Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-01-04 to 2013-04-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
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: Carbamic acid, N-(3-(triethoxysilyl)propyl)2-(ethyl-(4-nitrosophenyl) amino) ethyl ester
- Internal code: SAT 120009
- Batch No. 12/34#
- CAS No.: 1195231-94-7
- Purity: 92.8 wt% (NMR), 93.3 area% (HPLC)

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental 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 (lot no. 0636)
- 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 (lot no. 011012)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least five days) under laboratory conditions

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Based on the results observed in the preliminary test the following test item concentrations were selected for the main study:
12.5%, 25% and 50% (w/v).
The preparations were made immediately prior to each dosing.
AOO was used as vehicle and served as negative control.
No. of animals per dose:
5 mice / group
3 mice / prescreen test
Details on study design:
PRESCREEN TEST:
Before the initiation of the prescreen test, a solubility test was performed to define the vehicle and the maximum concentration which is technically
applicable to the animals. The maximum technically applicable concentration of the test item in the vehicle was found to be 50% in AOO (Acetone,
Merck, lot no. 42506714, expiry date: 07/2016; olive oil highly refined, Sigma, lot no. BCBH9319V, expiry date:03/2013).
In order to determine the highest tolerated and not excessively irritant test concentration a prescreen test was performed which was conducted
under the same conditions as the main study, except there was no assessment of lymph node proliferation. The mice were observed daily for any
clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to termination. Both ears
were observed for erythema and scored according to Table 1. Ear thickness measurements were performed on day 1 (pre-dose), day 3
(approximately 48 hours after the first dose) and day 6. Excessive local irritation was indicated by an erythema score ≥ 3 and/or ear swelling of
≥ 25%. Two animals were treated by topical application with the test item on three consecutive days at a concentration of 50% (suspended with AOO) to the entire dorsal surface of each ear. One further animal was treated with 100% AOO and served as negative control.
Immediately before the first application, approximately 48 hours after the first application and shortly before sacrificing the thickness of both ears of all animals was measured. During this period also all clinical signs were recorded.
Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge).
Neither signs of systemic toxicity nor signs of irritation at the application site could be detected in any animal.

PREPARATION OF ANIMALS:
The animals were randomly selected.
Identification was ensured by cage number and individual marking (tail).

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).

DOSE GROUPS:
3 test groups (3 different concentrations) and 1 negative control group (vehicle) were tested.

TEST REGIME:
- 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.
- 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 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, 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
Statistics:
Outlier tests according to Dixon, Grubbs and Nalimov were performed for the values measured for the number of disintegrations per minute (DPM).
If outliers were identified, these values were not included in the calculation of the stimulation indices. As at least four values per group are required for the
evaluation of the results, the outlier test was not repeated to detect further outliers.

Results and discussion

Positive control results:
The recent reliability check was performed in January 2013. The raw data of this study are kept in the BSL archives (BSL Project ID 125865 J).
- Positive-control substance: P-Phenylenediamine (CAS 106-50-3, Sigma, purity > 98%; Lot 041M0045V) 1%
- Vehicle:AOO (4:1 (v/v) acetone/olive oil)
- Species/strain: healthy CBA/CaOlaHsd mice
- Source: Harlan Winkelmann GmbH, 33178 Borchen, Germany
- Concentrations: 1% on three consecutive days.
The Stimulation Index was 14.8

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Each of the three tested concentrations exceeded the stimulation index of 3: - The stimulation index at a concentration of 12.5% was 41.0 - The stimulation index at a concentration of 25% was 35.9 - The stimulation index at a concentration of 50% was 32.0. All animals survived throughout the test period without showing any clinical signs. All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see: Any other information on results

Any other information on results incl. tables

TABLE: EAR THICKNESS - PRELIMINARY TEST (mm)

 

 

Concentration

 

Animal No.

Measurement of Ear Thickness (mm)

Day 1

Day 3

Day 6

left

right

left

right

left

right

SAT 120009 50% in AOO

1

0.19

0.19

0.18

0.19

0.18

0.20

SAT 120009 50% in AOO

2

0.20

0.19

0.19

0.19

0.19

0.20

Negative Control 100% AOO

3

0.18

0.19

0.17

0.18

0.19

0.18

 

TABLE: ABSOLUTE BODY WEIGHTS - PRELIMINARY TEST (g)

 

Concentration

Animal No.

Start of Study

End of Study

Weight Gain

SAT 120009 50% in AOO

1

22

21

-1

SAT 120009 50% in AOO

2

23

24

1

Negative Control 100% AOO

3

23

24

1

 

TABLE: RADIOACTIVE DETERMINATION OF THE POSITIVE CONTROL GROUP OF THE RECENT STUDY

 

POS

CPM

Test Item

Conc. [%]

Animal No

DPM

DPM - mean background

DPM/ Node

Stimulation Index

11

445.0

Negative

Control

 

11

930.0

913.6

456.8

 

12

284.0

 

12

587.0

570.6

285.3

 

13

664.0

 

13

1390.0

1373.6

686.8

 

14

678.0

 

14

1399.0

1382.6

691.3

 

15

432.0

 

15

896.0

879.6

439.8

 

MV

500.6

 

MV

1040.4

1024.0

512.0

1.0

SD

150.3

 

SD

312.9

312.9

156.4

 

16

9739.0

Phenylene-

diamine

1

16

20255.0

20238.6

10119.3

19.8

17

6607.0

 

17

13762.0

13745.6

6872.8

13.4

18

5829.0

 

18

12139.0

12122.6

6061.3

11.8

19

8307.0

 

19

17321.0

17304.6

8652.3

16.9

20

5884.0

 

20

12214.0

12197.6

6098.8

11.9

MV

7273.2

 

MV

15138.2

15121.8

7560.9

14.8

SD

1523.7

 

SD

3174.5

3174.5

1587.2

3.1

41

9.0

Background

Szinti and

TCA

 

 

18.0

 

 

 

42

6.0

 

 

13.0

 

 

 

43

8.0

 

 

17.0

 

 

 

44

8.0

 

 

17.0

 

 

 

45

8.0

 

 

17.0

 

 

 

MV

7.8

 

MV

16.4

0.0

0.0

0.0

SD

1.0

 

SD

1.7

 

 

 

 

TABLE: RADIOACTIVE DETERMINATION OF THE TEST SUBSTANCE GROUPS

 

POS

CPM

Test Item

Conc. [%]

Animal No

DPM

DPM - mean background

DPM/ Node

Stimulation Index

16

814.0

Negative

 Control

 

16

1657.0

1641.8

820.9

 

17

1949.0*

 

17

3987.0*

n.d.

n.d.

 

18

718.0

 

18

1466.0

1450.8

725.4

 

19

300.0

 

19

610.0

594.8

297.4

 

20

493.0

 

20

1004.0

988.8

494.4

 

MV

581.3

 

MV

1184.3

1169.1

584.5

1.0

SD

199.9

 

SD

407.8

407.8

203.9

 

1

18209.0

SAT 120009

in AOO

12.5

1

37579.0

37563.8

18781.9

32.1

2

25951.0

 

2

53908.0

53892.8

26946.4

46.1

3

31296.0

 

3

64897.0

64881.8

32440.9

55.5

4

19627.0

 

4

40986.0

40970.8

20485.4

35.0

5

20662.0

 

5

42472.0

42456.8

21228.4

36.3

MV

23149.0

 

MV

47968.4

47953.2

23976.6

41.0

SD

4841.9

 

SD

10087.2

10087.2

5043.6

8.6

6

12174.0**

SAT 120009

in AOO

25

6

25074.0**

n.d.

n.d.

n.d.

7

17854.0

 

7

36791.0

36775.8

18387.9

31.5

8

21924.0

 

8

45941.0

45925.8

22962.9

39.3

9

22412.0

 

9

46350.0

46334.8

23167.4

39.6

10

18872.0

 

10

38942.0

38926.8

19463.4

33.3

MV

20265.5

 

MV

42006.0

41990.8

20995.4

35.9

SD

1943.9

 

SD

4211.3

4211.3

2105.6

3.6

11

19667.0

SAT 120009

in AOO

50

11

40682.0

40666.8

20333.4

34.8

12

26146.0*

 

12

54469.0*

n.d.

n.d.

n.d.

13

17835.0

 

13

37189.0

37173.8

18586.9

31.8

14

16710.0

 

14

34243.0

34227.8

17113.9

29.3

15

18274.0

 

15

37516.0

37500.8

18750.4

32.1

MV

18121.5

 

MV

37407.5

37392.3

18696.2

32.0

SD

1059.0

 

SD

2280.1

2280.1

1140.1

2.0

56

6.0

Background

Szinti and

TCA

 

 

12.0

 

 

 

57

8.0

 

 

17.0

 

 

 

58

8.0

 

 

16.0

 

 

 

59

10.0

 

 

20.0

 

 

 

60

5.0

 

 

11.0

 

 

 

MV

7.4

 

MV

15.2

0.0

0.0

0.0

SD

1.7

 

SD

3.3

 

 

 

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

 

TABLE: ABSOLUTE BODY WEIGHTS (g)

 

Concentration

Animal No.

Start of Study

End of Study

Weight Gain

SAT 120009
12.5% in AOO

1

19

19

0

2

21

21

0

3

21

22

1

4

21

22

1

5

17

17

0

SAT 120009
25% in AOO

6

17

18

1

7

23

23

0

8

18

18

0

9

18

19

1

10

17

19

2

SAT 120009
50% in AOO

11

21

23

2

12

19

21

2

13

20

23

3

14

16

17

1

15

17

19

2

 

Negative

Control

100% AOO

 

16

17

18

1

17

20

21

1

18

20

20

0

19

21

23

2

20

21

23

2

 

TABLE: CLINICAL OBSERVATION

 

Time of Observation

Systemic Effects

Local Effects

Group 1, animals no. 1-5 / test item at a concentration of 12.5% in AOO

Day 1

nsf

nsf

Day 2

nsf

nsf

Day 3

nsf

nsf

Day 4

nsf

nsf

Day 5

nsf

nsf

Day 6

nsf

nsf

Group 2, animals no. 6-10 / test item at a concentration of 25% in AOO

Day 1

nsf

nsf

Day 2

nsf

nsf

Day 3

nsf

nsf

Day 4

nsf

nsf

Day 5

nsf

nsf

Day 6

nsf

nsf

Group 3, animals no. 11-15 / test item at a concentration of 50% in AOO

Day 1

nsf

nsf

Day 2

nsf

nsf

Day 3

nsf

nsf

Day 4

nsf

nsf

Day 5

nsf

nsf

Day 6

nsf

nsf

Group 4, animals no. 16-20 / negative control AOO

Day 1

nsf

nsf

Day 2

nsf

nsf

Day 3

nsf

nsf

Day 4

nsf

nsf

Day 5

nsf

nsf

Day 6

nsf

nsf

 

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The EC3 value (derived by linear interpolation) could not be calculated. Each of the tested concentrations exceeded the stimulation index of 3.
Consequently, according to OECD 429 the test item SAT 120009 is considered to be a dermal sensitiser.
Since it was not possible to calculate the EC3 value, the test item cannot be classified into any of the sub-categories (category 1A or B) according to Commission Regulation (EU) No 286/2011 and UN-GHS (Globally Harmonized Classification System). Therefore, according to Commission Regulation (EU) No 286/2011 and UN GHS , the test item SAT 120009 is classified into Category 1 and has an obligatory labelling requirement for skin sensitisation.
Executive summary:

SUMMARY

On the basis of the test results given below and in conformity with the criteria given inCommission Regulation (EU) No 286/2011 (CLP Regulation) the substance should be:

classified into category 1

X

unclassified

 

On the basis of the test results given below and in conformity with the criteria given in UN-GHS (Globally Harmonized Classification System) the substance should be:

classified into category 1

X

unclassified

 

Based on the results of the prescreen test the test item was assessed for sensitising properties at concentrations of 12.5%, 25% and 50% (w/v), each diluted with AOO 4:1 (v/v).

At the daily clinical observation the animals did not show any visible clinical symptoms and no case of mortality was observed.

Species/strain: Mice, CBA/CaOlaHsd

Number of animals:  20/main test

Vehicle: AOO (4:1 (v/v) acetone/olive oil)

Summary Results

Each of the three tested concentrations exceeded the stimulation index of 3.

The stimulation index at a concentration          of       12.5% was     41.0

The stimulation index at a concentration          of       25%    was     35.9

The stimulation index at a concentration          of       50%    was     32.0

Conclusion

The EC3 value (derived by linear interpolation) could not be calculated.

Each of the tested concentrations exceeded the stimulation index of 3. Consequently, according to OECD 429 the test item SAT 120009 is considered to be a dermal sensitiser.

Since it was not possible to calculate the EC3 value, the test item cannot be classified into any of the sub-categories (category 1A or B) according to Commission Regulation (EU) No 286/2011 and UN-GHS (Globally Harmonized Classification System). Therefore, according to Commission Regulation (EU) No 286/2011 and UN GHS , the test item SAT 120009 is classified into Category 1 and has an obligatory labelling requirement for skin sensitisation.