Registration Dossier

Administrative data

Description of key information

OECD TG 429 (GLP) from a structural analogue: not sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen
- Age at study initiation: 8 weeks
- Weight at study initiation: 17.1 g - 21.5 g
- Housing: single
- Diet (e.g. ad libitum): Kliba Labordiet, Kaiseraugst, Switzerland
- Water (e.g. ad libitum): tap water
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
other: DAE 4:3:3 (Dimethylacetamid 99+%, acetone and ethanol in a ratio of 4:3:3 (volume parts))
Concentration:
3, 10 and 30 %
No. of animals per dose:
6
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Several solvents were tested (acetone, DAE 4:3:3, propylene glycol, AOQ (acetone/olive oil 4:1 v%)) to establish a suitable vehicle tor the test. Finally DAE 4:3:3 (= Dimethylacetamid 99+%, acetone and ethanol in a ratio of 4:3:3 (volume parts)) was chosen as the vehicle, because good homogeneity was achieved as suspension. The highest concentration which could be prepared and applied was 30 %.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT: The study comprised three treatment groups, a vehicle control group and a concurrent untreated control group. Each group consisted of 6 mice.


TREATMENT PREPARATION AND ADMINISTRATION:
- 25 µl of the test substance per ear was applied on the dorsal part of both ears for 3 consecutive days. The animals were sacrificed on study day 5 by cervical dislocation.
- Ear and Lymph nodes (left and right auricular) weight were determined for each animal. Cell suspension were prepared form the lymph nodes and counted.
- Calculations: Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The indices of lymph node weight, cell count and ear weight were calculated as the ratio of the test group mean values for these parameters divided by those of the vehicle control group.
- Criteria used to consider a positive response: If a test substance does not show a statistically significant increase in cell count and/or lymph node weight as compared to the vehicle control in the presence of statistically significantly increased ear weights as indication of skin irritation, it is considered not to be a sensitizer. If at least one concentration tested causes a concentration dependent statistically significant increase in cell count and/ or lymph node weight without being accompanied by a statistically significant increase in ear weight, the test substance is considered to be a sensitizer. If statistically significant increases in ear weights are running in parallel to the increase in cell count and/or lymph node weight, it cannot be ruled out, that the lymph node response was caused by irritation and not by skin sensitization. Then, for identification of the relevance of the statistical evaluation, a comparison of the results to the present test to appropriate historical control values is performed. If one or a combination of the measured parameters change statistical significance, evaluation on basis of the criteria described above may be possible. If the statistical comparison with the historical control does not yield results useful for evaluation, further investigations may be necessary to differentiate between irritation and sensitization response. If a test substance does not elicit a statistical significant increase in lymph node weight and/or cell count but shows a clear concentration related increase in response, further investigating of the sensitization potential at higher concentrations should be considered.
Positive control substance(s):
other: Studies performed with positive control substance Alpha-Hexylcinnamaldehyde techn. 85% twice a year in the Iaboratory were used for evaluation
Statistics:
WILCOXON - Test for Lymph node weight, cell count and ear weight.
Parameter:
SI
Remarks on result:
other: The lymph node cell counts were only statistically increased in mice treated with the 3% test substance concentration, which do not display a concentration related change.

Lymph node weights and cell counts: Treatment of the mice with 30%, 10% and 3% test substance preparations induced no statistically significant increase in Lymph node weights as compared to the vehicle control group.

The lymph node cell counts were only statistically increased in mice treated with the 3% test substance concentration. Both parameters do not display concentration related changes.

 

TestGroup

 

Treatment

Lymph Node Weight [mg]

Mean

S.D.

Index1

0

untreated

5.3

0.6

0.97

1

vehicle DAE

5.5

0.4

1.00

2

3%in DAE

5.4

0.5

0.98

3

10% in DAE

5.4

1.2

0.99

4

30% in DAE

4.9

0.8

0.89

 

 

Test Group

 

Treatment

Cell Count [Counts/Lymph Node Pair]

Mean

S.D.

Index1

0

untreated

8,945,000

1,643,213

0.96

1

vehicle DAE

9,275,000

1,065,440

1.00

2

3%in DAE

10,837,333

1,080,936

1.17#

3

10% in DAE

11,706,667

2,867,507

1.26

4

30% in DAE

9,561,333

1,757,784

1.03

 

S.D.=Standard deviation

1test group xItest group 1 (vehicle control)

#=statistically significant for the value p 0.05

##= statistically significant for the value p 0.01

Ear weights: Treatment of the mice with 30% test substance preparations induced statistically significant increase in ear weights as compared to the vehicle control group. The ear weights of the mice treated with 10% or 3% test substance preparations were not statistically significantly increased. Due to observed rests of test substance on the ears of the mice applied with the 30% and 10% test substance concentrations, it remains unclear, whether the increased ear weights of the 30% test substance concentration were partly due to rests of test substance or fully attributable to irritating properties.

 

Test Group

 

Treatment

Ear Weight [mg]

Mean

S.D.

Index1

0

untreated

30.0

1.3

0.96

1

vehicle DAE

31.2

1.1

1.00

2

3%in DAE

32.4

1.5

1.04

3

10%in DAE

32.9

3.7

1.05

4

30% in DAE

33.9

3.0

1.09#

 

S.D.=Standard deviation

1test group x / test group 1 (vehicle control)

#=statistically significant for the value p ≤ 0.05

##= statistically significant for the value p ≤ 0.01

Body weights: The expected body weight gain was generally observed in the course of the study.

Other findings: No abnormalities were observed during general observation. Almost all ears of the animals, which were treated with the 10% and 30% test substance concentrations, exhibited yellow rests of test substance on days 1, 2 and 5. The ears of the mice, treated with the 10% and 3% test substance concentration were slight yellow or yellow colored on day 2 and 5.

Historical control data:

Animal No.

LymphnodeWeight

[mg)

Cell Counts

Ear Weight

[mg]

7

4.8

8,502,000

28.6

8

4.8

11,304,000

29.7

9

3.6

6,323,000

29.9

10

4.4

7,946,000

28.0

11

3.9

8,308,000

30.0

12

4.7

9,060,000

27.7

Conclusion: Based on the results discussed above and applying the evaluation criteria the test substance is considered not to possess a relevant sensitizing potential. This evaluation is corroborated by the lack of a concentration relation of lymph node response, especially at the 10% and 30 % concentrations and the experience, that even sensitizers with moderate potency, e.g. the positive control hexylcinnamaldehyde produce significant lymph node responses at the concentrations used in the present study.

Interpretation of results:
GHS criteria not met
Conclusions:
From the results of the study it is concluded, that the test substance does not have a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.
Executive summary:

The skin sensitizing potential of the test substance was assessed using the Murine Local Lymph Node Assay.

Groups of 6 female CBA/Ca mice each were treated with 3%, 10% and 30% w/w preparations of the test substance in DAE 4:3:3 (= NN-Dimethylacetamid 99+%, acetone and ethanol in a ratio ot 4:3:3) or with the vehicle alone. The study used 3 test groups and 2 control groups. Each test animal was applied with 25 µL per ear of the respective test substance preparation to the dorsum of both ears for three consecutive days. One control group was treated with 25 µL per ear of the vehicle alone, the other control group remained untreated to serve as a control for the immunological status of the animals.

Three days after the last application the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring the cellular content (indicator of cell proliferation) and weight of each animal's pooled lymph nodes. Moreover, a defined area with a diameter of 0.8 cm was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation.

No signs of systemic toxicity were noticed. The test substance did not induce a statistically significant and biologically relevant increase in lymph node weights when applied as 3%, 10% or 30% preparations in DAE 4:3:3. The Iymph node cell counts were only statistically increased in mice treated with the 3% test substance concentration. Both parameters do not display concentration related changes. Treatment of the mice with 30% test substance preparations induced a statistically significant increase in ear weights as compared to the vehicle control group. Due to observed rests of test substance on the ears of the mice applied with the 30% and 10% test substance

concentrations, it remains unclear, whether the increased ear weights of the 30% test substance concentration were partly due to rests of test substance or fully attributable to irritating properties.

A 30% suspension was the highest concentration of the test substance preparation suitable for application.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitization potential of the test item was not evaluated. However, reliable, experimental data of an structural analogue are available.

The skin sensitising potential of the structurally analogue substance was assessed using the Murine Local Lymph Node Assay performed according to GLP and OECD Guidelines 429 and 406. Groups of 6 female CBA/Ca mice each were treated with 25 μl per ear of 3 %, 10 % and 30 % w/w preparations of the test substance in DAE 4:3:3 (= NN-Dimethylacetamid 99+%, acetone and ethanol in a ratio of 4:3:3) or with the vehicle alone to the dorsum of both ears for three consecutive days. 2 control groups were used. One control group was treated with 25 μl per ear of the vehicle alone; the other control group remained untreated to serve as a control for the immunological status of the animals. Three days after the last application the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring the cellular content (indicator of cell proliferation) and weight of each animal's pooled lymph nodes. Moreover, a defined area with a diameter of 0.8 cm was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation. The mean indices (fold of change as compared to the vehicle control) for lymph node weight, cell count and ear weight were determined.

No signs of systemic toxicity were noticed. The test substance did not induce a statistically significant and biologically relevant increase in lymph node weights when applied as 3 %, 10 % or 30 % preparations in DAE 4:3:3. The lymph node cell counts were only statistically increased in mice treated with the 3 % test substance concentration. Both parameters do not display concentration related changes. Treatment of the mice with 30 % test substance preparations induced a statistically significant increase in ear weights as compared to the vehicle control group. Due to observed rests of test substance on the ears of the mice applied with the 30 % and 10 % test substance concentrations, it remains unclear, whether the increased ear weights of the 30 % test substance concentration were partly due to rests of test substance or fully attributable to irritating properties. A 30 % suspension was the highest concentration of the test substance preparation suitable for application.

From the results of the study it is concluded, that the test item does not have a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.

Based on the physico-chemical, structural as well as toxicoligical similarities, the same outcome is assumed for the actual substance.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008, as amended for the 13th time in Regulation (EU) No 2017/776.