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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:
23 - 29 Nov 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
OGYÉI, Országos Gyógyszerészeti és Élelmezés-egészségügyi Intézet, Budapest, Hungary
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
3,5-di-tert-butyl-4-hydroxybenzyl alcohol
EC Number:
201-815-6
EC Name:
3,5-di-tert-butyl-4-hydroxybenzyl alcohol
Cas Number:
88-26-6
Molecular formula:
C15H24O2
IUPAC Name:
2,6-di-tert-butyl-4-(hydroxymethyl)phenol
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, dry conditions. Protect from heat and direct sunlight

FORM AS APPLIED IN THE TEST (if different from that of starting material)
liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
Ola Hsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop ZRT., Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 11 weeks
- Weight at study initiation: 18.4 - 21.7 g
- Housing: 4 animals per cage, in Type II. Polypropylene / polycarbonate cages with deep wood sawdust bedding
- Diet: ssniff® Rat/Souris-Elevage E complete diet for rats and mice (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 14 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 – 70
- Photoperiod (hrs dark / hrs light): 12 / 12
- IN-LIFE DATES: From: 23 To: 29 Nov 2016

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
2.5, 5, 10 and 25 %
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
In the pre-screening test, three concentrations (5, 10 and 25% dissolved in dimethylformamide (DMF)) were selected, and 25 μl of each dose formulation were applied to the ears of each animal, 2 mice for each concentration, once a day for 3 consecutive days. Animals were observed for clinical signs of systemic toxicity and local irritation at the application site. Furthermore, body weights and ear thickness measurements were performed (before initial application and on Day 6, ear thickness was additionally measured on Day 3). None of the animals showed any abnormalities or any signs of signifcant irritation (indicated by an erythema score ≥ 3 and /or an increase of more than 25% in ear thickness). 1 out of 2 animals of the 5% treatment group showed a body weight decrease of 9%. Due to the incidental finding of the effect on body weight in only that dose group, this effect was not considered as treatment related. No mortality was observed.
Based on these results and considering that 25% was the maximum attainable concentration, 25 % (w/v) was selected as the highest test concentration for the main study. This concentration was expected not to induce systemic toxicity, nor to induce an increase in ear thickness exceeding 25% or to induce dermal erythema with a score of 3 or more, or more than 5% body weight loss.

- Compound solubility: 25% (maximum attainable concentration)
- Irritation: no
- Systemic toxicity: no
- Ear thickness measurements: yes, less than 25% increase in ear thickness
- Erythema scores: 0 (each test group, each time point)

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by ß-scintillation
- Criteria used to consider a positive response: A substance is regarded as a sensitizer in the LLNA, if the Stimulation Index (SI) is equal to or exceeding 3.

TREATMENT PREPARATION AND ADMINISTRATION:
25 μl of each dose formulation were applied to the dorsal skin of each ear of each animal once a day (using a MICROMAN (Model M100, Gilson)) for 3 consecutive days. On day 6 20 μCi 3H-methyl thymidine, contained in 250 μL of 1 x PBS (= 80 μCi/mL) was administered intravenously to each mouse via the tail vein with 1 mL sterile syringes. 5 h (± 30 minutes) after administration local lymph nodes were collected, minced, washed and pooled. The pooled lymph node cells (LNC) were treated with 3 mL of 5% Trichloroacetic acid (TCA) at 2 - 8 °C overnight (approximately 18 h) before determination of the ammount of 3H-methyl thymidine incorporation on day 7 (measured by Tri-Carb 3100TR, Liquid Scintillation Analyzer).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Mean values and standard deviations were calculated.

Results and discussion

Positive control results:
The positive control substance (25% hexyl cinnamic aldehyde (Batch number: MKBS3936V, SIGMA-ALDRICH, Germany) in AOO) induced a positive reaction, determined by a DPM/animal of 10966.4 compared to 804.4 DPM/animal in the vehicle control group, leading to a SI of 13.6. No abnormal clinical signs, erythema on the ears or body weight changes were observed. The lymph nodes were however much larger than those in the respective vehicle control group.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.7
Test group / Remarks:
2.5 % test group
Key result
Parameter:
SI
Value:
3.2
Test group / Remarks:
5 % test group
Remarks on result:
other: incidental finding, not treatment-related (no dose-dependency)
Key result
Parameter:
SI
Value:
1.6
Test group / Remarks:
10 % test group
Key result
Parameter:
SI
Value:
1.4
Test group / Remarks:
25 % test group
Parameter:
SI
Value:
13.6
Test group / Remarks:
positive control group
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA:
No significant lymphoproliferation (SI ≥ 3) was observed for the test item at treatment concentrations of 2.5, 10 and 25 %(w/v). A borderline result was observed in the 5% (w/v) test group. Due to missing dose-response, the increase SI value was considered as incidental and not treatment-related. Appearance of the lymph nodes was normal in both vehicle control groups and in the test groups.

DETAILS ON STIMULATION INDEX CALCULATION: SI = DPM/mouse of a treated group divided by the DPM/mouse of the respective negative control group (DPM/mouse = Group DPM/4; Group DPM = measured DPM group - average DPM background; Average DPM background = 25.5)

EC3 CALCULATION: Based on the obtained results no linear regression (using SI values) was performed for dose-response analysis and no EC3 value of the test item was calculated.

CLINICAL OBSERVATIONS:
No mortality or symptoms of systemic toxicity were observed in any treatment group. No signs of irritation (indicated by an erythema score ≥ 3) or any other local effect were observed in any treatment group. Lymph node appearance was not altered compared to that of the vehicle control group. Topical application of the test substance led to a slight increase in the mean DPM values of pooled lymph nodes. The following values (DPM/mouse) were obtained: 499.1, 848.1, 1574.1, 820.6 and 714.6 in vehicle control (DMF), 2.5, 5, 10 and 25% test groups.

BODY WEIGHTS:
Body weights decreased by ≥ 5 % were observed in the hexyl cinnamic aldehyde (HCA) group (1/4 animals, -6%), in the DMF group (2/4 animals, -7 % or -5 %), in the 5 % (w/v) test group (1/4 animals, -11 %) and in the 2.5 % (w/v) test group (2/4 animals, -8 % or -5 %). The observed effects were not dose-related and thus considered as significant only in one case (5 % (w/v) test group).

Any other information on results incl. tables

Table 1: Stimulation index in mice after application of the vehicles (AOO and DMF), test substance (2.5, 5, 10, 25% in DMF) or positive control substance (25% HCA in AOO)

Compound

Concentration [%]

DPM/ mouse

Stimulation index

Judgement

DMF

100

499.1

1.0

-

Test substance

2.5

848.1

1.7

Negative

5

1574.1

3.2

Positive, but incidential

10

820.6

1.6

Negative

25

714.6

1.4

Negative

AOO

100

804.4

1.0

-

HCA

25

10966.4

13.6

Positive

AOO = Acetone: Olive oil 4:1 (v/v) mixture

DMF = N,N -Dimethylformamide

HCA = Hexyl cinnamic aldehyde

- = Not applicable

Table 2: Body weight after application of the vehicle (AOO/DMF), test substance (2.5, 5, 10, 25% in DMF) or positive control substance (25% HCA in AOO)

Compound

Concentration [%]

Animal ID No.

Body weight

Day 1 [g]

Day 6 [g]

Change [%]

DMF

100

1

19.6

19.7

1

2

21.4

19.8

-7

3

18.9

18.8

-1

4

20.8

19.7

-5

Mean ± SD

20.2 ± 1.1

19.5 ± 0.5

-3

Test substance

2.5

5

20.5

18.8

-8

6

21.1

21.1

0

7

19.5

21.0

8

8

21.7

20.6

-5

Mean ± SD

20.7 ± 0.9

20.4 ± 1.1

-2

5

9

21.1

18.7

-11

10

19.5

18.9

-3

11

21.5

21.6

0

12

20.3

21.4

5

Mean ± SD

20.6 ± 0.9

20.2 ± 1.6

-2

10

13

21.0

21.3

1

14

21.6

21.7

0

15

19.7

20.7

5

16

19.4

20.9

8

Mean ± SD

20.4 ± 1.0

21.2 ± 0.4

4

25

17

21.4

20.9

-2

18

19.9

20.8

5

19

19.2

20.3

6

20

20.9

20.5

-2

Mean ± SD

20.4 ± 1.0

20.6 ± 0.3

1

AOO

100

21

21.2

21.3

0

22

21.1

20.8

-1

23

18.7

18.7

0

24

20.5

20.3

-1

Mean ± SD

20.4 ± 1.2

20.3 ± 1.1

0

HCA

25

25

19.7

20.7

5

26

21.3

20.0

-6

27

18.4

18.8

2

28

20.7

21.0

1

Mean ± SD

20.0 ± 1.3

20.1 ± 1.0

0

AOO = Acetone: Olive oil 4:1 (v/v) mixture

DMF = N,N -Dimethylformamide

HCA = Hexyl cinnamic aldehyde

SD = Standard deviation

Applicant's summary and conclusion

Interpretation of results:
other: CLP/ EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
CLP not classified