challenge
On the day prior to challenge dose administration, the test, HCA test, challenge control, and HCA challenge control animals were weighed and the hair was removed from the right side of the animals. On the day following clipping (Day 28), chambers were applied as indicated in Text Table 5.



Text Table 5 Challenge Dosing


Group Test Substance Dose Volume (mL) Concentration (%) Site No. of Animals
Males Females
Test Test Substance 0.3 25a 2 10 10
Test Test Substance 0.3 15a 4 10 10
Challenge Control Test Substance 0.3 25a 2 5 5
Challenge Control Test Substance 0.3 15a 4 5 5
HCA Test HCA 0.3 2.5b 2 5 5
0.3 1.0b 4
HCA Control HCA 0.3 2.5b 2 5 5
0.3 1.0b 4
a The vehicle used was mineral oil.
b The vehicle used was acetone.

Rechallenge
On the day prior to rechallenge dose administration, the test and rechallenge control animals were weighed and the hair was removed from the right side of the animals. On the day following clipping (Day 35), chambers were applied as indicated in Text Table 6.

Text Table 6 Rechallenge Dosing


Group Test Substance Dose Volume (mL) Concentration (%) Site No. of Animals
Males Females
Test Test Substance 0.3 1.5a 6 10 10
Rechallenge Control Test Substance 0.3 1.5a 2 5 5
a The vehicle used was mineral oil.

In-life Procedures, Observations, and Measurements
The in-life procedures, observations, and measurements listed below were performed for main study animals.
Positive control substance(s):
yes
Remarks:
HCA Preparation HCA dosing formulations were prepared at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared, protected from light, and dispensed on the day of dosing.
Positive control results:
Challenge Phase
Following challenge with 2.5% w/v HCA in acetone, dermal scores of 1 or 2 were noted in 5/10 HCA test animals at the 48-hour scoring interval. Dermal reactions in the HCA control
animals were limited to a dermal score of 1 in 1/10 animals by the 48-hour interval. Group mean dermal scores were higher in the HCA test animals (0.5 to 1.0) compared to the HCA control animals (0.5 and 0.2).
Following challenge with 1.0% w/v HCA in acetone, dermal scores of 1 or 2 were noted in 9/10 HCA test animals at the 48-hour scoring interval. Dermal reactions in the HCA control
animals were limited to a score of 1 in 2/10 animals at the 48-hour interval. Group mean dermal scores were higher in the HCA test animals (0.7 to 1.7) compared to the HCA control animals (0.6 and 0.3).

Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25%
No. with + reactions:
8
Total no. in group:
20
Clinical observations:
Mean score 0.3
Remarks on result:
other: Please see remarks section
Remarks:
Following challenge with 25% Test Substance in mineral oil, a dermal score of 1 was noted in 5/20 test animals at the 24-hour scoring interval and a dermal score of 2 was noted in 15/20 test animals by the 48-hour scoring interval. Dermal reactions in the remaining test animals and in all challenge control animals were scores of 0 or ±. Group mean dermal scores were higher in the test animals (0.3 to 1.5) as compared to challenge control animals (0.2 and 0.1).
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
25%
No. with + reactions:
15
Total no. in group:
20
Clinical observations:
Mean score 1.5
Remarks on result:
other: please see remarks section
Remarks:
Following challenge with 25% Test Substance in mineral oil, a dermal score of 1 was noted in 5/20 test animals at the 24-hour scoring interval and a dermal scoreof 2 was noted in 15/20 test animals by the 48-hour scoring interval. Dermal reactions in the remaining test animals and in all challenge control animals were scores of 0 or ±. Group mean dermal scores were higher in the test animals (0.3 to 1.5) as compared to challenge control animals (0.2 and 0.1)
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
15%
No. with + reactions:
13
Total no. in group:
20
Clinical observations:
Mean score 1.0
Remarks on result:
other: Please see remarks section
Remarks:
Following challenge with 15% Test Substance in mineral oil, dermal scores of 1 or 2 were noted in 10/20 test animals at the 24-hour scoring interval and in 18/20 test animals at the 48-hour scoring interval. Dermal reactions in the remaining test animals and for all challenge control animals were scores of 0 or ±. Group mean dermal scores were higher in the test animals (1.0 to 1.8) as compared to challenge control animals (0.1 and 0.0).
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
15%
No. with + reactions:
18
Total no. in group:
20
Clinical observations:
Mean score 1.8
Remarks on result:
other: Please see remarks section
Remarks:
Following challenge with 15% Test Substance in mineral oil, dermal scores of 1 or 2 were noted in 10/20 test animals at the 24-hour scoring interval and in 18/20 test animals at the 48-hour scoring interval. Dermal reactions in the remaining test animals and for all challenge control animals were scores of 0 or ±. Group mean dermal scores were higher in the test animals (1.0 to 1.8) as compared to challenge control animals (0.1 and 0.0).
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
other: Challenge control
Dose level:
25%
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
Mean score 0.2
Remarks on result:
other: Please see remarks section
Remarks:
Following challenge with 25% Test Substance in mineral oil, a dermal score of 1 was noted in 5/20 test animals at the 24-hour scoring interval and a dermal score of 2 was noted in 15/20 test animals by the 48-hour scoring interval. Dermal reactions in the remaining test animals and in all challenge control animals were scores of 0 or ±. Group mean dermal scores were higher in the test animals (0.3 to 1.5) as compared to challenge control animals (0.2 and 0.1).
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
other: Challenge control
Dose level:
25%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
Mean score 0.1
Remarks on result:
other: Please see remarks section
Remarks:
Following challenge with 25% Test Substance in mineral oil, a dermal score of 1 was noted in 5/20 test animals at the 24-hour scoring interval and a dermal score of 2 was noted in 15/20 test animals by the 48-hour scoring interval. Dermal reactions in the remaining test animals and in all challenge control animals were scores of 0 or ±. Group mean dermal scores were higher in the test animals (0.3 to 1.5) as compared to challenge control animals (0.2 and 0.1).
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
other: Challenge control
Dose level:
15%
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
Mean score 0.1
Remarks on result:
other: Please see remarks section
Remarks:
Following challenge with 15% Test Substance in mineral oil, dermal scores of 1 or 2 were noted in 10/20 test animals at the 24-hour scoring interval and in 18/20 test animals at the 48-hour scoring interval. Dermal reactions in the remaining test animals and for all challenge control animals were scores of 0 or ±. Group mean dermal scores were higher in the test animals (1.0 to 1.8) as compared to challenge control animals (0.1 and 0.0).
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
other: Challenge control
Dose level:
15%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Mean score 0
Remarks on result:
other: Please see remarks section
Remarks:
Following challenge with 15% Test Substance in mineral oil, dermal scores of 1 or 2 were noted in 10/20 test animals at the 24-hour scoring interval and in 18/20 test animals at the 48-hour scoring interval. Dermal reactions in the remaining test animals and for all challenge control animals were scores of 0 or ±. Group mean dermal scores were higher in the test animals (1.0 to 1.8) as compared to challenge control animals (0.1 and 0.0).
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
1.5%
No. with + reactions:
17
Total no. in group:
20
Clinical observations:
Mean score 1.2
Remarks on result:
other: Please see remarks section
Remarks:
Following rechallenge with 1.5% Test Substance in mineral oil, dermal scores of 1 or 2 were noted in 15/20 test animals at the 24-hour scoring interval and in 16/20 test animals at the 48-hour scoring interval. Dermal reactions in the remaining test animals were scores of 0 or ± and were limited to a score of 0 for all rechallenge control animals. Group mean dermal scores were higher in the test animals (1.2) as compared to rechallenge control animals (0.0).
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
1.5%
No. with + reactions:
16
Total no. in group:
20
Clinical observations:
Mean score 1.2
Remarks on result:
other: Please see remarks section
Remarks:
Following rechallenge with 1.5% Test Substance in mineral oil, dermal scores of 1 or 2 were noted in 15/20 test animals at the 24-hour scoring interval and in 16/20 test animals at the 48-hour scoring interval. Dermal reactions in the remaining test animals were scores of 0 or ± and were limited to a score of 0 for all rechallenge control animals. Group mean dermal scores were higher in the test animals (1.2) as compared to rechallenge control animals (0.0).
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
other: Rechallenge Control
Dose level:
1.5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Mean score 0
Remarks on result:
other: Please see remarks section
Remarks:
Following rechallenge with 1.5% Test Substance in mineral oil, dermal scores of 1 or 2 were noted in 15/20 test animals at the 24-hour scoring interval and in 16/20 test animals at the 48-hour scoring interval. Dermal reactions in the remaining test animals were scores of 0 or ± and were limited to a score of 0 for all rechallenge control animals. Group mean dermal scores were higher in the test animals (1.2) as compared to rechallenge control animals (0.0).
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
other: Rechallenge Control
Dose level:
1.5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Mean score 0
Remarks on result:
other: Please see remarks section
Remarks:
Following rechallenge with 1.5% Test Substance in mineral oil, dermal scores of 1 or 2 were noted in 15/20 test animals at the 24-hour scoring interval and in 16/20 test animals at the 48-hour scoring interval. Dermal reactions in the remaining test animals were scores of 0 or ± and were limited to a score of 0 for all rechallenge control animals. Group mean dermal scores were higher in the test animals (1.2) as compared to rechallenge control animals (0.0).

Mortality

No mortality occurred during the study.

 

Range-Finding Phases

The dermal grading system is presented in the attached Appendix 3.

 

Range-Finding Phases

(See attached Table 1) (See attached Table 2)

Based on the potential for the material to be a sensitizer, 2 range-finding phases were performed at the same time prior to the induction phase to assess 8 different test substance concentrations. The 100% as received concentration was considered appropriate for conducting the induction phase because it was the highest concentration with potential to cause mild irritation. Dermal irritation at the 100% concentration was limited to erythema grade 1 (slight but confluent or moderate patchy erythema) in 1/4 animals by 48 hours after application. The 75% and 50% concentrations had scores of ± (slight patchy erythema) present at 24 hours after application in 4/4 and 3/4 animals (respectively) that continued through 48 hours. For each concentration, the erythema increased to a grade 1 by 48 hours. The 25% concentration resulted in 2/4 animals with ± scores by 48 hours and this concentration was considered acceptable for challenge because it was the next highest non-irritating (no grade 1 erythema scores) concentration. The 20%, 12.5%, 5%, and 1% concentrations did not cause any dermal irritation.

 

Main Phase

The Dermal Grading System is presented in the attached Appendix 3.

 

Induction Phase

(See attached Table 3)

Dermal grade scores of ± (slight patchy erythema) or 1 (slight, but confluent or moderate patchy erythema) were present in 2 test animals during Induction 1. Dermal irritation increased by Induction 2 with scores ranging from ± to maximized erythema grade 3 (notable dermal lesions) for all animals. During Induction 3, there were 3 animals with an erythema score of 1, 8 animals with an erythema score of 2 (moderate, confluent erythema), and 9 animals had maximized erythema grade 3 at 48 hours. Additional dermal irritation was observed during Induction 2 and Induction 3 and consisted of blanching, superficial lightening, desquamation, eschar, and/or edema for 16/20 and 18/20 test animals, respectively.

 

Challenge Phase

(See attached Table 4)

Following challenge with 25% Test Substance in mineral oil, a dermal score of 1 was noted in 5/20 test animals at the 24-hour scoring interval and a dermal score of 2 was noted in 15/20 test animals by the 48-hour scoring interval. Dermal reactions in the remaining test animals and in all challenge control animals were scores of 0 or ±. Group mean dermal scores were higher in the test animals (0.3 to 1.5) as compared to challenge control animals (0.2 and 0.1).

Following challenge with 15% Test Substance in mineral oil, dermal scores of 1 or 2 were noted in 10/20 test animals at the 24-hour scoring interval and in 18/20 test animals at the 48-hour scoring interval. Dermal reactions in the remaining test animals and for all challenge control animals were scores of 0 or ±. Group mean dermal scores were higher in the test animals (1.0 to 1.8) as compared to challenge control animals (0.1 and 0.0).

Following challenge with 2.5% w/v HCA in acetone, dermal scores of 1 or 2 were noted in 5/10 HCA test animals at the 48-hour scoring interval. Dermal reactions in the HCA control

animals were limited to a dermal score of 1 in 1/10 animals by the 48-hour interval. Group mean dermal scores were higher in the HCA test animals (0.5 to 1.0) compared to the HCA control animals (0.5 and 0.2).

Following challenge with 1.0% w/v HCA in acetone, dermal scores of 1 or 2 were noted in 9/10 HCA test animals at the 48-hour scoring interval. Dermal reactions in the HCA control

animals were limited to a score of 1 in 2/10 animals at the 48-hour interval. Group mean dermal scores were higher in the HCA test animals (0.7 to 1.7) compared to the HCA control animals (0.6 and 0.3).

 

Rechallenge Phase

(See attached Table 5)

Following rechallenge with 1.5% Test Substance in mineral oil, dermal scores of 1 or 2 were noted in 15/20 test animals at the 24-hour scoring interval and in 16/20 test animals at the 48-hour scoring interval. Dermal reactions in the remaining test animals were scores of 0 or ± and were limited to a score of 0 for all rechallenge control animals. Group mean dermal scores were higher in the test animals (1.2) as compared to rechallenge control animals (0.0).

Body Weights

(see attached Appendix 4)

Two test females (Animal Nos. 6311 and 6315) had slight body weight loss between Day 27 and Day 34, following challenge administration. The body weight loss was not significant and overall these 2 females gained weight during the study duration.

No other Test Substance-related effects on body weight were observed in the test animals during the study. Overall weight gain in the animals throughout the study interval was indicative of good health in the test and control animals.

 

Clinical Observations

(see attached Appendix 5)

Clinical observations of yellow fecal staining of the abdominal or urogenital areas were noted in 1 test female (Animal No. 6314) and 1 challenge control animal (Animal No. 6331) on Day 0.

These observations are not considered Test Substance-related because they occurred prior to dose administration and are common findings in animals in solid-bottom caging.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Based on the results of this study, Test Substance is considered to be a contact sensitizer in guinea pigs. The criterion for sensitization (dermal scores ≥ 1 in at least 15% of the test animals) was met. The results of the HCA positive control study demonstrated that a valid test was performed and indicated that the test design would detect potential contact sensitizers.
Executive summary:

SUMMARY

The dermal sensitization potential of Test Substance was evaluated in Hartley-derived albino guinea pigs. Ten male and 10 female guinea pigs were topically treated with Test Substance, as received, once per week, for 3 consecutive weeks. Following a 2-week rest period, a challenge was performed whereby the 20 test and 10 previously untreated (naïve) challenge control guinea pigs were topically treated with 25% Test Substance in mineral oil and 15% Test Substance in mineral oil. A rechallenge was performed 1 week later, 20 test and 10 untreated rechallenge control guinea pigs were topically treated with 1.5% Test Substance in mineral oil.

An α-Hexylcinnamaldehyde (HCA) positive control group consisting of 10 HCA test and

10 HCA control guinea pigs was included in this study. The animals were treated as above with the HCA test animals receiving 5% w/v HCA in ethanol for induction and 2.5% and

1.0% w/v HCA in acetone for challenge.

 

Test Substance

Following challenge with 25% Test Substance in mineral oil, a dermal score of 1 was noted in 5/20 test animals at the 24-hour scoring interval and a dermal score of 2 was noted in 15/20 test animals by the 48-hour scoring interval. Dermal reactions in the remaining test animals and in all challenge control animals were scores of 0 or ±. Group mean dermal scores were higher in the test animals (0.3 to 1.5) as compared to challenge control animals (0.2 and 0.1).

Following challenge with 15% Test Substance in mineral oil, dermal scores of 1 or 2 were noted in 10/20 test animals at the 24-hour scoring interval and in 18/20 test animals at the 48-hour scoring interval. Dermal reactions in the remaining test animals and for all challenge control animals were scores of 0 or ±. Group mean dermal scores were higher in the test animals (1.0 to 1.8) as compared to challenge control animals (0.1 and 0.0).

Following rechallenge with 1.5% Test Substance in mineral oil, dermal scores of 1 or 2 were noted in 15/20 test animals at the 24-hour scoring interval and in 16/20 test animals at the 48-hour scoring interval. Dermal reactions in the remaining test animals were scores of 0 or ± and were limited to a score of 0 for all rechallenge control animals. Group mean dermal scores were higher in the test animals (1.2) as compared to rechallenge control animals (0.0).

α-Hexylcinnamaldehyde (HCA)

Following challenge with 2.5% w/v HCA in acetone, dermal scores of 1 or 2 were noted in 5/10 HCA test animals at the 48-hour scoring interval. Dermal reactions in the HCA control

animals were limited to a dermal score of 1 in 1/10 animals by the 48-hour interval. Group mean dermal scores were higher in the HCA test animals (0.5 to 1.0) compared to the HCA control animals (0.5 and 0.2).

Following challenge with 1.0% w/v HCA in acetone, dermal scores of 1 or 2 were noted in 9/10 HCA test animals at the 48-hour scoring interval. Dermal reactions in the HCA control

animals were limited to a score of 1 in 2/10 animals at the 48-hour interval. Group mean dermal scores were higher in the HCA test animals (0.7 to 1.7) compared to the HCA control animals (0.6 and 0.3).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

LLNA

INTRODUCTION

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.


METHOD

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50% w/w, this concentration was selected as the highest dose level to be 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 concentration of 1.5, 15 and 50% w/w. A further group of five animals was treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using alpha-hexyclinnamaldehyde tech., 85% at a concentration of 25% v/v in acetone/olive oil 4:1.


RESULTS

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

Stimulation index for 1.5, 15 and 50% were 0.76, 4.81 and 8.18, respectively.

The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 9%.

 

CONCLUSION

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

The positive control alpha-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.

Guinea Pigs-Modified Buehler Design test  

SUMMARY

The dermal sensitization potential of Test Substance was evaluated in Hartley-derived albino guinea pigs. Ten male and 10 female guinea pigs were topically treated with Test Substance, as received, once per week, for 3 consecutive weeks. Following a 2-week rest period, a challenge was performed whereby the 20 test and 10 previously untreated (naïve) challenge control guinea pigs were topically treated with 25% Test Substance in mineral oil and 15% Test Substance in mineral oil. A rechallenge was performed 1 week later, 20 test and 10 untreated rechallenge control guinea pigs were topically treated with 1.5% Test Substance in mineral oil.

An α-Hexylcinnamaldehyde (HCA) positive control group consisting of 10 HCA test and

10 HCA control guinea pigs was included in this study. The animals were treated as above with the HCA test animals receiving 5% w/v HCA in ethanol for induction and 2.5% and

1.0% w/v HCA in acetone for challenge.

 

Test Substance

Following challenge with 25% Test Substance in mineral oil, a dermal score of 1 was noted in 5/20 test animals at the 24-hour scoring interval and a dermal score of 2 was noted in 15/20 test animals by the 48-hour scoring interval. Dermal reactions in the remaining test animals and in all challenge control animals were scores of 0 or ±. Group mean dermal scores were higher in the test animals (0.3 to 1.5) as compared to challenge control animals (0.2 and 0.1).

Following challenge with 15% Test Substance in mineral oil, dermal scores of 1 or 2 were noted in 10/20 test animals at the 24-hour scoring interval and in 18/20 test animals at the 48-hour scoring interval. Dermal reactions in the remaining test animals and for all challenge control animals were scores of 0 or ±. Group mean dermal scores were higher in the test animals (1.0 to 1.8) as compared to challenge control animals (0.1 and 0.0).

Following rechallenge with 1.5% Test Substance in mineral oil, dermal scores of 1 or 2 were noted in 15/20 test animals at the 24-hour scoring interval and in 16/20 test animals at the 48-hour scoring interval. Dermal reactions in the remaining test animals were scores of 0 or ± and were limited to a score of 0 for all rechallenge control animals. Group mean dermal scores were higher in the test animals (1.2) as compared to rechallenge control animals (0.0).

α-Hexylcinnamaldehyde (HCA)

Following challenge with 2.5% w/v HCA in acetone, dermal scores of 1 or 2 were noted in 5/10 HCA test animals at the 48-hour scoring interval. Dermal reactions in the HCA control

animals were limited to a dermal score of 1 in 1/10 animals by the 48-hour interval. Group mean dermal scores were higher in the HCA test animals (0.5 to 1.0) compared to the HCA control animals (0.5 and 0.2).

Following challenge with 1.0% w/v HCA in acetone, dermal scores of 1 or 2 were noted in 9/10 HCA test animals at the 48-hour scoring interval. Dermal reactions in the HCA control

animals were limited to a score of 1 in 2/10 animals at the 48-hour interval. Group mean dermal scores were higher in the HCA test animals (0.7 to 1.7) compared to the HCA control animals (0.6 and 0.3).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

A GLP compliant, Klimish Grade 1, OECD 429 local lymph node sensitisation assay.

A GLP compliant, Klimish Grade 1, OECD 406 Guinea Pigs-Modified Buehler Design test  

The substance may be classified as skin sensitizer Sub-category1B