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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:
24 Jan - 25 Feb 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Vertberate study predates requirement to test in-vitro before vertebrate studies

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2-Ethylhexyl benzoate
- Physical state: clear colourless liquid
- Analytical purity: 99.734%
- Impurities (identity and concentrations): 2-Ethyl-4-methyl-1-pentyl benzoate 0.209%, C8 benzoate 0.023%, 2-Ethylhexyl phthalate 0.004%, unknown impurities 0.03%
- Lot/batch No.: 9915-133-2
- Sample ID: 9915-140-1
- Expiration date of the lot/batch: 2000-12-04
- Storage condition of test material: room temperature in light, in brown glass bottle

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: D. Hall (Newchurch, Staffs, UK)
- Age at study initiation: 4-7 weeks
- Weight at study initiation: 362-435 g
- Housing: the animals were housed in groups of five in suspended metal cages with wire mesh floors.
- Diet: vitamin C enriched guinea-pig diet (Harlan Teklad 9600 FD2 SQC), ad libitum. In addition, hay was given thrice weekly.
- Water: drinking water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-20
- Humidity (%): 38-58
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: Alembicol D
Concentration / amount:
Preliminary study:
Intradermal: 0.1-10%; epicutaneous: 25-100%
Main study:
Induction:
Intradermal: 7.5%; epicutaneous: 100%
Challenge:
37.5 and 75%
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: Alembicol D
Concentration / amount:
Preliminary study:
Intradermal: 0.1-10%; epicutaneous: 25-100%
Main study:
Induction:
Intradermal: 7.5%; epicutaneous: 100%
Challenge:
37.5 and 75%
No. of animals per dose:
Preliminary study
6
Main study
5 (controls), 10 (in test group)
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: single injection (intradermal) and 48 h (epicutaneous)

- Test groups:
Intradermal (3 pairs of injections):
Injection 1: a 1:1 mixture (v/v) FCA/water
Injection 2: test substance in Alembicol D
Injection 3: test substance in a 1:1 mixture (v/v) FCA/water
Epicutaneous: test substance

- Control group:
Intradermal (3 pairs of injections):
Injection 1: a 1:1 mixture (v/v) FCA/water
Injection 2: Alembicol D
Injection 3: Alembicol D in a 1:1 mixture (v/v) FCA/water
Epicutaneous: Alembicol D

- Site: flank
- Frequency of applications: every 7 days
- Duration: Day 0-8
- Concentrations: intradermal 7.5%, epicutaneous 100%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 20
- Exposure period: 24 h
- Test groups: test substance
- Control group: test substance
- Site: left flank
- Concentrations: 37.5 and 75% (v/v)
- Evaluation (hr after challenge): 48 and 72 h

OTHER:
The preliminary investigations indicated that the neat test substance applied topically did not produce skin irritation. Therefore, six days after the injections, the injection site was clipped and shaved free of hair and the site was pre-treated by gentle rubbing with 0.5 mL per site of 10% (w/w) sodium lauryl sulphate in petrolatum.
Challenge controls:
The control group is actually a challenge control.
Positive control substance(s):
yes
Remarks:
hexyl cinnamic aldehyde (induction: 10% intradermal; epicutaneous 100%; challenge: 100%)

Results and discussion

Positive control results:
The positive control substance (10% hexyl cinnamic aldehyde in Alembicol D (intradermal), 50 and 100% (challenge)) induced positive reactions in 9/10 animals (90%), thus confirming the sensitivity and reliability of the experimental technique.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0% (induction); 37.5 and 75% (challenge)
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0% (induction); 37.5 and 75% (challenge). No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
7.5 and 100% (induction: intradermal and epicutaneous); 37.5 and 75% (challenge)
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
slight erythema at application site (75% of the test material) in 1/10 animals
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 7.5 and 100% (induction: intradermal and epicutaneous); 37.5 and 75% (challenge). No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: slight erythema at application site (75% of the test material) in 1/10 animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0% (induction); 37.5 and 75% (challenge)
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0% (induction); 37.5 and 75% (challenge). No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
7.5 and 100% (induction: intradermal and epicutaneous); 37.5 and 75% (challenge)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 7.5 and 100% (induction: intradermal and epicutaneous); 37.5 and 75% (challenge). No with. + reactions: 0.0. Total no. in groups: 10.0.

Any other information on results incl. tables

No signs of ill health or toxicity were observed.

After intradermal injections with FCA necrosis was recorded in test and control animals. Slight irritation was seen in test animals receiving intradermal 7.5% of the test substance in Alembicol D and in control animals.

After topical application, slight erythema was observed in 5/10 animals and well defined erythema in 4/10 animals; no erythema was seen in the control animals. After challenge, slight erythema was noted for one test animal at the 24 h reading only. The reaction was considered to be a background irritation.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
CLP: not classified