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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:
December 2017 - April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Test data is required for classification purposes.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
Qualifier:
according to
Guideline:
other: Procedures and facilities comply with the requirements of Directive 2010/63/EU [7] and the national legislation defined in the animal protection law concerning the protection of animals used for experimental and other scientific procedures
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The GLP certificate is included within the attached study report
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Specific details on test material used for the study:
Analytical purity – 97.60% (BaSeO4)
Physical state – Rhombohedral crystals
Colour – White
Molecular weight – 280.32 g/mol
Expiry date – 24th April 2019
Storage conditions – Room temperature
Stability under test conditions - Acceptable

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
healthy CBA/CaOlaHsd mice
Sex:
female
Details on test animals and environmental conditions:
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes.
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.

Housing and Feeding Conditions
• Full barrier in an air-conditioned room
• Temperature: 22 ± 3 °C
• Relative humidity: 55 ± 10%
• Artificial light, sequence being 12 hours light, 12 hours dark
• Air change: at least 10 x / hour
• Free access to Altromin 1324 maintenance diet for rats and mice
• Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
• The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
• Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
• Adequate acclimatisation period (at least five days) under laboratory conditions

Clinical Observation
Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application.

Weight Assessment
The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR).

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
Pretest concentrations: 12.5 %, 25%, 50% and 100% Barium Selenate in DMSO
Main test: 0.75%, 1.5% and 3% Barium selenate in DMSO
No. of animals per dose:
2
Details on study design:
Topical Application

Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear.
Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1.

Administration of 3H-Methyl Thymidine

Five days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250µL of 3H-methyl thymidine, diluted with PBS to a working concentration of 80 µCi/mL.

Preparation of Cell Suspension

Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, weighed, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4° C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of Incorporated 3H -Methyl Thymidine

The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.
Positive control substance(s):
other: Phenylenediamine in DMSO
Statistics:
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.
EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation, EC3=c+[(3-d)/(b-d)]x(a c), between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.
A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).
On the basis of the test results, the test substance may be classified into one of the following categories in conformity with the criteria given in Commission Regulation (EU) No 286/2011 as well as in GHS - Globally Harmonised System of Classification and Labelling of Chemicals, seventh revised edition, 2017.

Results and discussion

Positive control results:
Refer to table in “Any other information on results incl. tables”

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Value:
0.8
Test group / Remarks:
Test group 1 (0.75%)
Remarks on result:
other: Acceptable, EC3 is <3
Key result
Parameter:
EC3
Value:
1.6
Test group / Remarks:
Test group 2 (1.5%)
Remarks on result:
other: Acceptable, EC3 is <3
Key result
Parameter:
EC3
Value:
0.8
Test group / Remarks:
Test group 3 (3%)
Remarks on result:
other: Acceptable, EC3 is <3
Cellular proliferation data / Observations:
Not calculated as stimulation indices of all concentrations were below 3.

Any other information on results incl. tables

Radioactive Determination of the Test Substance– Main Study–Test Groups 1 and 2

POS

CPM

Test Item

Conc. [%]

Animal number

DPM

DPM mean background

DPM / node

Simulation index

16

876.0

Negative control

100

411

1766.0

1716.8

858.4

 

17

1083.0

412

2187.0

2137.8

1068.9

 

18

1328.0

413

2684.0

2634.8

1317.4

 

19

554.0

414

1115.0

1065.8

532.9

 

20

1450.0

415

2923.0

2873.8

1436.9

 

MV

1058.2

MV

2135.0

2085.8

1042.9

1.0

SD

320.7

SD

648.3

648.3

324.2

 

26

373.0

Barium Selenate in DMSO

0.75

16

719.0

669.8

334.9

0.3

27

1980.0

17

2181.0

2131.8

1065.9

1.0

28

1039.0

18

2089.0

2039.8

1019.9

1.0

29

1351.0

19

2751.0

2701.8

1350.9

1.3

30

623.0

20

1246.0

1196.8

598.4

0.6

MV

893.6

MV

1797.2

1748.0

874.0

0.8

SD

349.3

SD

722.3

722.3

361.1

0.3

31

1178.0

Barium Selenate in DMSO

1.5

21

2365.0

2315.8

1157.9

1.1

32

2090.0

22

4207.0

4157.8

2078.9

2.0

33

2823.0

23

5734.0

5684.8

2842.4

2.7

34

1649.0

24

3300.0

3250.8

1625.4

1.6

35

923.0

25

1837.0

1787.8

893.9

0.9

MV

1732.6

MV

3488.6

3439.4

1719.7

1.6

SD

676.3

SD

1384.5

1384.5

692.2

0.7

76

56.0

Background Szinti and TCA

 

 

108.0

 

 

 

77

9.0

 

 

17.0

 

 

 

78

32.0

 

 

61.0

 

 

 

79

11.0

 

 

21.0

 

 

 

80

20.0

 

 

39.0

 

 

 

MV

25.6

 

MV

49.2

0.0

0.0

0.0

SD

17.2

 

SD

33.3

 

 

 

If not noted individually, the results include both lymph nodes of an animal.

POS = position in counter;  CPM = counts per minute;  Conc. = concentration;  DPM = disintegrations
per minute; SD = standard deviation; MV = mean value; Szinti = scintillation fluid; TCA = trichloro-
acetic acid


 

Radioactive Determination of the Test Substance– Main Study–Test Group 3

POS

CPM

Test Item

Conc. [%]

Animal number

DPM

DPM mean background

DPM / node

Simulation index

16

153.0

Negative control

100

401

286.0

270.2

135.1

 

17

165.0

402

309.0

293.2

146.6

 

18

310.0*

403

575.0*

n.d.

n.d.

 

19

169.0

404

313.0

297.2

148.6

 

20

173.0

405

333.0

317.2

158.6

 

MV

165.0

MV

310.3

294.5

147.2

1.0

SD

7.5

SD

16.7

16.7

8.3

 

1

177.0

Background Selenate in DMSO

3

1

338.0

322.2

161.1

1.1

2

145.0

2

274.0

258.2

129.1

0.9

3

344.0*

3

636.0*

n.d.

n.d.

n.d.

4

98.0

4

180.0

164.2

82.1

0.6

5

103.0

5

190.0

174.2

87.1

0.6

MV

130.8

MV

245.5

229.7

114.9

0.8

SD

32.3

SD

64.7

64.7

32.3

0.2

66

8.0

Background Szinti and TCA

 

 

14.0

 

 

 

67

8.0

 

15.0

 

 

 

68

8.0

 

16.0

 

 

 

69

10.0

 

19.0

 

 

 

70

8.0

 

15.0

 

 

 

MV

8.4

MV

15.8

0.0

0.0

0.0

SD

0.8

SD

1.7

 

 

 

* = outlier, failed Grubbs, Nalimov and Dixon; n.d. = not determined

If not noted individually, the results include both lymph nodes of an animal.

POS = position in counter;  CPM = counts per minute;  Conc. = concentration;  DPM = disintegrations
per minute; SD = standard deviation; MV = mean value; Szinti = scintillation fluid; TCA = trichloro-
acetic acid

Radioactive Determination of the Positive-Control Groupof the Current Study (14 March 2018)

POS

CPM

Test Item

Conc. [%]

Animal number

DPM

DPM mean background

DPM / node

Simulation index

16

153.0

Negative control

100

401

286.0

270.2

135.1

 

17

165.0

402

309.0

293.2

146.6

 

18

310.0*

403

575.0*

n.d.

n.d.

 

19

169.0

404

313.0

297.2

148.6

 

20

173.0

405

333.0

317.2

158.6

 

MV

165.0

MV

310.3

294.5

147.2

1.0

SD

7.5

SD

16.7

16.7

8.3

 

21

177.0

Background Selenate in DMSO

1

406

2360.0

2344.2

1172.1

8.0

22

145.0

407

3172.0

3156.2

1578.1

10.7

23

344.0*

408

1739.0

1723.2

861.6

5.9

24

98.0

409

1808.0

1792.2

896.1

6.1

25

103.0

410

3431.0

3415.2

1707.6

11.6

MV

130.8

MV

2502.0

2486.2

1243.1

8.4

SD

32.3

SD

692.2

692.2

346.1

2.4

66

8.0

Background Szinti and TCA

 

 

14.0

 

 

 

67

8.0

 

15.0

 

 

 

68

8.0

 

16.0

 

 

 

69

10.0

 

19.0

 

 

 

70

8.0

 

15.0

 

 

 

MV

8.4

MV

15.8

0.0

0.0

0.0

SD

0.8

SD

1.7

 

 

 

If not noted individually, the results include both lymph nodes of an animal.

POS = position in counter;  CPM = counts per minute;  Conc. = concentration;  DPM = disintegrations
per minute; SD = standard deviation; MV = mean value; Szinti = scintillation fluid; TCA = trichloro-
acetic acid

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Unclassified
Conclusions:
The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3.
Consequently, according to OECD 429 the test item Barium Selenate as described in this report is expected to have no sensitising properties and therefore should not be regarded as a dermal sensitiser.
The results of radioactivity determination are supported by the means of the lymph node weights per group, which showed no relevant difference compared to the negative control.
According to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System) the test item Barium Selenate has no obligatory labelling requirement for skin sensitisation and is unclassified.
Executive summary:

The potential for Barium Selenate to induce sensitisation was analysed using the LLNA (Local Lymph Node Assay - LLNA) method. The in vivo test system used mice; strain - CBA/CaOlaHsd, sex - female (nulliparous and non-pregnant).

The main study was started with test item concentrations of 3%, 6.25% and 12.5% based on the results observed in the pre-screen test. However, severe signs of systemic toxicity or mortality were found in all animals of the 12.5% dose group. In the 6.25% dose group two animals had to be euthanised due to animal welfare reasons and after showing severe signs of systemic toxicity. No toxic effects were observed in any animal of the 3% dose group. As the results obtained with the dose groups 6.25% and 12.5% were insufficient for skin sensitisation evaluation, they were excluded for further evaluation.

However, to provide sufficient data for skin sensitisation assessment of the test item Barium Selenate according to OECD 429, the main study was extended by two dose groups with test item concentrations of 0.75 % and 1.5%.

Accordingly, the three dose groups with test item concentrations of 0.75% (test group 1), 1.5% (test group 2) and 3% (test group 3) were used for evaluation. Stimulation indices of 0.8, 1.6 and 0.8 were obtained for the dose groups 0.75, 1.5 and 3% respectively. The EC3 value could not be calculated due to the stimulation indexes being below 3 and as such the test item is not considered to be a dermal sensitiser.