3,7,11,15-tetramethylhexadecane-1,2,3-triol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 10 July 1996 and 16 August 1996.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

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:

guinea pig maximisation test

Justification for non-LLNA method:

An LLNA study was not conducted because adequate data from other methods (guinea pig maximisation tests) were available and sufficient for classification purposes.

Test material

Test material information

Constituent 1

Reference substance name:

3,7,11,15-tetramethylhexadecane-1,2,3-triol

EC Number:

277-923-2

EC Name:

3,7,11,15-tetramethylhexadecane-1,2,3-triol

Cas Number:

74563-64-7

Molecular formula:

C20H42O3

IUPAC Name:

3,7,11,15-tetramethylhexadecane-1,2,3-triol

Test material form:

liquid: viscous

Details on test material:

Substance name: Phytantriol
Storage conditions: In the dark between 2 °C and 8 °C, under nitrogen
Lot No.: 508007

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

Young female nulliparous, non-pregnant albino guinea pigs of the Dunkin Hartley strain were supplied by D. Hall, Darley Oaks, Burton-on-Trent, Staffordshire, England. The animals were ordered in the weight range 300 - 350g and were delivered on 5th July, 1996. The animals used for the initial range-finding study were acclimatised for 5 days and those selected for the main study were acclimatised for 18 days.
The animals were housed in groups of up to 5 in stainless steel cages and identified by the number of the cage to which they were allocated and within each cage by ear tattoo.
A pelleted diet, SQC FDI guinea pig diet with added vitamin C (Special Diets Services, Witham, Essex, England) and mains drinking water were freely available. Certificates of analysis for both diet and drinking water are held on file at Quintiles England Limited.
The animal rooms were air conditioned with temperature within the range 20 - 22° C and relative humidity within the range 52 - 69% during the acclimatisation and study periods.
Fluorescent lighting was controlled to give an artificial cycle of 12 hours light (0600-1800 hours)/12 hours dark per day.

Study design: in vivo (non-LLNA)

Induction

Route:

intradermal and epicutaneous

Vehicle:

other: Ethanol

Concentration / amount:

Intradermal: 5% v/v solution of test article in light liquid paraffin or in emulsion with Freund's Complete Adjuvant.
Epicutaneous: 50% v/v concentration of the test article in ethanol

Challenge

Route:

epicutaneous, occlusive

Vehicle:

other: Ethanol

Concentration / amount:

25% v/v in ethanol

No. of animals per dose:

Five animals per dose

Details on study design:

Intradermal Injection Concentration Ranging Study
Before the start of the main study, an injection concentration range-finding study was performed to determine a suitable concentration of the test article for the intradermal injection stage of the main study.
This ranging study was performed in one animal which, 7 days, previously, had been pre-treated with 4 intradermal injections of a 1: 1 mixture of Freund’s Complete Adjuvant (FCA) and water.
0.1 ml aliquots of 50%, 25%, 10%, 5%, 1% and 0.5% v/v concentrations of the test article in light liquid paraffin were injected intradermally into the flanks of one guinea pig. The animal was examined on the day of dosing and then daily thereafter for a further 5 days and the response at each injection site was noted.
The results of this range-finding study indicated that a concentration of 5% v/v test article in light liquid paraffin would not provoke an unacceptable irritant response and was therefore selected for use for the intradermal injection stage of the main study.

Topical Irritancy Ranging Study
The potential of the test article to cause skin irritation was assessed by means of a topical concentration ranging study using 4 animals that were in the weight range 435 - 485 g and which had been previously treated with 1:1 FCA/water as described above. The concentrations used were undiluted test article and 50%, 25% and 12.5% v/v concentrations of the test article in ethanol.
An area 8 x 5 cm was clipped free of fur over the back and flanks of 4 animals and 4 patches of Whatman No 3 filter paper, 2cm x 2cm, each saturated with a different concentration of the test article were placed onto the skin, 2 patches on each flank. A strip of 5cm wide "Blenderm" surgical tape was placed over the patches to act as an occlusive barrier and the patches held in place for 24 hours by encircling the trunk of each animal with "Elastoplast" elastic adhesive bandage.
Twenty four and 48 hours after removing the patches and dressings the animals were examined under a standard light source designed to comply with the requirements of BS 950 Part 1 (artificial daylight for the assessment of colour) and any reaction at the treated sites was assessed.

Responses were assessed and scored using the following system :-
No reaction = 0
Discrete or patchy erythema = 1
Moderate and confluent erythema = 2
Intense erythema & swelling = 3

The results of this preliminary topical range-finding study indicated that a 50% v/v concentration of the test article was minimally irritant and therefore suitable for use for the topical induction stage of the main study.
A 25% v/v concentration of the test article was the maximum non-irritant concentration and was selected for use for the challenge application.


Main Study Induction
Thirty healthy animals that were within the weight range 464 - 566 g were selected for the study and randomly allocated to a group of 20 test animals and a group of 10 control animals using a stratified bodyweight procedure. The dorsal area between the shoulders of each animal was clipped free of fur and 3 pairs of intradermal injections made within this area. The dose volume of each injection was 0.1 ml and each pair of injections consisted of :-

A) Test Group
1) 50% v/v Freund’s Complete Adjuvant (FCA) emulsified in water.
2) 5% v/v test article in light liquid paraffin
3) 5% v/v concentration of the test article in a 1 : 1 emulsion of FCA and water

B) Control Group
1) 50% v/v Freund’s Complete Adjuvant emulsified in water
2) Light liquid paraffin
3) 50% concentration of light liquid paraffin in a 1 : I emulsion of FCA and water.

Twenty four hours after administration of the intradermal injections all animals were examined for signs of irritation in the treated area. All test animals exhibited moderate degree of irritation at the injection sites whilst all control animals exhibited minimal to moderate skin irritation at the injection site.
Six days after intradermal induction, the area surrounding the injection sites of all animals was clipped free of fur. At the sponsor’s request, 24 hours before the topical induction stage of the main study, the animals in the control group were pre-treated with 0.5 ml of 10% sodium lauryl sulphate in light liquid paraffin in order to mimic the irritant response expected to be produced by the test article in the test group.

The following day, patches of Whatman No. 3 filter paper, 4 x 2cm, each saturated with a 50% v/v concentration of the test article in ethanol were placed over the injection sites of all animals in the test group to boost the injection process. A strip of 5cm wide "Blenderm" surgical tape was placed over the patch to act as an occlusive barrier and the whole assembly held in position by wrapping the trunk of each animal with a length of "Elastoplast" elastic adhesive bandage. The prepared skin sites on all control animals was similarly treated with the filter paper being saturated with the vehicle, ethanol.
The patches and dressings were removed after 48 hours.
Twenty four hours after removal of the patches all animals were again examined for signs of irritation in the treated area. At this examination, all test and control animals exhibited minimal to moderate skin irritation at the treated site.

Main Study Challenge
Fourteen days after the topical induction application, the fur was clipped from the back and flanks of all animals. Patches of Whatman No.3 filter paper, 2cm x 2cm, were saturated with 25% v/v concentration of test article (the maximum non-irritant concentration) and placed on the left flank of all test and control animals. The right flank of each test and control animal was similarly treated with a patch soaked with the vehicle, ethanol. The patches were occluded and secured using the method previously described for topical application.
The patches and dressings were removed after a contact period of 24 hours.
Twenty four and 48 hours after removing the dressings the animals were examined and any reaction scored

Positive control substance(s):

yes

Remarks:

Mercaptobenzothiazole (MBT)

Results and discussion

Positive control results:

Mercaptobenzothiazole (MBT) was used as a positive control as this is known to be a sensitiser. The material was administered as a 10% w/v concentration in light liquid paraffin for the intradermal injection phase. Seven days after administration of the intradermal injections, MBT was administered topically to animals in the test group at a concentration of 10% w/v in acetone whilst animals in the control group were treated with acetone alone. Fourteen days after this topical application, a 10% w/v concentration of MBT in acetone was applied to the left flank of all test and control animals whilst acetone alone was applied to the right flank. Both sites were then occluded for 24 hours. Skin sites were examined and the skin responses were scored 24 and 48 hours after the end of the occlusion period.
Four of the 10 test animals exhibited positive responses to challenge with 10% MBT at the 24 and the 48 hour observations, resulting in a response incidence of 40%. None of the test animals responded to challenge with the vehicle at any of the observations.
None of the control animals responded to challenge with either 10% MBT or the vehicle at any of the observations.
These results confirm that Mercaptobenzothiazole (MBT) is a moderate sensitiser under the conditions of this study and the test system is therefore considered to be validated.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25%

No. with + reactions:

8

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

8

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

10% w/v MBT

No. with + reactions:

4

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

10% w/v MBT

No. with + reactions:

4

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

Following the challenge application, 8 out of the 20 test guinea pigs reacted positively to 25% v/v test article at the 24 and/or the 48 hour observation, resulting in a response incidence of 40%. One of test animals exhibited a weak positive response (erythema score of 1) to the vehicle, ethanol, at the 24 hour examination only. None of the remaining test animals reacted positively to challenge with the vehicle at any examination.

None of the control animals reacted positively to challenge with either 25% v/v test article or the vehicle (ethanol) at any examination, resulting in a response incidence of 0%.

As no positive responses were exhibited by any of the control animals, the single isolated weak positive response by one animal in the test group to ethanol may have been due to non-specific irritation.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

It is considered that the test article exhibited a moderate sensitising potential in the guinea pig under the conditions of the study.

Executive summary:

The skin sensitisation potential of the test substance was assessed according to OECD Test Guideline 406 using a guinea pig maximisation test. There was a positive response incidence of 40 % at the 24 and / or 48 h examination. The test substance is classified as a category 1B skin sensitizer; classification into sub-category 1B is appropriate based on the following criteria for the guinea pig maximisation test: ≥ 30% responding at > 1% intradermal induction dose.