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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:
19th August 1991 to 14th November 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
May, 1981
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The test was conducted in 1991.

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of allyl (2-methylbutoxy)acetate and allyl (3-methylbutoxy)acetate
EC Number:
916-328-0
Molecular formula:
C10H18O3
IUPAC Name:
Reaction mass of allyl (2-methylbutoxy)acetate and allyl (3-methylbutoxy)acetate
Specific details on test material used for the study:
Note: The test reports refers to a substance (CAS No. 67634-00-8) that is typically 71-85% of the multi-constituent substance reaction mass of allyl (2-methylbutoxy)acetate and allyl (3-methylbutoxy)acetate (EC No. 916-328-0). The other component (CAS No. 67634-01-9) is very structurally similar and  no difference in toxicity is expected (the ECHA C&L database classifications do not show any new hazards that CAS # 67634-00-8  does not have; checked 11-12-18).

SOURCE OF TEST MATERIAL
- Batch No.of test material:184114
- Expiration date of the lot/batch:October 24, 1991
- Purity:99.3%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the original container, protected from light, at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: stable for at least 2 hours in ethanol

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test article and vehicle were placed into a glass beaker on a tared Mettler PM 200 balance. A weight/wei~ht dilution was prepared using a homogenizer. Homogeneity of the test art1cle in vehicle (ethanol) was maintained during treatment using a magnetic stirrer. The preparations were made immediately prior to each dosing.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Himalayan
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:BRL, Biological Research Laboratories Ltd., Wtil ferstrasse 4, CH-4414 Fullinsdorf
- Age at study initiation: 5 weeks
- Weight at study initiation: Control and test group: 305 - 346 g; Pretest: 309 - 341 g
- Housing:Individually in Makrolon type-3 cages with standard softwood bedding ( 11 Lignocel11, Schill AG, CH-4132 Muttenz).
- Diet: Diet Pelleted standard Kliba 342, Batche 63/91 guinea pig breeding/ maintenance diet (11 Kliba 11 , KlingentalmOhle AG, CH-4303 Kaiseraugst), ad libitum
- Water: Community tap water from FOllinsdorf, ad libitum. Once weekly additional supply of ascorbic acid (1 g/1) via the drinking water
- Acclimation period: One week under test conditions after veterinary examination

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 40-70%
- Air changes:10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light/12 hours dark

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: ethanol
Concentration / amount:
5%
Day(s)/duration:
7 days
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
other: ethanol
Concentration / amount:
100% undiluted
Day(s)/duration:
48 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: ethanol
Concentration / amount:
30% (posterior left flank)
Day(s)/duration:
24 hours
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
other: ethanol
Concentration / amount:
10% (anterior right flank)
Day(s)/duration:
24 hours
No.:
#3
Route:
epicutaneous, occlusive
Vehicle:
other: ethanol
Concentration / amount:
3% (posterior right flank)
Day(s)/duration:
24 hours
No. of animals per dose:
Two females were used for the intracutaneous pretest and 4 females for the epicutaneous pretest.
Ten females for the control group.
20 females for the test group.
Details on study design:
RANGE FINDING TESTS:
Intradermal injections:
Intradermal injections (0.1 ml/site) were made into the clipped flank of two guinea-pigs at concentrations of 5, 3 and 1% of the test article in ethanol. The resulting dermal reactions were assessed 24 hours later. For intracutaneous induction application a 5% test article dilution was selected (Appendix A).

Epidermal applications:
Patches of filter paper (2 x 2 em) was saturated with the undiluted test article and with concentrations of 30%, 10% and 3% of the test article in ethanol and applied to the clipped and shaved flanks of each of four guinea-pigs. The patches were covered by a strip of aluminum foil and firmly secured by elastic plaster wrapped around the trunk and covered with impervious adhesive tape. This procedure ensured the intensive contact of the test article. The dressings were removed after an exposure period of 24 hours and the reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema on a numerical basis according to Draize scale.

The allocation of the different test sites on the animals was altenated in order to minimize site to site variation in responsiveness.

MAIN STUDY
Induction
Intradermal injections:
An area of dorsal skin from the scapular region (approximately 6 x 8 em) was clipped free of hair. Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 4 x 6 em area in the clipped region as follows:

Test group:
1) Freund's complete adjuvant 50:50 with physiological saline.
2) The test article, diluted to 5% with ethanol.
3) The test article diluted to 5% by emulsion in a 50:50 mixture of ethanol and Freund's complete adjuvant: physiological saline (1:1).

Control Group:
1) Freund's complete adjuvant 50:50 with physiological saline.
2) ethanol.
3) 50:50 mixture of ethanol and Freund's complete adjuvant: physiological saline (1:1).

Epidermal applications:

One week after the injections, the scapular area (approximately 6 x 8 em) was again clipped and shaved free of hair. A 2 x 4 em patch of filter paper was saturated with the undiluted test article (100 %) and placed over the injection sites of the test animals. The patch was covered by aluminum foil and firm.ly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The dressings were left in place for approximately 48 hours. The epidermal application procedure described ensured intensive contact of the test article. The guinea-pigs of the control group were treated as described above with the
omission of test article (ethanol only). Reaction sites were assessed for erythema and oedema 24 and 48 hours after removal of the dressing, using the numerical grading system according to Oraize.

First Challenge
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 em area on the left and right flank of each guinea-pig. Four patches (2 x 2 em) of filter paper were saturated with non-irritant concentrations of 30% (posterior left flank), 10% (anterior right flank), 3% (posterior right flank) and the vehicle only (ethanol, applied to the anterior left flank) using the same method as for the epidermal application. The dressings were removed approximately 24 hours later. The sites were assessed for erythema and oedema 24 and 48 hours after removal of the dressing, using the numerical scoring system according to Draize. The control animals were treated in the same way as described above. Erythema and oedema reactions were described in the tables under Appendix A.
Positive control substance(s):
yes
Remarks:
A positive control group (Formaldehyde-solution) is included for sensitivity check of the guinea pig strain (see Appendix E). The most recent test was run from June 17 to July 1B, 1991 (RCC Project 30347B).

Results and discussion

Positive control results:
According to the procedures used in this experiment (run from June 17 to July 18, 1991), clear positive results were observed in the (HCHO) treated animals
after the epidermal challenge application. Eight out of 10 animals gave a positive response (Appendix E)

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
30%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
30%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
3%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
3%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
All dose levels
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
All dose levels
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
15%
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
15%
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In a guinea pig maximisation test, allyl (3-methylbutoxy) acetate was not sensitising.
Executive summary:

In a dermal sensitization study (B-158-967) with allyl (3-methylbutoxy) acetate (99.3%) in ethanol, young adult Himalayan female guinea pigs were tested in a maximisation test. For induction, 30% allyl (3-methylbutoxy) acetate in ethanol (intradermal injections) and 100% allyl (3-methylbutoxy) acetate in ethanol (topical application) was used. For challenge, 30, 10 and 3% allyl (3-methylbutoxy) acetate in ethanol was used for topical application. The evaluation of skin reactions after challenge was carried out at 24 and 48 hrs. The positive control was formaldehyde.

The body weight of animals increased during the study and was not affected by the treatment. The positive control, formaldehyde, gave the appropriate response. No deaths occurred. No systemic symptoms were observed in the animals. Application area around the injection sites 1, 2 and 3 was found to show erythema and oedema from day 2 to 6; necroses from day 7 to 11; encrustation from day 12 to 15 and exfoliation from day 16 to 21. No positive reactions were evident after the first challenge application at 24 or 48 hours, neither when treated with ethanol alone nor when treated with the 30%, 10% and 3% test article dilutions.

Based on these results, the substance is not sensitising.