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Toxicological information

Skin sensitisation

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guideline
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".

Test material

Constituent 1
Chemical structure
Reference substance name:
Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid
EC Number:
270-470-1
EC Name:
Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid
Cas Number:
68441-66-7
Molecular formula:
not available due to complexity of the substance
IUPAC Name:
Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid
Test material form:
liquid
Details on test material:
100% product
Date of Manufacture September 2021
Date of Expiry December 01, 2024
Appearance Colourless liquid
Storage Condition
(at JRF)
As per the instruction received from the Sponsor on storage of the test item,
the test item was stored:
Storage Temperature : Room temperature (15 to 30 °C)
Storage Condition : Protected from light
Storage Container : Kept in original container as supplied by the
Sponsor
Storage Location : Test Item Control Office, JRF

In vivo test system

Test animals

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



Study design: in vivo (non-LLNA)

Inductionopen allclose all
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
Challenge
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

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
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

Applicant's summary and conclusion

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)