3-methoxyoestra-1,3,5(10)-trien-17-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Estrone

EC Number:

200-164-5

EC Name:

Estrone

Cas Number:

53-16-7

Molecular formula:

C18H22O2

IUPAC Name:

3-hydroxyestra-1,3,5(10)-trien-17-one

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels. The formulations were stirtred with magnetic stirrer immediately prior to dosing.

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: approx. 11 weeks old
- Weight at study initiation:
- Housing: group housed
- Diet (e.g. ad libitum): pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 d
A health inspection was performed prior to treatment, to ensure that the animals were in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0ºC (actual range: 17.6 - 21.6ºC),
- Humidity (%): 40-70% (actual range: 21 – 64%)
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

Pre-Screen test: 25% and 50%
Main test: 10%, 25% and 50%

No. of animals per dose:

Pre-Screen test: 2
Main Test: 5

Details on study design:

PRE-SCREEN TESTS:
- tested at 25% and 50% concentration
- Compound solubility:
- Irritation: Slight irritation along with white test substance remnants (which did not hamper scoring) were observed on both ears of all animals treated at a 25 and 50% concentration between Days 1 and 3
- Systemic toxicity: no
- Ear thickness measurements: no


MAIN STUDY

- Criteria used to consider a positive response:
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group.
If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM.

TREATMENT PREPARATION AND ADMINISTRATION:
On the induction days 1,2 and 3 the dorsal surface of both ears was topically treated (25 µl/ear) with the test substance concentration.

On day 6 the draining (auricular) lymph node of each ear was excised after injection of 3H-methyl thymidine via tail vein to the mice 5 h before and tissue was processed for radioactivity measurements.

Additionally, clinical signs (daily), body weights (day 1 and 6) and signs of irritation or other local effects were observed.

Positive control substance(s):

other:

Results and discussion

Positive control results:

reliability check:
The SI values calculated for the substance concentrations 5, 10 and 25% were 1.2, 2.2 and 3.7 respectively. An EC3 value of 18.0% was calculated using linear interpolation.
The calculated EC3 value was found to be in the expected range of 2 and 20%. The results of 6 monthly HCA reliability checks of the recent years were 13.8, 13.9, 16.0, 11.9, 16.9 and 10.7%.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.6

Variability:

+- 0.4

Test group / Remarks:

5 females/10 %-group

Results 2

Key result

Parameter:

SI

Value:

1.7

Variability:

+- 0.5

Test group / Remarks:

5 females/25 %-group

Results 3

Parameter:

SI

Value:

0.9

Variability:

+- 0.2

Test group / Remarks:

5 females/50 %-group

Results 4

Parameter:

SI

Value:

1

Variability:

+- 0.2

Test group / Remarks:

vehicle control group

Results 5

Parameter:

other: mean disintegrations per minute (DPM)/animal

Value:

449

Test group / Remarks:

10 %-group

Results 6

Parameter:

other: mean disintegrations per minute (DPM)/animal

Value:

470

Test group / Remarks:

25 %-group

Results 7

Parameter:

other: mean disintegrations per minute (DPM)/animal

Value:

268

Test group / Remarks:

50 %-group

Results 8

Parameter:

other: mean disintegrations per minute (DPM)/animal

Value:

283

Test group / Remarks:

vehicle control group

Any other information on results incl. tables

No mortalities occurred, no symptoms of systemic toxicity, no changes in body weights and body weight gain were observed.

A slight irritation (grade 1) of the ears was seen in all animals treated with 25 and 50%, but this was considered not to have a toxicologically significant effect on the activity of the nodes. No oedema were observed.

All auricular lymph nodes were considered normal in size, no macroscopic abnormalities of the surrounding area were noted in any of the animals.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

not skin sensitizing

Executive summary:

To determine the skin-sensitizing properties of estrone the mouse local lymph node assay was performed on female CBA/J mice (5/group) according to OECD guideline 429. The main study was conducted with the following test substance concentrations: 0 (vehicle control), 10%, 25% and 50% formulated in propylene glycol.

All animals treated with 25 and 50% showed slight irritation of the ears, but this had no toxicological significant effect on the activity of the lymph nodes. White test substance remnants were present on the ears of all animals at 10, 25 and 50%, which did not hamper scoring of the skin irritation reactions.

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10,

25 and 50% were 449, 470 and 268 DPM respectively. The mean DPM/animal value for the vehicle

control group was 283 DPM.The mean DPM/animal values were 283 (vehicle control), 449 (10%), 470 (25%) and 268 (50%). The SI values calculated were 1.6 (10%), 1.7 (25%) and 0.9 (50%). Since there was no indication that the test substance elicits an SI above or equal to 3 when tested up to 50%, estrone was considered not to be a skin sensitizer.

A regularly performed reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at the laboratory is an appropriate model for testing for contact hypersensitivity.