Registration Dossier

Administrative data

Description of key information

GPMT: non-sensitizing

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 Dec 1982 - 8 Jan 1983
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Principles of method if other than guideline:
Procedures based on the method described by Bertil Magnusson, M.D., and Albert M. Kligman, M.D. Ph.D. in "The Identification of Contact Allergens by Animal Assay: The Guinea Pig Maximization Test," Journal of Investigative Dermatology, 57, 268-276 and in Allergic Contact Dermatitis in the Guinea Pig, Identification of Contact Allergens, Thomas, Springfield, Illinois, 1970.
GLP compliance:
no
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study performed before LLNA guideline was available.
Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
Supplier: Hazleton-Dutchland, Inc., Denver, Pennsylvania
Date Received: December 1, 1982
Weight Range at Initiation of Treatment (grams): Males: 300-361 Females: 289-341
Animal Identification: Each animal was identified with a monel ear tag bearing its unique animal number.
Selection: Animals were selected randomly for study. Those of questionable health or with outlying body weights were eliminated prior to selection.

Husbandry:
Equilibration Period: 14 days
Housing: Individually in suspended stainless steel cages.
Food: Charles River Vitamin C-Fortified Guinea Pig Diet, -ad libitum.
Water: Automatic watering system, ad libitum (Elizabethtown Water Company).
Environmental Condition: 1. Temperature: 65-75F is considered an acceptable temperature range for guinea pigs; room temperature was monitored twice daily and maintained within this range to the maximum extent possible.
2. Humidity: 30-70% is considered an acceptable humidity range for guinea pigs; humidity was monitored twice daily and maintained within this range to the maximum extent possibl e.
3. Light Cycle: 12 hours light, 12 hours dark (controlled by an automatic timer).
Route:
intradermal and epicutaneous
Vehicle:
unchanged (no vehicle)
Concentration / amount:
1. Induction (Intradermal):
Site One:
Adjuvant: 10 mls of FCA was added to 10 mls deionized water, to produce a 0.5 g/ml (50%) mixture.
Site Two: Test or control material
Vehicle Control: 100% - Primary Amyl Acetate (no mixture required)
Positive Control: 0.01 g of DNCB was added to 10 mls propylene glycol to produce a 0.001 g/ml (0.1 %) mixture.

2. Topical:
Vehicle Control: Primary Amyl Acetate - 100% (no mixture required) for induction and challenge phase.
Positive Control:
Induction: 0.01 g of.DNCB was added to 80% ethanol which was brought to a total volume of 10 ml to produce a 0.001 g/ml (0.1%) mixture.
Challenge: 0.01 g of DNCB was added to 80% ethanol which was brought to a total volume of 10 ml to produce a 0.001 g/ml 0.1%) mixture.

Fresh mixtures were prepared prior to each administration.
No.:
#1
Route:
epicutaneous, semiocclusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
Vehicle Control: Primary Amyl Acetate - 100% (no mixture required) for induction and challenge phase.
Positive Control:
Induction: 0.01 g of.DNCB was added to 80% ethanol which was brought to a total volume of 10 ml to produce a 0.001 g/ml (0.1%) mixture.
Challenge: 0.01 g of DNCB was added to 80% ethanol which was brought to a total volume of 10 ml to produce a 0.001 g/ml 0.1%) mixture.

Fresh mixtures were prepared prior to each administration.
No. of animals per dose:
DNCB (Positive control) 10 guinea pigs (5M, 5F)
DNCB (Irritation control) 10 guinea pigs (5M, 5F)

Primary amy acetate 20 guinea pigs (10M, 10F)
Primary amy acetate (irritation control 10 guinea pigs (5M, 5F)
Details on study design:
Dosing Procedure:
1. Induction Phase:
On the day prior to the injections, the hair in the shoulder region (approximately 4x6 cm) was clipped short with an electric clipper. Substances were then administered by intradermal injection, using a 1.0 cc syringe and a 25/26 gauge needle, in the clipped shoulder area. A row of three injections were made on each side, for a total of six injections. The injections consisted of the following :
Two sites with 0.1 ml of FCA/water emulsion
Two sites with 0.1 ml of test or control material
Two sites with 0.1 ml of test or control material/FCA emulsion

2. Induction Phase - Topical Application:
One week after the intradermal injections, the topical application was performed. The hair in the shoulder area was re-clipped.

The vehicle (Primary Amyl Acetate) without the test substance, and the positive control material was applied to control animals in the same manner as the test substance. The patches were left in place for 48 hours after which they were removed and the skin wiped free of any excess material.

3. Challenge Phase:
Two weeks after the topical application, the challege treatment was administered. The hair was removed from a 5x5 cm area on the flank, by clipping as described previously.
Patches were applied to the flanks using the same procedure as for topical application on Day 7, except that a 2x2 cm piece of filter paper was used and allowed to remain on the animal for 24 hours.

4. Irritation Control Challenge:
In order to differentiate dermal reactions produced by irritation from those produced by sensitization, ten animals (previously untreated) were subjected to the same challenge procedures as the animals which were dosed during the induction phase.

Experimental Evaluation:
Viability Check: Twice Daily
Observations: Animals were observed prior to treatment and weekly during the study for general health and unusual observations were recorded. (Animals which were not considered to be in good health prior to treatment were not placed on study) .
Evaluation of Dermal Responses:
Approximately 21 hours a:fter removing the patch, the challenge area was gently clipped. Readings were made on all animals 24 and 48 hours after the removal of the patches.

At each interval the treated site was evaluated for erythema and edema or other evidence of dermal irritation according to the scoring system used. Adjacent areas of untreated skin was used for comparison. Special notations were made of necrosis, eschar, or other evidence of irreversibi l e alteration of tissue structure. Any abnormal pharmacologic signs were also noted.

Redness or edema at the challenge site at any of the observations which was greater than that seen in the vehicle and/or irritation control animal s was considered an allergic response. Number (percentages) of animals reacting, rather than intensity of reactions, is the criterion for categorizing materials as sensitizers and assessing sensitization potency.

Sensitizers are categorized, based on percentage of animals affected as weak (Grade 1) to extreme (Grade V) sensitizers.
Challenge controls:
See above
Positive control substance(s):
yes
Remarks:
2,4-dinitrochlorohenzene (DNCB)
Positive control substance(s):
other: 2,4-dinitrochlorobenzene
Statistics:
No additional information available.
Positive control results:
All of the ten animals treated with DNCB (Group 1A ) exhibited dermal responses at challenge to a non-irritating concentration, as confirmed by a lack of response by control animals (Group IB) to the same concentration. This positive response to a known sensitizer demonstrates the susceptibility of this group of animals to sensitization.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100 %
No. with + reactions:
1
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100 %. No with. + reactions: 1.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100 %. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.1 %
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 0.1 %. No with. + reactions: 10.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.1 %
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 0.1 %. No with. + reactions: 9.0. Total no. in groups: 10.0.

Mortality

All animals survived throughout the study.

Dermal Responses

Dermal scores of 1 or greater (in the absence of dermal response in irritation control animals) are considered clearly indicative of sensitization. Scores of + (barely perceptible erythema) are considered equivocal, although a high percentage of scores o f + in treated animals with no dermal response in irritation control animals is considered suggestive of sensitization.

Several (17 of 20) of the animals treated with primary amyl acetate (Group IIA) exhibited barely perceptible erythema (scores of +) at 24 hours, one exhibited a dermal score of 1, and 4 exhibited edema. However, 5 of the 10 irritation control animals (Group IIB) also exhibited slight dermal erythema (scores of 2 or 1) at 24 hours. At 48 hours, only two test animals had minimal erythema (scores o f +) and no responses were present in irritation control animals. Although it is possible that this material may possess a very slight sensitization potential, the response seen is considered more indicative of transient irritation than of sensitization.

Interpretation of results:
GHS criteria not met
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A guideline conform sensitization test is available for the reaction mass of 2-methylbutyl acetate and pentyl acetate. The negative results are supported by a patch test in 25 human volunteers. Additionally, data are also provided for isoamylacetate, a minor constituent of the reaction mass.

In a Guinea Pig Maximization Test conducted equivalent to the OECD Guideline 406 (DOW 1983, data published by Ballantyne et al., 1986) an intradermal induction was performed in the shoulder region (approx. 4*6 cm) by a row of 3 injection, each shoulder site (2 sites with 0.1 ml of FCA/water emulsion, 2 sites with 0.1 ml of test or control material, and 2 sites with 0.1 ml of test or control material/FCA emulsion) of test animals (Hartley guinea pig, 10 animals per sex). A topical application (second epidermal induction) of unchanged test substance was then conducted one week later, whereas the patches were left in place for 48 hours after which they were removed and the skin wiped free of any excess material. A challenge treatment (undiluted test substance) was administered 2 weeks after the topical application, whereas the patches were applied to the flanks using the same procedure as for topical application, except that they were allowed to remain on the animal for 24 hours. Approximately 21 hours after removing the patch, the challenge area was gently clipped, and readings were made on all animals 24 and 48 hours after the removal of the patches. In order to differentiate dermal reactions produced by irritation from those produced by sensitization, 5 animals per sex (previously untreated) were subjected to the same challenge procedures as the animals which were dosed during the induction phase. 2,4-Dinitrochlorohenzene (DNCB) was used as positive control (5 animals per sex).

10/10 animals of the positive control group showed a positive reaction 24 and 48 hours after challenge. 17/20 animals in the test substance group exhibited barely perceptible erythema 24 hours after challenge and 1 exhibited a dermal score of 1. 4 animals exhibited edema. After 48 hours, only barely perceptible erythema remained in two animals. Of the control animals, 5/10 also exhibited slight dermal erythema at the 24h reading, and 1 had a dermal score of 1. Considering the comparable results in the irritation control animals and their transient nature, the effects are indicative of transient irritation rather than of sensitization.

Results of a human patch test performed by Kligman et al. (1973) are cited by Opdyke et al. (see section on human data). 25 volunteers received five occlusive applications of 8% isoamyl acetate (not further specified) in petrolatum for 48h each, with a rest period of 24 hours in between applications. The concentration during induction was selected to produce brisk dermatitis after 24h. During challenge, a non-irritant concentration in petrolatum is occlusively applied for 48h. Evaluation of the skin sites revealed no sensitizing potential, which supports the results obtained in the animal study described above.

CAS No. 123-92-2

In an open epicutaneous test, (Klecak, 1985) guinea pigs (male/female; at least 6 per test dose) were given repeated daily (3 - 4 weeks) epicutaneous applications of isoamyl acetate (not further specified; 0.1 ml undiluted or at concentrations of 30, 10, 3 and 1% or lower). The challenge period consisted of 2 (day directly after the end of the induction period and 14 days later) epicutaneous administrations of isoamyl acetate (0.025 ml of the minimal irritating and some lower primary nonirritating concentrations). The evaluations after 24, 48 and/or 72 hours showed a negative result.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The registered substance was not sensitizing in the guinea pig maximization test. This is supported by the results of a human patch test. Therefore, no classification is required according GHS (REGULATION (EC) No 1272/2008 (CLP))