Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 July - 21 July 2010
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Diisoamyl phthalate
- Physical state: colourless to yellow clear liquid
- Analytical purity: 99.98%

- Lot/batch No.: 7920-1-1
- Expiration date of the lot/batch: 12 August 2012
- Storage condition of test material: ca. room temperature in the dark
- Other:

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
-Source: United Kingdom
- Age at study initiation: approximately 10- 12 weeks
- Weight at study initiation: 18.0 to 24.7 g
- Housing: individually in polycarbonate cages with woodflake bedding.
- Diet (e.g. ad libitum): free access
- Water (e.g. ad libitum): freely available
- Acclimation period: at least 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23°C
- Humidity (%): 40 to 70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours continuous dark per 24 hours

IN-LIFE DATES: From: To: 12 July to 21 July 2010

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0 (vehicle alone), 25 and 50 %v/v and as supplied (ie undiluted)
No. of animals per dose:
Groups of 4 mice were treated at one of three concentrations of the test substance
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: A vehicle trial was conducted and Diisoamyl phthalate showed that it formed a pale yellow clear solution at 50% v/v in acetone:olive oil (4:1 v/v), which was satisfactory for dose administration.
- Irritation: no signs of irritation were seen over the dosed area during the study.
- Lymph node proliferation response: no data

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Pooled treatment group approach
- Criteria used to consider a positive response: the test substance is regarded as a sensitizer if at least one concentration of the test substance
results in a three-fold greater increase in 3HTdR incorporation compared to control values

TREATMENT PREPARATION AND ADMINISTRATION:

Preliminary test: Three females were treated with one of three concentrations of the test substance. The mice were treated by daily application of 25 μl of appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3).

Main test: Groups of four mice were treated at one of three concentrations of the test substance. The mice were treated by daily application of 25 μl of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3). A further group of four mice will receive the vehicle alone in the same manner and a further group of four mice will be shamed dosed.

In the main phase of the study, five days following the first topical application of test substance (Day 6) all mice were treated with 250 μl of phosphate buffered saline containing 3H-methyl Thymidinea (3HTdR: 80 μCi/mL) giving a nominal 20 μCi to each mouse.

A single cell suspension of lymph node cells (LNC) was prepared by gentle mechanical disaggregation through a stainless steel gauze (200 mesh size). The pooled LNC were then washed by adding 10 mL phosphate buffered saline, pelleted at 190 x g for 10 minutes and resuspended. The cells were washed twice again and resuspended in 3 mL trichloroacetic acid (TCA: 5%) following the final wash. After overnight incubation with 5% TCA at 4°C, the precipitate was recovered by centrifugation and resuspended in 1 mL 5% TCA and transferred to 10 mL Ultima gold scintillation fluid on Day 7. 3HTdR incorporation was measured by β-scintillation counting.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
Responses to the positive control substance hexyl cinnamic aldehyde (HCA), in contemporaneous studies demonstrated the reliability and sensitivity of this assay to detect skin sensitization potential in this laboratory.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 25% v/v 2.1 50% v/v 2.7 As supplied 6.4
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 25% v/v 5101.1 50% v/v 6644.9 As supplied 9025.7

Any other information on results incl. tables

The test substance is regarded as a sensitizer if at least one concentration of the test substance results in a three-fold greater increase in 3HTdR incorporation compared to control values.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
Diisoamyl phthalate (Diisopentyl phthalate) is regarded as a potential skin sensitizer.