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Diss Factsheets

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03-24-2020 to 06-25-2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
OECD Guideline for Testing of Chemicals No. 429, July 22, 2010
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Reaction mass of 1-Hexanol, 2-ethyl-, reaction products with 1,6-diisocyanatohexane and Hexane, 1,6-diisocyanato-, homopolymer
EC Number:
939-549-4
Molecular formula:
Unspecified
IUPAC Name:
Reaction mass of 1-Hexanol, 2-ethyl-, reaction products with 1,6-diisocyanatohexane and Hexane, 1,6-diisocyanato-, homopolymer
Specific details on test material used for the study:
Physical state / color: Liquid / colourless to slightly yellow
Storage conditions: Room temperature, under nitrogen

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd / SPF
Sex:
female
Details on test animals and environmental conditions:
Age on day 0: 8 - 12 weeks
Sex: Female
Supplier: Envigo RMS GmbH
Kreuzelweg 53
NL-5961 NM Horst
Arrival in the test facility: Acclimatization period at least 5 days before the first test
substance application
Identification: The single housed animals were identified by cage cards
Body weight on day 0: 18.6 g – 22.3 g (pretest)
17.2 g – 20.9 g (main test)

Room temperature / relative humidity: The animals were housed in fully air-conditioned rooms. Central air-conditioning guaranteed a range of 20 – 24°C for temperature and of 45 – 65% for relative humidity. There were no deviations from these ranges, which influenced the results
of the study.
Day / night rhythm: 12 h / 12 h (6.00 a.m. – 6.00 p.m. / 6.00 p.m. – 6.00 a.m.)
Type of cage: Polycarbonate cages type MII with mesh wire tops supplied by BECKER & Co., Castrop-Rauxel, Germany
Enrichment: PLEXX mouse tunnel (red, transparent) and nest building material Nestlets (Ancare, Typ NES 3600) (PLEXX b.v.; AB Elst, Netherlands)

Number of animals per cage: 1 animal
As group housing may increase oral exposure due to grooming of the animals and may interfere with clear observations of each individual animal, animals were single housed for the duration of the test.
Feeding: Mouse and rat maintenance diet “GLP”, Granovit AG, Kaiseraugst, Switzerland; ad libitum
Drinking water: Tap water ad libitum
Bedding: Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data).

Study design: in vivo (LLNA)

Vehicle:
methyl ethyl ketone
Concentration:
10% and 50% (first pretest)
0.5%, 1.0% and 2.5% (2nd pretest)
0.05%, 0.1% and 0.25% (main study)
No. of animals per dose:
5
Details on study design:
Conduct of the study
The study comprised four test groups. Each group consisted of 5 mice.
Randomization: Prior to first application, the animals were distributed to the individual groups, received animal numbers and were allocated to the respective cages according to the randomization instructions of Win Rando Version 3.2.
Body weight determination: Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
Signs and symptoms: Obvious signs of systemic toxicity and/or local inflammation at the application sites were noted for each animal in the raw data.
Mortality: A check for moribund and dead animals was made twice each workday (beginning and end) and once on Saturdays, Sundays
and on public holidays.
Form of application: Epicutaneous application is simulating dermal contact with the compound, which can occur under practical use conditions.
Application volume: 25 μL per ear
Site of application: Dorsal surface of both ears
Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site

³H-thymidine injection
On study day five (about 66 to 72 hours after the last application of test substance to the ears), about 20 μCi 3H-thymidine* in 250 μL sterile saline were injected into the tail vein of the mice.

Terminal procedures
The animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation under Isoflurane anesthesia.

Determination of ear weight: Immediately after the death of each animal, a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each animal. These measurements serve for
detecting a potential inflammatory ear swelling.
Removal and weight determination of the lymph nodes:
Immediately after removal of the ear punches, the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.
Preparation of cell suspension and determination of cell count:
After weight determination, the pooled lymph nodes of each animal were stored in phosphate-buffered saline (PBS) in an ice
water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully
passing the lymph nodes through an iron mesh (mesh size 200 μm) into 8 mL phosphate-buffered physiological saline. For
determination of cell counts, an aliquot of each suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was
determined using a Casy® Counter.

Measurement of 3H-thymidine incorporation of the lymph node cells:
The remaining cell suspensions were washed twice with PBS and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate was transferred to scintillation fluid and incorporation of 3H-thymidine into the cells was measured in a β-scintillation counter.

Data evaluation
Table(s) and/or figure(s) of measured parameters presented in the report were produced using a PC-based tabular calculation software. The mean and individual data were not always rounded, but the significant digits were produced by changing the display format.
Consequently, calculation of mean values by using the individual data presented in the report will in some instances yield minor variations in value.

Positive control substance(s):
other: Alpha-Hexylcinnamaldehyde, techn. 85%
Statistics:
Mean values and standard deviations of the measured parameters were calculated per test group from the individual values. The stimulation indices of 3H-thymidine incorporation, cell count, lymph node weight and ear weight measurements were calculated by dividing the mean values per test group and/or single animal values by the mean of the vehicle treated group.
Statistical analysis of 3H-Thymidine incorporation, cell count, lymph node weight and ear weight were performed by the WILCOXON test.

Results and discussion

Positive control results:
Result of last run including a periodic positiv control:
1%, 5%, 15% Alpha-Hexylcinnamaldehyde, techn. 85%
Stimulation Index (3H-Thymidine incorporation): 3.06, 7.70 and 11.13 (ratio of test group values to control group values >=3.0 indicates a positive result.
Stimulation index (cell counts): 1.59, 2.26 and 3.00
EC 3: ca. 1
EC 1.5: ca. 1

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks:
3H-thymidine incorporation
Value:
1
Test group / Remarks:
vehicle MEK
Parameter:
SI
Remarks:
3H-thymidine incorporation
Value:
2.55
Test group / Remarks:
0.05% in MEK
Parameter:
SI
Remarks:
3H-thymidine incorporation
Value:
9.1
Test group / Remarks:
0.10% in MEK
Parameter:
SI
Remarks:
3H-thymidine incorporation
Value:
22.44
Test group / Remarks:
0.25% in MEK
Parameter:
SI
Remarks:
cell counts
Value:
1
Test group / Remarks:
vehicle MEK
Parameter:
SI
Remarks:
cell counts
Value:
1.55
Test group / Remarks:
0.05% in MEK
Parameter:
SI
Remarks:
cell counts
Value:
2.24
Test group / Remarks:
0.10% in MEK
Parameter:
SI
Remarks:
cell counts
Value:
3.53
Test group / Remarks:
0.25% in MEK
Parameter:
EC3
Remarks:
3H-thymidine incorporation
Value:
0.053
Remarks on result:
other: %
Parameter:
other: EC1.5
Remarks:
cell count
Value:
0.048
Remarks on result:
other:
Remarks:
%

Any other information on results incl. tables

Pretest / Irritation Screening

In the pretest two mice each were treated with test-substance concentrations of 10% and 50% (1st pretest) and 0.5%, 1.0% and 2.5% (2nd pretest) on three consecutive days. Overall, no relevant signs of systemic toxicity were observed in the pretests. However, reduced general condition was observed in the animals treated with the 10% and 50% concentrations on study day 2.

After application of the 10% and 50% concentrations, the animals showed severe increases (>25%) in ear weights (compared to historical vehicle values) and ear thickness measurements as indication of excessive ear skin irritation. In addition, well-defined erythema (day 2) and slight scales on the ear skin (day 5) were observed at the 10% concentration. Moderate to severe erythema (day 2) and moderate scales on the ear skin (day 5) were noted in animals treated with the 50% concentration. After treatment with the 1.0% and 2.5% concentrations, the animals showed severe increases (≥25%) in ear thickness measurements as indication of excessive ear skin irritation. Slight scales on the ear skin were observed at the 2.5% concentration on study day 5. The 0.5% concentration did not show relevant signs of local ear skin irritation. Considerably increased lymph node weights were observed in all concentrations without a clear dose-response relationship.

 

Test Group

 

Treatment

 

Animal No.

Lymph Node Weight

[mg/Lymph Node Pair]

 

Mean

 

S.D.

 

Stimulation Index1(SI) per animal

 

Stimulation Index1(SI) per test group

 

vehicle

historical control

 

 

5.3

0.3

 

1.00

 

1

 

10% in MEK

1

2

23.8

17.5

20.7

4.5

4.49

3.30

3.90

 

2

 

50% in MEK

3

4

22.1

21.8

22.0

0.2

4.17

4.11

4.14

 

3

 

0.5% in MEK

5

6

17.7

18.1

17.9

0.3

3.34

3.42

3.38

 

4

 

1.0% in MEK

7

8

19.6

20.9

20.3

0.9

3.70

3.94

3.82

 

5

 

2.5% in MEK

9

10

19.5

24.7

22.1

3.7

3.68

4.66

4.17

 

Test Group

 

Treatment

 

Animal No.

EarWeight

[mg/animal]

 

Mean

 

S.D.

 

Stimulation Index1(SI) per animal

 

Stimulation Index1(SI) per test group

 

vehicle

historical control

 

 

30.9

1.3

 

1.00

 

1

 

10% in MEK

1

2

38.0

40.4

39.2

1.7

1.23

1.31

1.27

 

2

 

50% in MEK

3

4

42.9

41.8

42.4

0.8

1.39

1.35

1.37

 

3

 

0.5% in MEK

5

6

30.4

31.9

31.2

1.1

0.98

1.03

1.01

 

4

 

1.0% in MEK

7

8

34.0

33.5

33.8

0.4

1.10

1.08

1.09

 

5

 

2.5% in MEK

9

10

35.9

36.9

36.4

0.7

1.16

1.19

1.18

1 test group x / vehicle MEK (historical control)

 

Test Group

 

Treatment

 

Animal No.

Ear Thickness [µm]

d 0

d 2

d 5

left

right

Mean

left

right

Mean

left

right

Mean

 

1

 

10% in MEK

1

2

200

205

200

200

200

203

260

250

260

250

260

250

270

280

270

280

270

280

 

2

 

50% in MEK

3

4

200

200

200

200

200

200

270

260

270

260

270

260

340

360

350

360

345

360

 

3

 

0.5% in MEK

5

6

200

205

200

205

200

205

225

225

225

225

225

225

240

245

235

240

238

243

 

4

 

1.0% in MEK

7

8

200

200

200

200

200

200

225

225

225

225

225

225

250

250

250

255

250

253

 

5

 

2.5% in MEK

9

10

205

200

205

200

205

200

245

240

245

245

245

243

270

275

265

270

268

273

 

Test Group

 

Treatment

 

Animal No.

Difference d 0 to d 2

[µm]

Ear swelling

d 2 (%)

Difference d 0 to d 5

[µm]

Ear

swelling d 5 (%)

 

1

 

10% in MEK

1

2

60

48

30

23

70

78

35

38

 

2

 

50% in MEK

3

4

70

60

35

30

145

160

73

80

 

3

 

0.5% in MEK

5

6

25

20

13

10

38

38

19

18

 

4

 

1.0% in MEK

7

8

25

25

13

13

50

53

25

26

 

5

 

2.5% in MEK

9

10

40

43

20

21

63

73

30

36

 

Test Group

 

Treatment

 

Animal No.

Body Weight d0 [g]

Body Weight d5 [g]

Body Weight Change d5-d0 [g]

Individual

Mean

S.D.

Individual

Mean

S.D.

Individual

Mean

S.D.

 

1

 

10% in MEK

1

2

18.9

18.6

18.8

0.2

19.9

19.2

19.6

0.5

1.0

0.6

0.8

0.3

 

2

 

50% in MEK

3

4

21.3

19.3

20.3

1.4

20.5

19.3

19.9

0.8

-0.8

0.0

-0.4

0.6

 

3

 

0.5% in MEK

5

6

20.1

18.7

19.4

1.0

19.9

19.8

19.9

0.1

-0.2

1.1

0.4

0.9

 

4

 

1.0% in MEK

7

8

20.0

22.1

21.1

1.5

19.8

21.9

20.9

1.5

-0.2

-0.2

-0.2

0.0

 

5

 

2.5% in MEK

9

10

22.3

21.0

21.7

0.9

23.3

21.9

22.6

1.0

1.0

0.9

0.9

0.1

Nature and duration of clinical findings of individual animals:

Test Group

1

Treatment

10% in MEK

Animal Numbers

1

2

reduced general condition

d2

d2

 

Test Group

2

Treatment

50% in MEK

Animal Numbers

3

4

reduced general condition

d2

d2

 

Test Group

3

Treatment

0.5% in MEK

Animal Numbers

5

6

 

-

-

 

Test Group

4

Treatment

1.0% in MEK

Animal Numbers

7

8

 

-

-

 

Test Group

5

Treatment

2.5% in MEK

Animal Numbers

9

10

 

-

-

 

Nature and duration of local findings of individual animals

Test Group

1

Treatment

10% in MEK

Animal Numbers

1

2

well-defined erythema

d2

d2

scales on the ear skin, slight

d5

d5

 

Test Group

2

Treatment

50% in MEK

Animal Numbers

3

4

moderate to severe erythema

d2

d2

scales on the ear skin, moderate

d5

d5

 

Test Group

3

Treatment

0.5% in MEK

Animal Numbers

5

6

 

-

-

 

Test Group

4

Treatment

1.0% in MEK

Animal Numbers

7

8

 

-

-

 

Test Group

5

Treatment

2.5% in MEK

Animal Numbers

9

10

scales on the ear skin, slight

d5

d5

 

Main test

3H-thymidine incorporation, cell count and lymph node weight

When applied as 0.10% and 0.25% concentrations in MEK, the test substance induced biologically relevant (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3) and concentration-dependent increases of 3H-thymidine incorporation into the cells from the auricular lymph nodes. The increase of the 0.05% concentration failed to reach the cutoff value. The increases of 3H-thymidine incorporation were statistically significant at all concentrations.

Concomitantly, all concentrations induced biologically relevant and statistically significant responses (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. The increase of the 0.05% concentration lies at the border of of biological relevance. In addition, statistically significant increases in lymph node weights were noted at all concentrations.

 

 

Test Group

 

Treatment

³H-thymidine incorporation [DPM/Lymph Node Pair]

Mean

S.D.

Stimulation Index1

1

vehicle MEK

120.9

45.4

1.00

 

2

0.05% in MEK

308.1

59.2

2.55

##

3

0.10% in MEK

1,099.6

340.3

9.10

##

4

0.25% in MEK

2,712.2

445.9

22.44

##

 

 

 

Test Group

 

Treatment

Cell Counts [Counts/Lymph Node Pair]

Mean

S.D.

Stimulation Index1

1

vehicle MEK

9,952,800

1,624,907

1.00

 

2

0.05% in MEK

15,420,800

1,836,718

1.55

##

3

0.10% in MEK

22,309,600

4,161,851

2.24

##

4

0.25% in MEK

35,161,600

7,514,526

3.53

##

 

 

 

Test Group

 

Treatment

Lymph Node Weight [mg/Lymph Node Pair]

Mean

S.D.

Stimulation Index1

1

vehicle MEK

5.2

0.9

1.00

 

2

0.05% in MEK

7.0

1.0

1.34

##

3

0.10% in MEK

9.3

1.3

1.77

##

4

0.25% in MEK

14.3

0.8

2.73

##

1vs. mean of test group 1 (vehicle control)

## = statistically significant for the value p ≤ 0.01

Ear weights

The test-substance concentrations did not cause any increases in ear weights (no increase to stimulation index (SI) of ≥ 1.25), demonstrating the absence of relevant ear skin irritation.

 

Test Group

 

Treatment

Ear Weight [mg/animal]

Mean

S.D.

Stimulation Index1

1

vehicle MEK

31.1

1.8

1.00

2

0.05% in MEK

30.0

1.1

0.97

3

0.10% in MEK

30.6

0.9

0.98

4

0.25% in MEK

31.4

1.3

1.01

1 vs. mean of test group 1 (vehicle control)

## = statistically significant for the value p ≤ 0.01

Body weights

The expected mean body weigh gain was generally observed in all test groups during the study period.

Test Group

Treatment

Animal No.

Body Weight d0 [g]

Body Weight d5 [g]

Body Weight Change d5-d0 [g]

Individual

Mean

S.D.

Individual

Mean

S.D.

Individual

Mean

S.D.

 

 

1

17.7

 

 

19.1

 

 

1.4

 

 

 

 

2

17.2

 

 

17.8

 

 

0.6

 

 

1

vehicle MEK

3

19.5

18.8

1.4

19.5

19.1

1.1

0.0

0.2

0.8

 

 

4

19.2

 

 

18.4

 

 

-0.8

 

 

 

 

5

20.6

 

 

20.6

 

 

0.0

 

 

 

 

6

20.0

 

 

21.2

 

 

1.2

 

 

 

 

7

19.6

 

 

19.4

 

 

-0.2

 

 

2

0.05% in MEK

8

19.4

19.3

1.0

20.3

19.7

1.3

0.9

0.4

0.6

 

 

9

17.5

 

 

17.7

 

 

0.2

 

 

 

 

10

19.9

 

 

19.8

 

 

-0.1

 

 

 

 

11

19.8

 

 

20.9

 

 

1.1

 

 

 

 

12

18.7

 

 

19.4

 

 

0.7

 

 

3

0.10% in MEK

13

19.2

19.3

0.5

20.1

20.2

0.9

0.9

0.9

0.4

 

 

14

19.1

 

 

19.4

 

 

0.3

 

 

 

 

15

19.9

 

 

21.4

 

 

1.5

 

 

 

 

16

19.2

 

 

21.0

 

 

1.8

 

 

 

 

17

20.4

 

 

22.1

 

 

1.7

 

 

4

0.25% in MEK

18

18.1

19.5

1.1

19.0

20.7

1.1

0.9

1.1

0.7

 

 

19

20.9

 

 

21.0

 

 

0.1

 

 

 

 

20

19.0

 

 

20.2

 

 

1.2

 

 

Clinical signs

No signs of systemic toxicity were noticed in all animals during general observation.

Local findings

No local findings were observed during the observation period.

Applicant's summary and conclusion

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
Basonat HA 3000 exhibits a skin sensitizing potential in the Local Lymph Node Assay under the test conditions chosen.
Executive summary:

The skin sensitizing potential of Basonat HA 3000 was assessed using the radioactive Local Lymph Node Assay. The assay simulates the induction phase for skin sensitization in mice. It determines the response of cells in the auricular lymph nodes to repeated application of the test substance to the dorsal skin of the ears. Groups of 5 female CBA/CaOlaHsd mice each were treated with 0.05%, 0.10% and 0.25% (w/w) preparations of the test substance in methyl ethyl ketone (MEK) or with the vehicle alone. Each test animal was treated with 25 μL per ear of the appropriate test-substance preparation or the vehicle alone, applied to the dorsal surfaces of both ears on three consecutive days. Three days after the last application, about 20 μCi 3H-thymidine in 250 μL sterile saline were

injected into the tail vein of the mice. About 5 hours after the 3H-thymidine injection, the mice were sacrificed, and the auricular lymph nodes were removed. Lymph node response was evaluated measuring 3H-thymidine incorporation (indicator of cell proliferation). The cell count and weight of each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear, and for each animal, the weight of the pooled punches was determined to obtain an indication of possible ear skin irritation. No signs of systemic toxicity or irritation were noticed in all animals during general observation.

When applied as 0.10% and 0.25% concentrations in MEK, the test substance induced biologically relevant (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3) and concentration-dependent increases of 3H-thymidine incorporation into the cells from the auricular lymph nodes. The increase of the 0.05% concentration failed to reach the cutoff value. The increases of 3H-thymidine incorporation were statistically significant at all concentrations. Concomitantly, all concentrations induced biologically relevant and statistically significant responses (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. The increase of the 0.05% concentration lies at the border of of biological relevance. In addition, statistically significant increases in lymph node weights were noted at all concentrations.

The test-substance concentrations did not cause any increases in ear weights (no increase to stimulation index (SI) of ≥ 1.25), demonstrating the absence of relevant ear skin irritation.

Thus, it is concluded that Basonat HA 3000 exhibits a skin sensitizing potential in the Local Lymph Node Assay under the test conditions chosen. The threshold concentration for sensitization induction was around 0.05%. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation was calculated by linear regression from the results of the 0.05% and 0.10% concentrations to be 0.053%. The EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by semi-logarithmical regression from the results of the 0.05% and 0.10%

concentrations to be 0.048%.