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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-06-20 to 2012-06-26
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

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 lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Sodium selenite
- Molecular formula (if other than submission substance): Na2SeO3

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: ELEVAGE JANVIER; Route des Chènes Secs B.P. 4105; 53940 LE GENEST-ST-ISLE, France
- Age at study initiation: 10 weeks
- Weight at study initiation: 20.5 – 22.6 g
- Housing: Group caging - mice were provided with glass tunneltubes; Type II. polypropylene/ polycarbonate
- Diet (e.g. ad libitum): ssniff SM R/M-Z+H Autoclavable complete diet for rats and mice - ad libitum
- Water (e.g. ad libitum): tap water from the municipal supply from 500 ml bottle, ad libitum.
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 15-20 air exchange/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily light

IN-LIFE DATES: From: To:

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
preliminary experiment: 100, 50, 10, 5, 2.5, 1, 0.5 and 0.1 % (w/v)
main experiment: 1, 0.5 and 0.1 % (w/v)
No. of animals per dose:
Preliminary experiment: 2 females per group
Main experiment: 4 females per group
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: examined in a short Preliminary Compatibility Test; the formulation (suspension) using Propylene glycol (PG)
as vehicle was suitable for the test.
- Irritation: ear thickness and the revealing ear punch weights were determinded, on day 1,3 and 6
- Lymph node proliferation response: not assessed, according to guideline

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Determination of cellular proliferation; incorporation of ³H-methyl thymidine is measured by β-scintillation counting as
disintegrations per minute (DPM).
- Criteria used to consider a positive response: A stimulation index of 3 or greater is an indication of a positive result.

TREATMENT PREPARATION AND ADMINISTRATION:
During the assay, each mouse was topically dosed on the dorsal surface of each ear with 25 μl of the appropriate formulation applied using a pipette. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
A significant lymphoproliferative response (stimulation index value of 12.8) was noted for the positive control chemical and this result confirmed
the validity of the assay.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
3.2
Test group / Remarks:
0.5 % (w/v)
Key result
Parameter:
SI
Value:
2
Test group / Remarks:
0.25 % (w/v)
Key result
Parameter:
SI
Value:
2.1
Test group / Remarks:
0.1 % (w/v)

Any other information on results incl. tables

The highest dose group of 1% (w/v) was excluded from the evaluation as during the experiment one animal was found dead on Day 4.

DPM, DPN and Stimulation Index Values for all Groups

Test Group Name

Measured DPM/group

Group DPM

(background corrected)

No. of Nodes

DPN

Stimulation Index Values

Background (5 (w/v) % TCA )

35.5

 

-

 

 

Negative control PG

1079

1043.5

8

130.4

1.0

Sodium selenite 1% (w/v) in PG*

2391

2355.5

6

392.6

3.0

Sodium selenite

0.5% (w/v) in PG

3405

3369.5

8

421.2

3.2

Sodium selenite 0.25% (w/v) in PG

2163

2127.5

8

265.9

2.0

Sodium selenite 0.1% (w/v) in PG

2256

2220.5

8

277.6

2.1

Positive control 25 % HCA in PG

13396

13360.5

8

1670.1

12.8

* Due to the mortality at 1% (w/v) makes this DPM data uninterpretable. The numbers are presented in the table, but are not used in study interpretation.

DPN = DPM divided by the number of lymph nodes

Individual ear thickness for all animals (preliminary test)

Animal Number

Test Group Name

Ear thickness on Day 1 (mm)

Ear thickness on Day 3 (mm)

Ear thickness on Day 6 (mm)

Ear weight

 on Day 6 (mg)

9375

100 (w/v) %

0.23 / 0.23

 

 

 

9394

100 (w/v) %

0.21 / 0.22

 

 

 

9430

50 (w/v) %

0.22 / 0.22

 

 

 

9437

50 (w/v) %

0.22 / 0.22

 

 

 

9441

10 (w/v) %

0.20 / 0.20

 

 

 

9410

10 (w/v) %

0.23 / 0.23

 

 

 

9545

5 (w/v) %

0.23 / 0.22

 

 

 

9611

5 (w/v) %

0.21 / 0.22

 

 

 

9616

2.5 (w/v) %

0.20 / 0.21

 

 

 

9580

2.5 (w/v) %

0.21 / 0.21

 

 

 

9426

1 (w/v) %

0.23 / 0.24

0.23 / 0.23

0.26 / 0.27

24.4

9442

1 (w/v) %

0.22 / 0.21

0.23 / 0.22

0.18 / 0.17

23.6

9445

0.5 (w/v) %

0.22 / 0.21

0.23 / 0.23

0.26/ 0.24

23.6

9448

0.5 (w/v) %

0.21 / 0.21

0.21 / 0.22

0.27 / 0.21

23.5

9379

0.1 (w/v) %

0.21 / 0.20

0.25 / 0.22

0.22 / 0.22

13.8

9393

0.1 (w/v) %

0.22/0.23

0.22/0.23

0.22/0.22

14.2

**: Based on the historical control data, the maximum normal weight is 21.1 mg. Positive response is over 26.3 mg.

Ear thickness of the animals was measured using by a thickness gauge on Days 1, 3 and 6 in survived animals. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the survived experimental animals. The ear thickness data and the revealing ear punch weights were within the historical control range in the 0.1 %(w/v) dose group. The ear thickness data on Day 6 in the 1 and 0.5 % (w/v) treated group animals were slightly higher than measured on Days 1 or 3, and the ear punch weights were also slightly higher than the normal range, but this was considered as a negative effect. Although these results could be an indication of irritation, there were no visible signs of any irritation, so the highest dose (1 %) was considered to be acceptable for the main test. In order to make sure that there will be three acceptable dose groups, an additional dose group was used in the main experiment.

Applicant's summary and conclusion

Interpretation of results:
ambiguous
Conclusions:
Under the conditions of the present assay Sodium selenite was shown to have a sensitization potential (sensitizer) in the Local Lymph Node Assay, according to study author. However, the positive response at the highest concentration was slight and metals are known to have a lower predictivity in the LLNA than organic molecules.
Executive summary:

In an OECD Guideline 429 study (Skin Sensitisation: Local Lymph Node Assay), groups of four female CBA/J Rj mice were treated with 1, 0.5, 0.25 and 0.1% (w/v) Sodium selenite in PG.

The solutions of the test item were applied on the dorsal surface of ears of experimental animals (25 μl/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

The highest dose group of 1% (w/v) was excluded from the evaluation as during the experiment one animal was found dead on Day 4. The guideline requires the maximum dose to have no systemic toxicity and 4 animals alive, these criteria were not met.

No mortality, signs of systemic toxicity or body weight loss were observed during the study for the remaining three dose groups. No treatment related effects were observed on animal body weights in any treated groups. Very slight erythema (score 1) was observed in 1 % (w/v) test item treated group animals on Day 3 and in one or two animals in positive control group on Days 2 and 3.

Stimulation index values of the test item were 3.2, 2.0 and 2.1 at treatment concentrations of 0.5, 0.25 and 0.1% (w/v), respectively.

A significant lymphoproliferative response (stimulation index value of 12.8) was noted for the positive control (α-Hexylcinnamaldehyde (25% (w/v) dissolved in PG)), which confirmed the validity of the assay.

According to study author, under the conditions of the present assay Sodium selenite (Batch No.: 114307) tested in Propylene Glycol as vehicle, was shown to have a sensitization potential (sensitizer) in the Local Lymph Node Assay. However, the positive response at the highest concentration was slight and metals are known to have a lower predictivity in the LLNA than organic molecules.

However, the following factors were not taken into account by the study author:

  1. The substance Sodium selenite is dermally relatively toxic
  2. Inflammatory processes might be a possible confounding factor for a false positive result
  3. The effect on ear weights as observed in the preliminary range finding study were not considered as possible irritation by the study authors.

Further discussion is outlined in the endpoint summary.