2-hydroxymethyl-9-methyl-6-(1-methylethyl)-1,4-dioxaspiro[4.5]decane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well-documented experiment according to GLP and EC and OECD guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxymethyl-9-methyl-6-(1-methylethyl)-1,4-dioxaspiro[4.5]decane

EC Number:

408-200-3

EC Name:

2-hydroxymethyl-9-methyl-6-(1-methylethyl)-1,4-dioxaspiro[4.5]decane

Cas Number:

63187-91-7

Molecular formula:

C13H24O3

IUPAC Name:

[9-methyl-6-(propan-2-yl)-1,4-dioxaspiro[4.5]decan-2-yl]methanol

Test material form:

other: liquid

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Himalayan albino, SPF

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Species: Himalayan albino, SPF-quality
- Source: BRL Ltd., Basel, Switzerland
- Age at study initiation: approx. 9 weeks
- Weight at study initiation: 332-457 g
- Housing: 2 per cage, metal cages with wire-mesh floors and equipped with automated drinking system
- Diet: standard guinea pig diet including ascorbic acid (1600 mg/kg) LC23-B, 4mm pellets (Hope Farms, Woerden, The Netherlands); ad libitum. In addition hay was provided once a week.
- Water (e.g. ad libitum): Tap water diluted with decalcified water; ad libitum
- Acclimation period: at least 5 days before start of treatment under test conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 55%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (artificial fluorescent light)
Fluctuations from these optimal conditions were noted, but were considered to not have affected the study integrity.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

propylene glycol

Induction 2

Concentration / amount:

Intradermal injection (induction): 0.1 mL/site at a concentration of 2% (w/w) in propylene glycol.
Epidermal application: (induction): 0.5 mL of the undiluted test susbtance.
Epidermal application: (challenge): 0.05 mL at a concentration of 25%, 10% or 5% in polyethylene glycol.

Challenge

Challenge 1

Route:

epicutaneous, semiocclusive

Vehicle:

propylene glycol

Challenge 2

Concentration / amount:

Intradermal injection (induction): 0.1 mL/site at a concentration of 2% (w/w) in propylene glycol.
Epidermal application: (induction): 0.5 mL of the undiluted test susbtance.
Epidermal application: (challenge): 0.05 mL at a concentration of 25%, 10% or 5% in polyethylene glycol.

No. of animals per dose:

Preliminary study: 5 females
Main study experimental group: 20 females
Main study control group: 10 females

Details on study design:

RANGE FINDING TESTS:
The objective of this experiment was to identify the test substance concentrations suitable for the induction and challenge phases of the main study, and to detect any effects of systemic toxicity.

Intradermal injections: Four intradermal injections (0.1 mL/site) were made into the clipped shoulder region of one guinea pig at a concentration of 5% (w/w) of the test substance in propylene glycol. The resulting dermal reactions (erythema, necrosis and area affected) were assessed 24 and 48 hours later.

Epidermal applications: The intradermally injected animal was also treated epidermally at the shaved left flank with 0.5 mL of the undiluted test substance using a Metalline patch (Lohman, Neuwied, Germany) mounted on Micropore tape (3M, St. Paul, USA) and held in place with Coban elastic bandage (3M, St. Paul, USA). After 24 hours, the dressing and residual test substance were removed using a moistened tissue. The treated skin was assessed for erythema and oedema 24 and 48 hours after bandage removal on a numerical basis. For more details on scoring system, see below.
Four other animals were shaved on the left flank and exposed to 0.05 mL of a 100%, 50%, 25% and 10% (w/w) test substance concentration in propylene glycol, occlusively administered by means of Square chambers (v.d. Bend, Brielle, The Netherlands) mounted on Micropore tape and fixed in place by means of Coban elastic bandage. This procedure ensured the intensive contact of the test substance even if it is insoluble in the vehicle used. After 24 hours, the dressing and residual test substance were removed using a moistened tissue. The reaction sites were assessed for erythema and oedema on a numerical basis (see below for details on scoring system), 24 and 48 hours after bandage removal. Immediately after the 24 hour reading, the treated areas were shaved.

MAIN STUDY:
A. INDUCTION EXPOSURE
Intradermal injections: on day 1 an area of the dorsal skin from the scapular region (approx. 4 x 6 cm) was clipped free of hair. Three pairs of intradermal injections (A, B and C, two injections of each type on either side of the animal’s back; 0.1 mL/site) were made at a border of a 2 x 4 cm area in the clipped region.
Injections A: test substance diluted to 2% (w/w) with polyethylene glycol
Injections B: Freunds’ Complete Adjuvant (FCA, Difco, Detroit, USA), 50:50 with distilled water for injection.
Injections C: test substance, at twice the concentration used in (A), emulsified in a 50:50 mixture of Freunds’ Complete Adjuvant.

Epidermal applications:
Seven days after the intradermal injections, the scapular area (approx. 6 x 8 cm) was clipped and shaved free of hair. A 2 x 4 cm patch of Metalline mounted on Micropore tape was applied with 0.5 mL of the undiluted test substance and placed between the injection sites of the tested animals. The Micropore tape was firmly secured, wrapped around the trunk of the animal and secured with Coban elastic bandage. After 48 hours, the dressing and residual test substance were removed using a moistened tissue. The epidermal application procedure described ensured intensive contact of the test substance even if it is insoluble in the vehicle used. The guinea pigs of the control group were treated as described above by the intradermal and epidermal inductions with the omission of the test substance. Because the test substance was applied undiluted for the epidermal induction, a dry patch was administered to the test area of the control animals. Reaction sites were assessed on a numerical basis (see below for details on scoring) for erythema and oedema immediately after removal of the dressings.

B. CHALLENGE EXPOSURE
The test and control guinea pigs were challenged two weeks after the epidermal induction application. Hair was clipped and shaved from a 5 x 5 cm area on the left flank of each guinea pig. A volume of 0.05 mL of each of the following 3 test substance concentrations and vehicle were applied using Square chambers attached to Micropore tape:
1) 25% in polyethylene glycol
2) 10% in polyethylene glycol
3) 5% in polyethylene glycol
4) Polyethylene glycol

The patches were placed on the shaved area, the Micropore tape firmly secured around the trunk of the animal and held in place by Cogan elastic bandage. The dressings and residual test substance were removed after approx. 24 hours, using a moistened tissue. The sites were assessed on a numerical basis for redness and swelling 24 and 48 hours after removal of the dressings.

Positive control substance(s):

no

Results and discussion

Any other information on results incl. tables

Primary irritation experiments

 

No signs of systemic toxicity were observed during the primary irritation experiments. However, body weight loss was noted in one of the 5 animals.

 

Intradermal injections

 

Animal n°	Conc. % (w/w)	Skin readings after 24 / 48 hours
		Erythema	Necrosis	Diameter (mm)
440	5	* / *	yes / yes	5 / 5

*= erythema could not be determined due to necrosis

 

Epidermal application following intradermal injections

 

Animal n°	Conc. % (w/w)	Skin readings after 24h		Skin readings after 48h
		Erythema	Oedema	Erythema	Oedema
440	100	1	0	1s	1

 

 

Epidermal application only

Animal n°	Conc. % (w/w)	Skin readings after 24h		Skin readings after 48h
		Erythema	Oedema	Erythema	Oedema
436	100 50 25 10	1s 1s 1s 0s	1 1 1 0	1s 1s 1s 0s	1 1 1 0
437	100 50 25 10	1s 1s 1s 0s	1 1 0 0	1s 1s 0s 0s	1 1 0 0
438	100 50 25 10	2 1 0s 0s	1 0 0 0	1s 1s 0s 0s	1 1 0 0
439	100 50 25 10	1s 0s 0s 0s	1 1 0 0	1s 1s 0s 0s	1 1 0 0

 

   

Main study

 

Induction

 

The experimental animals showed very slight or well defined erythema after the 48 hours occluded epidermal induction exposure.

 

Challenge

 

Control group: One animal showed red spots and scaliness in response to the 25% test substance concentration.

 

Experimental group: three animals showed red spots and scaliness in response to the 25% test substance concentration. Taking into account the intensity of the responses and comparing these with the reactions seen in the control animals, none of the animals showed a positive skin reaction in response to the concentration tested.

N°	Induction		Challenge
	Day 10		Day 24				Day 25
	100%		25%	10%	5%	0%	25%	10%	5%	0%
	Er	Oe
Experimental group
406	2	0	0	0	0	0	0	0	0	0
407	2	0	0	0	0	0	0	0	0	0
408	2	0	0	0	0	0	0	0	0	0
409	2	0	0	0	0	0	0	0	0	0
410	2	0	1	0	0	0	0s	0	0	0
411	1	0	0	0	0	0	0	0	0	0
412	2	0	0	0	0	0	0	0	0	0
413	2	0	0	0	0	0	0	0	0	0
414	2	0	0	0	0	0	0	0	0	0
415	2	0	0	0	0	0	0	0	0	0
416	1	0	0	0	0	0	0	0	0	0
417	1	0	1	0	0	0	0	0	0	0
418	1	0	0	0	0	0	0	0	0	0
419	2	0	0	0	0	0	0	0	0	0
420	2	0	0	0	0	0	0	0	0	0
421	2	0	0	0	0	0	0	0	0	0
422	2	0	0	0	0	0	0	0	0	0
423	2	0	1	0	0	0	0s	0	0	0
424	2	0	0	0	0	0	0	0	0	0
425	2	0	0	0	0	0	0	0	0	0
Control group
426	0	0	0	0	0	0	0	0	0	0
427	0	0	0	0	0	0	0	0	0	0
428	0	0	0	0	0	0	0	0	0	0
429	0	0	1	0	0	0	1s	0	0	0
430	0	0	0	0	0	0	0	0	0	0
431	0	0	0	0	0	0	0	0	0	0
432	0	0	0	0	0	0	0	0	0	0
433	0	0	0	0	0	0	0	0	0	0
434	0	0	0	0	0	0	0	0	0	0
435	0	0	0	0	0	0	0	0	0	0

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Applying the EEC criteria for classification and labelling requirements of the Dangerous Substances Directive (67/548/EEC), the test substance need not be labelled as a kin sensitiser.

Executive summary:

The study assesses the potential of the test substance to induce delayed contact hypersensitivity (skin sensitisation) in the guinea pig after intradermal and epidermal exposures. The study is carried out according to GLP and OECD and EU guidelines.

 

In a preliminary experiment, the irritating effect of the test substance was examined. From this preliminary experiment, the slightly irritating and non-irritating test substance concentrations were determined, which were subsequently used in the main study.

 

The experimental animals were intradermally injected with a 2% concentration and epidermally exposed to undiluted test substance, while the control animals were similarly treated, but with the vehicle only, or with a dry patch. Immediately after the epidermal exposure, the skin irritation was scored. Two weeks after the epidermal application, all animals were challenged with test substance concentrations of 25%, 10% and 5%, and the vehicle. The challenge reactions were assessed 24 and 48 hours after bandage removal.

 

The epidermal exposure of the test substance in the induction phase resulted in very slight to well defined erythema. The epidermal exposure of the test substance in the challenge phase resulted in no positive sensitisation reactions in response to the test substance concentrations tested.

 

Under the conditions used in this study, the test substance induced no sensitisation. Applying the EEC criteria for classification and labelling requirements for dangerous substances (67/548/EEC), HR91/917 247 is not to be classified as a skin sensitiser.