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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:
01. Apr. 2003 - 02. May 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The maximisation test was considered to be the most suitable test type because a challange was investigated.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name : BENZOlC ACID ISONONYLESTER
Batch/Lot Number : 1276/00576
Expiry date : 31 December 2003
Description at use : Colourless liquid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
Young adult female guinea pigs of Dunkin-Hartley strain were ordered from Harlan Italy S .r.l., San Pietro al Natisone (UD), Italy. Animals were ordered nulliparous and non-pregnant, within the weight range of 250 to 300 grams and 4 to 5 weeks of age. They were supplied by Harlan Netherlands, Kreuzelweg 53, 5961 NM Horst, P.O. Box 6174 NL, 5960 AD Horst, Netherlands and appeared to be in an acceptable condition following arrival on 21 st March 2003. Animals were identified by tattoo on the ear following arrival and an acclimatisation period of at least 5 days was permitted before
undertaking any dosing procedure.
Animals were housed during the acclimatisation period in groups ofup to 10 animals in cages measuring 87x71x24 cm and during the study in groups of up to 5 animals, in stainless steel eages with a grid floor measuring 48 x 63 x 41 cm. Cages were suspended over metal trays whieh held an absorbent material. This was inspected daily and changed as necessary. Throughout the study, each cage was identified by a label, colour-coded according to group, recording the study number, animal numbers and details of treatment.
Controls for the animal room were set to maintain temperature within the range of 22 ± 2°C and relative humidity within the range of 55 ± 15%. Actual conditions achieved were recorded daily. The room was Iit by fluorescent light to give an artificial cycle of 12 hours light / 12 hours dark, and therewere approximately 15 to 20 air changes per hour.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Concentration / amount:
Induction - intradermal injection: 20 % test item in corn oil; 20 % test item in emulsified Freund´s complete adjuvants
Induction - topical application: 100 % test item
Challenge: 50 % test item
Challengeopen allclose all
Route:
epicutaneous, occlusive
Concentration / amount:
Induction - intradermal injection: 20 % test item in corn oil; 20 % test item in emulsified Freund´s complete adjuvants
Induction - topical application: 100 % test item
Challenge: 50 % test item
No. of animals per dose:
20 animals in treatment group
10 animals in control group
Details on study design:
Main study - Induction - Intradermal injection
Animals were allocated to treatment to give a test group of 20 animals and a control group of 10 animals. On the day of dosing (Day 1) the hair was clipped from the scapular region of each animal over an area approximately 20 x 40 mm. Three pairs of intradermal injections were made at the prepared skin site of each animal. All injections were made at the edge of the prepared site and the anterior and median injections were positioned close together and distant from the posterior injections. A volume of 0.1 ml was injected at each point. Animals of the test group were treated as follows:
Injection site Treatment
Anterior Emulsified Freund's complete adjuvant
Median 20% test item in corn oil
Posterior 20% test item in emulsified Freund's complete adjuvant
Animals of the control group were treated in the same manner except that the test item was replaced by the vehicle alone. Skin reaction at the injection sites was assessed approximately 24 hours after injection.
Main study - Induction - Topical application
Six days after injection (Day 7 of the study) the area surrounding the injection sites on each animal was clipped free of hair. A 0.5 ml aliquot of sodium lauryl sulphate at 10% concentration in petrolatum was spread evenly over the skin surrounding the injection site.
The next day (Day 8), animals of the test group were treated with the test item at 100% concentration by a gauze patch which was covered with 004 ml of the substance and then placed over the injection sites. This was covered with a strip of aluminium foil to serve as an occlusive barner and the animal was then wrapped with a length of elastic adhesive bandage to maintain the gauze patch in contact with the skin. All animals of
the test group were treated in this manner. Animals of the control group were similarly treated with the vehicle alone. After a contact period of 48 hours the dressings were removed and the treated sites gently cleaned by washing with warm water. Reaction to treatment was assessed approximately 24 hours after removal of the dressings
Challenge controls:
On Day 22, 3 weeks after preparing the animals for the first induction phase of the main study, all animals were prepared far challenge by clipping the flanks free of hair to expose areas approximate1y 50 mm x 50 nun on each flank. Patches of gauze measuring 20 mm x 20 mm were coated with 0.2 ml aliquots of the test item at 50% concentration. These were placed on the right flank of each animal, of both test and control groups, in the centre of the prepared skin site. The left flank of each animal was similarly treated with patches soaked with 0.2 ml of the vehicle alone
(corn oil). The treated sites were covered with a stJip of aluminium foil to act as an occlusive barrier and each animal then wrapped with a length of elastic bandage to keep the test item and vehicle in contact with the skin. After a contact period of 24 hours the dressings and patches were removed and the treated sites were washed with warm water. Approximately 22 hours after removal of the dressings and patches, the treated sites
were closely clipped to remove any hair that may have grown. This was a deviation from the study Protoco1 which indicated that animals be clipped approximately 21 hours after removal of dressing. Approximately 2 hours later, 24 hours after removal of the dressings, the treated sites were examined for any signs of reaction to treatment.

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Group:
positive control
Remarks on result:
not measured/tested

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The results obtained in this study indicate that the test item, BENZOIC ACID ISONONYLESTER, does not elicit a sensitisation response in the guinea pig there being no reaction observed at challenge.
Executive summary:

The potential of the test item, BENZOlC ACID ISONONYLESTER, to induce and elicit delayed dermal sensitisation was assessed by a guinea pig model using the maximisation test of Magnusson and Kligrnan. The concentrations of the test item used in the main study were detennined by the results of preliminary screening tests. The main sensitisation test was undertaken using a test group of 20 animals and a control group of 10 animals. In an attempt to induce sensitisation, test animals were intradermally injected with an emulsion of Freund's complete adjuvant and the test item at 20% concentration in both the selected vehicle (corn oil) and an emulsion of Freund's complete adjuvant. One week later this was boosted by topical application of the undiluted test item over the injection sites. Control group animals were treated in the same manner but the selected vehicle was used in place of the test item. Two weeks after the second induction stage, all animals were challenged by topical application of both the vehicle and the undiluted test item. At the challenge with the test item at 50% concentration no response to the test item was apparent in any animal of the test or control groups. No reaction to the vehicle alone was observed in any anima!. These results indicate that the test item, BENZOIC ACID ISONONYLESTER, does not elicit a sensitisation response in the guinea pig, there being no reaction observed at challenge. European Directives concerning the classification, packaging and labelling of dangerous substances (67/548/EEC and subsequent revisions) would indicate the following: Classification : Not required; Symbol : None indicated; R Phrase : None indicated.