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Diss Factsheets

Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 March 2022 - 14 June 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted independently by co-registrant.
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
Buehler test
Justification for non-LLNA method:
Justification is attached above under "Attached Justification".
Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
Test System : Guinea pig
Species (strain) : Cavia porcellus (Hartley)
Animal Source : Animal Breeding Facility-JRF
Preliminary : Irritancy test: 2 males + 2 females
Main study : Thirty [10 (5 Males and 5 Females) in the control and 20 (10 Males and
10 Females) in the treatment group]
Sex : Male and Female (Females were nulliparous and non-pregnant)
Initial Body Weight (g)
on Day 0 : Male: Minimum: 278.8, Maximum: 354.2
Female: Minimum: 303.3, Maximum: 359.7
Age of Animal on Day 0 : 5 to 8 weeks
The study was undertaken in compliance with the guidelines of the “Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International” and “Guidelines for Laboratory Animals Facility” issued by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India.
Compliance with these guidelines ensures the humane care of animals used throughout the experiment.
It further enhances the well-being of animals, which subsequently promotes a quality outcome of the experiment for the advancement of biological knowledge relevant to humans and animals.
The project proposal for the experimentation was approved by the “Institutional Animal Ethics
Committee (IAEC)”, JRF.

Acclimatisation
The guinea pigs were received into the experimental room post veterinary examination for health. They
were allowed to acclimatise to the laboratory conditions for 7 days before commencement of dosing.

Husbandry Practices
Caging : Solid polypropylene cages with labels were used. Autoclaved clean corn
cob was used as the bedding material.
Water Bottle : Each cage was supplied with a polypropylene water bottle with a
stainless-steel nozzle.
Housing : Five guinea pigs per cage per sex
Room Sanitation : Daily: 1. The rack was cleaned with a cloth, 2. The floor of the
experimental procedure room was swept, 3. All worktops and the floor
were mopped with a disinfectant solution.
Enrichment Material : Hut


Animal Identification
After receiving in the experimental room, guinea pigs were marked with a permanent ink marker
before randomisation and unique numbers on the ear using a tattoo machine post-randomisation.
Appropriate cage cards were attached to the cages indicating the study number, test item code, group number, sex, concentration, type of study, cage number, and animal number.

Feed and Water
Guinea pigs were provided feed and water ad libitum. The quality of feed and water is regularly
monitored at Jai Research Foundation. There were no known contaminants in the feed and water at levels that would have interfered with the experimental results obtained.
Feed : Teklad certified Global High Fiber Guinea pig diet manufactured by Envigo, USA.
Water : UV sterilised water filtered through Reverse Osmosis water filtration system, supplemented
with Vitamin-C (1 g/L).

Environmental Conditions
Animal Room : DCR - 208 Department of Toxicology
Temperature Range : 18 to 23 °C
Relative Humidity Range : 63 to 66%
Photoperiod : The photoperiod was 12 h of artificial light and 12 h of darkness, light
hours being 06:00 – 18:00 h.
Air Changes : Minimum 15 air changes/h



Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
0.2ml 25% (v/v)
Day(s)/duration:
days 0, 7 and 14, 6 h exposure. Observations at 24 and 48h post exposure
Adequacy of induction:
other: No skin reactions were observed in the guinea pigs at 24 and 48 h post-patch removal
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
0.2 ml 50% (v/v)
Day(s)/duration:
days 0, 7 and 14, 6 h exposure. Observations at 24 and 48h post exposure
Adequacy of induction:
other: No skin reactions were observed in the guinea pigs at 24 and 48 h post-patch removal
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
0.2 ml 75% (v/v)
Day(s)/duration:
days 0, 7 and 14, 6 h exposure. Observations at 24 and 48h post exposure
Adequacy of induction:
other: No skin reactions were observed in the guinea pigs at 24 and 48 h post-patch removal
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.2ml
Day(s)/duration:
days 0, 7 and 14, 6 h exposure. Observations at 24 post exposure.
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.2ml
Day(s)/duration:
Induction carried out on day 28, 6 h exposure period. Observations at 30 and 54 h post exposure.
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
20
Details on study design:
Randomisation
After acclimatisation, guinea pigs were randomised into two groups using in-house developed,
validated computer software (Gad and Weil, 1994).

Pilot Study and Dose Selection
Topical Irritancy Test
This test was carried out to determine the highest concentration of the test item required to produce mild skin irritation for induction and the highest non-irritating concentration for the challenge phase in the main study. Based on the solubility test, where the test item formed emulsion with distilled water, distilled water was used as a vehicle in this study. Patches loaded with 0.2 mL 25% (v/v), 50% (v/v) and 75% (v/v) in distilled water and 100% (undiluted) were topically
applied to the clipped flanks of four guinea pigs (2 males and 2 females). The patches were held in contact for 6 h by an occlusive dressing (with Medi-Tape 330, hypoallergenic surgical tape). The skin reactions were evaluated following the Draize method (Refer section 2.14) at 24 and 48 h post-patch removal.

No skin reactions were observed in the guinea pigs at 24 and 48 h post-patch removal at dose levels of 0.2 mL 25% (v/v), 50% (v/v) and 75% (v/v) in distilled water and 100% (undiluted)
Based on results of the pilot study, a volume of 0.2 mL undiluted (100%) was selected for
the topical induction applications (day 0, 7, and 14) and for the challenge exposure on day 28.


Buehler Test
Thirty guinea pigs were randomised into 2 groups by randomisation and comprised 5 males and 5 females in the control group and 10 males and 10 females in the treatment group.

Preparation of Application Site
Using a clipper, hair was removed from both the flanks of guinea pigs at approximately 24 h before the
treatment for topical induction and challenge application.

Induction Phase: Topical Application
Days 0, 7 and 14 - Treatment Group
Patches loaded with 0.2 mL undiluted (100%) were applied to the left flank and were held in
contact by an occlusive dressing for 6 h.

Days 0, 7 and 14 - Control Group
Patches loaded with 0.2 mL distilled water were applied to the left flank and were held in contact by an occlusive dressing for 6 h.
At the end of the exposure period (days 0, 7 and 14), dressing and patches were removed, and the residual test item was removed using cotton soaked in distilled water. Skin reactions were evaluated at 24h postpatch removal on days 1, 8 and 15 following the Draize method (Refer section 2.14).

Challenge Phase: Topical Application
Day 28 (Treatment and Control Groups)
Patches loaded with 0.2 mL undiluted (100%) were applied to the right flank of all guinea
pigs. The patches were held in contact by an occlusive dressing for 6 h. At the end of the exposure period, the dressing and patches were removed, and the residual test item was removed using cotton soaked in distilled water.

Observations
The guinea pigs were observed at least twice a day for mortality and morbidity, and clinical signs were
recorded once a day during study. Skin reactions were observed at 24 and 48 h from patch removal as
per the Magnusson and Kligman grading scale. The initial (day 0) and terminal body weight (day 30)
of guinea pigs were recorded.

The degree of sensitising potential was assigned according to the percentage of guinea pigs giving a
positive response in the treatment group. The skin sensitisation results were interpreted as per the
Globally Harmonized System of Classification and Labeling of Chemicals (GHS 2021)

Evaluation of Results
Body weight data were statistically analysed by Student’s t-test (Gad and Weil, 1994).


Terminal Procedures
At the end of the observation period, the guinea pigs were humanely killed by carbon dioxide
asphyxiation and were discarded without any necropsy.
Challenge controls:
10 (5 Males and 5 Females) in control group.
Days 0, 7 and 14 - Control Group
Patches loaded with 0.2 mL distilled water were applied to the left flank and were held in contact by an occlusive dressing for 6 h.
At the end of the exposure period (days 0, 7 and 14), dressing and patches were removed, and the residual test item was removed using cotton soaked in distilled water. Skin reactions were evaluated at 24 h postpatch removal on days 1, 8 and 15 following the Draize method
Key result
Reading:
1st reading
Hours after challenge:
30
Group:
test chemical
Dose level:
0.2ml
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No clinical sign related to the treatment was observed in any guinea pig
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
54
Group:
test chemical
Dose level:
0.2ml
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No clinical sign related to the treatment was observed in any guinea pig
Remarks on result:
no indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of this study, an indication of the classifications for CAS 68441-67-1 is as mentioned below:
Globally Harmonized System of Classification
and Labelling of Chemicals (GHS 2021) : Not considered as skin sensitiser
Executive summary:

Thirty Hartley strain guinea pigs were randomly divided into the control group (comprising 10 guinea pigs: 5 males and 5 females) and the treatment group (comprising 20 guinea pigs: 10 males and 10 females). Based on the results of the pilot study, a volume of 0.2 mL undiluted                                                                                                                           (100%) was selected for the topical induction applications (days 0, 7, and 14) and the challenge exposure on day 28.


The skin of guinea pigs was observed at 24 h, following the patch removal after induction applications (on days 0, 7, and 14), and skin reactions were graded as per the Draize Method (Draize et al., 1944). The skin reactions at 24 and 48 h post-patch removal, following the challenge application, were graded, as per the Magnusson and Kligman grading scale (Magnusson and Kligman, 1969).


No erythema or oedema was observed on the left flank on day 1, following the topical application (on day 0); on day 8, following the topical application (on day 7) and day 15, following the topical application (on day 14). No skin reaction was observed in guinea pigs from the control group.


Visual observation of the skin following the challenge application did not reveal any positive skin response at 30 and 54 h from the test item application in the treatment and control groups.


No clinical sign related to the treatment was observed during the study.


The mean body weight of the treatment group guinea pigs remained comparable to that of the control group.


A sensitization rate of 0% at 24 and 48 h post-patch removal from the time of challenge application was observed using a non-adjuvant method.


Based on the results of this study, an indication of the classifications for CAS 68441-67-1 is as mentioned below:


Globally Harmonized System of Classification and              :   Not considered as skin sensitiser


Labelling of Chemicals (GHS 2021)

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 Apr 2021 - 11 May 2021
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:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Identification: Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid
Batch (Lot) Number: 2019194336
Expiry date: 03 June 2022
Physical Description: Clear light yellow liquid
Purity/Composition: UVCB
Storage Conditions: At room temperature
Additional information
Test Facility test item number: 212207/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Chemical name (IUPAC, synonym or trade name): Trade names: HATCOL ® 1106, HATCOL ® 2926, HATCOL ® 3326
CAS number: 68441-66-7
Stability at higher temperatures: Yes, maximum temperature: 175°C, maximum duration: 10 minutes
Solubility in vehicle:
Acetone Not indicated
Fatty oils Not indicated
Stability in vehicle:
Acetone Stable
Fatty oils Stable
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
Test System
Species: Mouse
Strain: CBA/J
Condition: Inbred, SPF-Quality
Source: Janvier, Le Genest-Saint-Isle, France
Number of Animals: 20 females (nulliparous and non-pregnant). Five females per group.
Age at the Initiation of Dosing: Young adult animals (approximately 10 weeks old) were selected.
Weight at the Initiation of Dosing: 22.4 to 25.6 g.

Justification for Test System and Number of Animals
The CBA/J mouse was chosen as the animal model for this study as recognized by international guidelines as a recommended test system (e.g. OECD, FDA, MHLW). The test method and number of animals were based on the test guidelines.
The results of a reliability test with three concentrations of Alpha-Hexylcinnamaldehyde (CAS No. 101-86-0) in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously and using the same materials, animal supplier, animal strain and essential procedures are summarized in Appendix 4 of this report. For both scientific and animal welfare reasons, no concurrent positive control group was included in the study. An extensive data base is available with reliability checks performed each half year during at least the recent 9 years showing reproducible and consistent positive results.
The study plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. within the framework of Appendix 1 of project license AVD2360020172866 approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).

Animal Identification
At study assignment, each animal was identified using a tail mark with indelible ink.

Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

Selection, Assignment, Replacement, and Disposition of Animals
Animals were assigned to the study at the discretion of the coordinating biotechnician, with all animals within ± 20% of the sex mean body weights. Animals in poor health or at extremes of body weight range were not assigned to the study. Before the initiation of dosing, a health inspection was performed and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions. The disposition of all animals was documented in the study records.

Husbandry

Housing
On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. The room\(s) in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities. Each cage was clearly labeled.

Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 22°C with an actual daily mean relative humidity of 44 to 45%. A 12 hour light/12 hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) 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 analysis were provided by the supplier and are on file at the Test Facility.
It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap-water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.
It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment
For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA), except when interrupted by study procedures/activities.

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0 % w/w control (Acetone/Olive oil (4:1 v/v) (AcOO))
25 test item; % w/w
50 test item; % w/w
100 test item; % w/w
No. of animals per dose:
Five animals were treated with one test item concentration per group.
Details on study design:
Rationale for Vehicle
The vehicle was chosen from the vehicles specified in the test guideline (in order of preference): Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methylethylketone, propylene glycol, dimethylsulfoxide and 1% Pluronic© L92 in Elix water (in case an aqueous vehicle is suitable). The vehicle was selected on the basis of maximizing the solubility based on trial preparations performed at Charles River Den Bosch and on information provided by the Sponsor. Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study and these preparations were not used for dosing. Raw Data of these trials were retained by the Test Facility.

Test Item Characterization
The Sponsor provided to the Test Facility documentation of the identity, purity, composition, and stability for the test item. The characterization of the test item was conducted in a sponsor or sponsor subcontractor quality environment. A Certificate of Analysis or equivalent document was provided to the Test Facility and is presented in Appendix 3.
The Sponsor has appropriate documentation on file concerning the method of synthesis, fabrication or derivation of the test item, and this information is available to the appropriate regulatory agencies should it be requested.

Reserve Samples
For each batch (lot) of test item, a reserve sample (about 0.5 gram) was collected and maintained under the appropriate storage conditions by the Test Facility. The sample will be destroyed after the expiry date.

Test and Reference Item Inventory and Disposition
Records of the receipt, distribution, and storage of test item were maintained. With the exception of reserve samples, all unused Sponsor-supplied test item will be discarded or returned to the Sponsor after completion of the scheduled program of work. Records of the decisions made will be kept at the Test Facility.

Preparation of Test Item
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item.
No adjustment was made for specific gravity of the vehicle and no correction was made for the purity/composition of the test item, since the test method requires a logical concentration range rather than specific dose levels.
Any residual volumes were discarded.

Sample Collection and Analysis
Analysis of test item in vehicle for concentration, stability, homogeneity was not performed. Test item data showed that the test item is stable in Acetone and fatty oils.

Experimental Design

Justification of Route and Dose Levels
Dose route and dose concentrations used are in compliance with the OECD test guidelines for LLNA studies.

Pre-screen Test
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
Two test item concentrations were tested; a 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines.
The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult females per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation.

Main Study
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.

Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The formulations were stirred with a magnetic stirrer until dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.

Excision of the Nodes - Day 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After five hours, all animals were euthanized according to laboratories Standard Operating Procedures. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.

Tissue Processing for Radioactivity - Day 6
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 µm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

Radioactivity Measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2910TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

In - Life Procedures, Observations, and Measurements

Mortality/Moribundity Checks
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations

Postdose Observations
Postdose observations were performed once daily on Days 1-6 (on Days 1-3 at least 3 hours after dosing).
All the animals were examined for reaction to dosing. The onset, intensity and duration of these signs was recorded (if appropriate), particular attention being paid to the animals during and for the first hour after dosing.

Body Weights
Animals were weighed individually on Day 1 (predose) and 6 (prior to necropsy).

Irritation
Erythema and eschar formation observations were performed once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing), according to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded.
Erythema and eschar formation:
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth) 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema 4

Terminal Procedures
No necropsy was performed, since all animals survived until the end of the observation period.

ANALYSIS
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.
If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.
The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of items and mixtures, including all amendments.
The EC3 value (the estimated test item concentration that will give a SI =3) was determined, using linear interpolation (reference 1).

COMPUTERIZED SYSTEMS
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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by Charles River Den Bosch. In this study, performed in November 2020, females of the CBA/J mouse strain (Janvier, Le Genest-Saint-Isle, France) were checked for sensitivity to Alpha- Hexylcinnamaldehyde, technical grade (HCA). The females were approximately 10 weeks old at commencement of the study. The study was based on the OECD Guideline No. 429, EC No 440/2008, Part B.42 and EPA, OPPTS 870.2600 “Skin Sensitization”. Alpha- Hexylcinnamaldehyde, technical grade (CAS no. 101-86-0) was fabricated under lot no. MKCD3159 (Sigma- Aldrich, Steinheim, Germany). Concentrations used for this study were 5, 10 and 25% in Acetone/Olive oil (4:1 v/v; AcOO). The SI values calculated for the test item concentrations 5, 10 and 25% were 2.1, 3.6 and 9.0 respectively. An EC3 value of 8.0% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the last HCA reliability tests of the recent years were 16.3, 12.8, 9.0 and 10.9%.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.
The raw data, study plan and report from this study are kept in the Charles River Den Bosch archives. The test described above was performed in accordance with Charles River Den Bosch Standard Operating Procedures and the report was audited by the QA-unit.
Key result
Parameter:
SI
Value:
1.6
Test group / Remarks:
25% test item concentration.
Key result
Parameter:
SI
Value:
2.4
Test group / Remarks:
50% test item concentration
Key result
Parameter:
SI
Value:
4.7
Test group / Remarks:
100% test item concentration
Cellular proliferation data / Observations:
Pre - Screen Test

At a 50% and 100% test item concentration, no signs of systemic toxicity were noted and no irritation was observed and therefore a 100% concentration was selected as highest concentration for the main study.

Main Study

Skin Reactions / Irritation
No irritation was observed in any of the animals.

Systemic Toxicity
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Macroscopic Examination of the Lymph Nodes and Surrounding Area
All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size.
No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Radioactivity Measurements and SI Values
Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 50 and 100% were 282, 406 and 808 DPM, respectively. The mean DPM/animal value for the vehicle control group was 172 DPM. The SI values calculated for the test item concentrations 25, 50 and 100% were 1.6, 2.4 and 4.7, respectively.

Table 1         Pre-Screen Test: Body Weights and Skin Reactions

TS1  (%)

animal

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

bw

(g)2

erythema3

erythema

erythema

erythema

erythema

erythema

bw

(g)

left

right

left

right

left

right

left

right

left

right

left

right

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

50

81

23.7

0

0

0

0

0

0

0

0

0

0

0

0

24.5

 

82

22.7

0

0

0

0

0

0

0

0

0

0

0

0

21.3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

100

83

21.8

0

0

0

0

0

0

0

0

0

0

0

0

22.3

 

84

23.6

0

0

0

0

0

0

0

0

0

0

0

0

22.9

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1  TS = test item (% w/w).

2  Body weight (grams).

3  Grading erythema and eschar formation(Left = dorsal surface of left ear; right = dorsal surface of right ear):

    0 = No erythema

 

Table 2          Pre-Screen Test: Ear Thickness Measurements

TS1 (%)

animal

Day 1

Day 3

Day 6

left

right

left

right

left

right

(mm)

(mm)

(mm)

%2

(mm)

%2

(mm)

%2

(mm)

%2

 

 

 

 

 

 

 

 

 

 

 

 

50

81

0.220

0.225

0.225

2

0.230

2

0.225

2

0.225

0

 

82

0.210

0.210

0.245

17

0.240

14

0.230

10

0.235

12

 

 

 

 

 

 

 

 

 

 

 

 

100

83

0.215

0.220

0.250

16

0.245

11

0.240

12

0.240

9

 

84

0.215

0.220

0.240

12

0.245

11

0.240

12

0.245

11

 

 

 

 

 

 

 

 

 

 

 

 

Left (mm) = thickness of left ear in millimeters; right (mm) = thickness of right ear in millimeters.

1  TS = test item (% w/w).

2  Percent increase compared to Day 1 pre-dose value. A 25% value is used as the threshold for selection for

    use in the main study.

 

 

 

 

 

                                                                                                                                     

Table 3          Main Study: Body Weights and Skin Reactions

group

TS(%)

animal

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

bw

(g)2

erythema3

erythema

erythema

erythema

erythema

erythema

bw

(g)

left

right

left

right

left

right

left

right

left

right

left

right

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1

0

1

23.8

\0

0

0

0

0

0

0

0

0

0

0

0

25.3

 

 

2

23.2

0

0

0

0

0

0

0

0

0

0

0

0

24.5

 

 

3

25.1

0

0

0

0

0

0

0

0

0

0

0

0

24.9

 

 

4

22.5

0

0

0

0

0

0

0

0

0

0

0

0

23.0

 

 

5

24.1

0

0

0

0

0

0

0

0

0

0

0

0

24.2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2

25

6

24.1

0

0

0

0

0

0

0

0

0

0

0

0

23.7

 

 

7

23.0

0

0

0

0

0

0

0

0

0

0

0

0

23.1

 

 

8

23.5

0

0

0

0

0

0

0

0

0

0

0

0

23.9

 

 

9

24.8

0

0

0

0

0

0

0

0

0

0

0

0

25.8

 

 

10

25.4

0

0

0

0

0

0

0

0

0

0

0

0

26.3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3

50

11

25.6

0

0

0

0

0

0

0

0

0

0

0

0

25.6

 

 

12

23.9

0

0

0

0

0

0

0

0

0

0

0

0

23.0

 

 

13

22.4

0

0

0

0

0

0

0

0

0

0

0

0

21.1

 

 

14

24.9

0

0

0

0

0

0

0

0

0

0

0

0

23.5

 

 

15

23.4

0

0

0

0

0

0

0

0

0

0

0

0

22.1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4

100

16

25.3

0

0

0

0

0

0

0

0

0

0

0

0

24.9

 

 

17

23.0

0

0

0

0

0

0

0

0

0

0

0

0

22.2

 

 

18

24.3

0

0

0

0

0

0

0

0

0

0

0

0

23.2

 

 

19

24.5

0

0

0

0

0

0

0

0

0

0

0

0

24.0

 

 

20

23.4

0

0

0

0

0

0

0

0

0

0

0

0

23.7

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1  TS = test item (% w/w).

2  Body weight (grams).

3  Grading erythema and eschar formation(Left = dorsal surface of left ear; right = dorsal surface of right ear):

    0 = No erythema

Table4         Main Study: Relative Size Lymph Nodes, Radioactivity Counts (DPM) and Stimulation Index(SI)

group

TS1(%)

animal

Size nodes2

DPM3/ animal

mean

DPM ± SEM4

mean

SI ± SEM

left

right

 

 

 

 

 

 

 

 

1

0

1

n

n

91

172

±

29

1.0

±

0.2

 

 

2

n

n

193

 

 

3

n

n

144

 

 

4

n

n

267

 

 

5

n

n

164

 

 

 

 

 

 

 

 

 

 

 

 

2

25

6

n

n

258

282

±

48

1.6

±

0.3

 

 

7

n

n

358

 

 

8

n

n

236

 

 

9

n

n

142

 

 

10

n

n

417

 

 

 

 

 

 

 

 

 

 

 

 

3

50

11

n

n

206

406

±

76

2.4

±

0.4

 

 

12

n

n

616

 

 

13

n

n

547

 

 

14

n

n

352

 

 

15

n

n

307

 

 

 

 

 

 

 

 

 

 

 

 

4

100

16

n

n

534

808

±

127

4.7

±

0.7

 

 

17

n

n

1258

 

 

18

n

n

708

 

 

19

n

n

894

 

 

20

n

n

646

 

 

 

 

 

 

 

 

1  TS = test item (% w/w).

2  Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +,++ or +++: degree of enlargement, n: considered to be normal).

3    DPM= Disintegrations per minute.

4    SEM = Standard Error of the Mean.

 

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Remarks:
The substance could be considered as a weak sensitiser;. However, the LLNA has been found to generate false positive results when testing many fatty acids or alcohols, hydrolyzed products of esters
Conclusions:
These results indicate that the test item could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 63% was calculated.
The six-month reliability check with Alpha-Hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity (see Appendix 4).
Based on these results:
• According to the recommendations made in the test guidelines (including all amendments), Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid would be regarded as skin sensitizer.
• According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments), Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid should be classified as skin sensitizer (Category 1B).
• According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.
Executive summary:

The objective of this study was to evaluate whether Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid induces skin sensitization in mice after three epidermal exposures of the animals under the conditions described in this report.


The study was carried out based on the guidelines described in:


·        OECD, Section 4, Health Effects, No.429 (2010).


·        EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay".


·        EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.


The six-month reliability check with Alpha-Hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.


Test item concentrations selected for the main study were based on the results of a pre-screen test. Based on the results, the highest concentration required according to the guidelines was selected. 


In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 25, 50 or 100% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v) (AcOO)). Three days after the last exposure, all animals were injected with3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.


No irritation was observed in any of the animals and all auricular lymph nodes of the animals of the experimental and control group were considered normal in size.


Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 50 and 100% were 282, 406 and 808 DPM, respectively. The mean DPM/animal value for the vehicle control group was 172 DPM. The SI values calculated for the test item concentrations 25, 50 and 100% were 1.6, 2.4 and 4.7, respectively


These results indicate that the test item could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 63% was calculated.


Based on these results:


·        According to the recommendations made in the test guidelines (including all amendments), Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid would be regarded as skin sensitizer.


·        According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments), Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid should be classified as skin sensitizer (Category 1B).


·        According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.


 However, the LLNA has been found to generate false positive results when testing many fatty acids or alcohols, hydrolyzed products of esters. Weight of evidence indicates that this result is considered to be a false positive. 

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

A solubility assessment for the applicability of the in vitro skin sensitization assays DPRA, KeratinoSensTM, and h-CLAT assays was conducted. The solubility assessment showed that the substance was not compatible with any of the solvents used in the DPRA, KeratinoSensTM, and h-CLAT assays. As a result, the in vitro skin sensitization testing strategy cannot be initiated and in vivo skin sensitization studies are required to evaluate the skin sensitization potential of these products.


The local lymph node assay (LLNA; OECD 429) has become the standard test method to investigate the skin sensitization potential of a test substance in vivo. While the LLNA uses fewer animals and results in a refinement in the experimental design, it only measures the first step of the ACD process and is reported to give false positive results for some chemical functional groups.  However, the LLNA has been found to generate false positive results when testing many fatty acids or alcohols, hydrolyzed products of esters (Kreiling et al., 2008; Yamashita et al., 2015; Basketter et al., 2009; OECD 2021; Garcia et al., 2010; Ball et al., 2011;).  The results of the LLNA test conducted on the substance indicate that the test item could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 63% was calculated.


Literature review found that two esters (oleic acid ester, CAS # 70491-68-8; and Isopropyl myristate, CAS # 110-27-0) generated false positive results in the LLNA test, while guinea pig maximization tests (GPMT; OECD 406) correctly predicted the skin sensitization potentials of these esters (Basketter et al., 2009; Kimber et al., 2021; Kimber & Pemberton, 2014; Fiume et al., 2015; OECD, 2021).


Independently to the lead registrant and without their knowledge, a guinea pig (Buehler) study was conducted and demonstrated negative results. On the basis of the available evidence, it is concluded that LLNA results for the substance is very likely a false-positive, taking also into account no reports of a sensitization reaction in workers handling this ester in the plant.


References


Ball et al., 2011. Evaluating the sensitization potential of surfactants: Integrating data from the local lymph node assay, guinea pig maximization test, and in vitro methods in a weight-of-evidence approach.  Regulatory Toxicology and Pharmacology 60 (3): 389-400. https://www.sciencedirect.com/science/article/pii/S0273230011001036?via%3Dihub


Basketter et al., 2009. Application of a weight of evidence approach to assessing discordant sensitisation datasets: Implications for REACH.  Regulatory Toxicology and Pharmacology 55 (1): 90-96. https://www.sciencedirect.com/science/article/pii/S0273230009001081


Fiume et al., 2015. Safety Assessment of Alkyl Esters as Used in Cosmetics. International Journal of Toxicology 34 (2S): 5S-69S. https://journals.sagepub.com/doi/10.1177/1091581815594027?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed


Garcia et al., 2010. Comparative testing for the identification of skin-sensitizing potentials of nonionic sugar lipid surfactants. Regulatory Toxicology and Pharmacology 58 (2): 301-307. https://www.sciencedirect.com/science/article/pii/S027323001000108X?via%3Dihub


Kimber et al., 2021. The activity of methacrylate esters in skin sensitisation test methods II. A review of complementary and additional analyses. Regulatory Toxicology and Pharmacology 119: 104821. https://www.sciencedirect.com/science/article/pii/S0273230020302476


Kimber & Pemberton, 2014. Assessment of the skin sensitising potency of the lower alkyl methacrylate esters. Regulatory Toxicology and Pharmacology 70 (1): 24-36.  https://www.sciencedirect.com/science/article/pii/S0273230014001275?via%3Dihub


Kreiling et al., 2008. Comparison of the skin sensitizing potential of unsaturated compounds as assessed by the murine local lymph node assay (LLNA) and the guinea pig maximization test (GPMT). Food and Chemical Toxicology 46 (6): 1896-1904. https://www.sciencedirect.com/science/article/pii/S0278691508000410?via%3Dihub


OECD, 2021. Environment Directorate Chemicals and Biotechnology Committee. OECD Expert Group on Defined Approaches for Skin Sensitisation. Annex 6: Analysis of LLNA Reference data to conclude on predictivity of alternative methods for lipophilic chemical. https://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=ENV-CBC-MONO(2021)11/ann6 &doclanguage=en


Yamashita et al., 2015. Unsaturated fatty acids show clear elicitation responses in a modified local lymph node assay with an elicitation phase, and test positive in the direct peptide reactivity assay. The Journal of Toxicological Sciences 40 (6): 843-853. https://www.jstage.jst.go.jp/article/jts/40/6/40_843/_article

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid is not classified as skin sensitizer.