Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessement.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity: 98.9%
Lot number: SM/4051089
Expiration date: 9/2006
Physical Description: Clear colorless liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Jackson Laboratories, Bar Harbor, ME 04609
- Age at study initiation: 11 weeks at start of dosing; records of dates of birth for animals used in this study are retained in the Calvert archives.
- Weight at study initiation: 18-25 grams at the outset (Day 1) of the study
- Housing: Animals were group housed (5 per cage) upon receipt in compliance with National Research Council "Guide for the Care and Use of Laboratory Animals". The room in which the animals were kept was documented in the study records. No other species were kept in the same room.
- Diet (e.g. ad libitum): Animals had access to Certified Rodent Chow 7012C ad libitum. The lot number(s) andspecifications of each lot used are archived at Calvert. No contaminants were known to be present in the certified diet at levels that would be expected to interfere with the results of this study. Analysis of the diet was limited to that performed by the manufacturer, records of which are maintained in the Calvert archives.
- Water (e.g. ad libitum): Tap water was available ad libitum, via water bottles. The water is routinely analyzed for contaminants as per Calvert SOP's. No contaminants were known to be present in the water at levels that would be expected to interfere with the results of this study. Results of the water analysis are maintained in the Calvert archives.
- Acclimation period: Study animals were acclimated to their housing for six days prior to their first day of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 to 26.7 °C
- Humidity (%): 30-53
- Air changes (per hr): no information
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

Study design: in vivo (LLNA)

Vehicle:
other: Diethyl phthalate/ethanol in a 3:1 ratio
Concentration:
7.5%, 15%, 30%
No. of animals per dose:
5 animals
Details on study design:
Mice were treated on the dorsal surface of both ears, once per day on Days 1, 2, and 3. Approximately 24±2 hours between applications of test article was maintained. On Day 6 the mice were injected i.v, with 20 μCi of 3H_ thymidine in 250 μI of sterile saline. Five hours later the mice were euthanized by CO2 asphyxiation. Ear thickness measurements of each mouse were recorded and the draining auricular lymph nodes removed. At removal, the number of nodes collected per animal was recorded, and the nodes were examined for size/appearance and the data recorded. Any unexpected observations were noted in study records. A single cell suspension was prepared from the lymph nodes of each mouse. Cells were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) overnight at 2-8°C. The pellets were recovered by centrifugation and resuspended in 1 ml of TCA and transferred to a vial containing scintillation fluid. An additional 1 ml of TCA was used to rinse the tube, and it was also transferred to the scintillation fluid. Incorporation of 3H_ thymidine was measured in a ß-scintillation counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean disintegrations per minute (DPM) for each group was evaluated using SYSTAT version 9.01, developed by SPSS, Inc. Increases in 3H-thymidine incorporation relative to the vehicle-treated control were derived for each group and recorded as stimulation indices (SI). The criterion for a positive response is that one or more concentrations of a test article elicits a 3-fold or greater increase in isotope incorporation relative to the vehicle control.
Body weight data and ear thickness measurements were also evaluated.
Individual DPM values were analyzed by log transformation (base 10) of the data. The evaluation of the equality of means for the DPM, body weight and ear thickness data was made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means are found, a Dunnett's test was used to determine the degree of significance from the control means.

Results and discussion

Positive control results:
The positive control, 35% (v/v) HCA, resulted in a stimulation index (SI) of 6.66. A 3-fold or greater increase in proliferative activity relative to the concurrent vehicle treated control is considered a positive response. In addition, the response with the positive control in this study was also statistically significant (p<0.01) when compared to the vehicle control group.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Exposure to the test substance at 7.5, 15 and 30% (w/v) resulted in stimulation indices of 1.95, 5.70, and 4.24, respectively. Statistically significant differences were also found at 15 and 30% (P < 0.01 and P < 0.05, respectively).The EC3 was calculated to be 9.6%.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The mean DPM for each treatment group was compared to the vehicle control group. Since the data indicated that the test article was positive, the EC3 was calculated to be 9.6.

Any other information on results incl. tables

Group

Treatment

Dose

DPM

(mean ± sem)

S1

(Test/control Ratio)

Results**

1

Vehicle*

-

228 ± 28

-

-

2

04-230(6)

7.5%

444 ± 228

1.95

-

3

04-230(6)

15%

1299 ± 336****

5.70

+

4

04-230(6)

30%

966 ± 235***

4.24

+

5

HCA

35%

1518±221****

6.66

+

* Diethyl phthalate/ethanol in a ratio of 3:1

**Test/control ratio of 3.0 or greater represents a positive result

***Statistically significant difference when compared to the vehicle control group (Group 1) (p<0.05)

****Statistically significant difference when compared to the vehicle control group (Group 1) (p<0.01)

Applicant's summary and conclusion