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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:
August 10, 2011 - August 24, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD guidelines and GLP.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test Item: 1,1,3,3-tetramethylbutyl peroxyneodecanoate
CAS No.: 51240-95-0
Batch No.: 1102442046
Purity: 71.6 % (dose calculation not adjusted to purity)
Storage: In the freezer at -20 ± 5°C
Stability in solvent: Not indicated by the sponsor
Expiration Date: December 01, 2011

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS: mice, CBA/CaCrl
- Source: Charles River UK, Manston Road, Margate, Kent CT9 4LT
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 16.0 - 20.2 g
- Housing: group caging
- Diet: pelleted standard diet (Harlan Laboratories B.V., 5960 AD Horst, The Netherlands), ad libidum
- Water: tap water, (Gemeindewerke, 64380 Rossdorf, Germany), ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-65 %
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.
- Bedding: granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5, 10, and 25% (v/v)
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:

A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was a 50 % (v/v) solution in acetone:olive oil (4+1 v/v). Due to the properties of the test item, higher concentrations could not be used.
To determine the highest non-irritant test concentration that does at the same time not induce signs of systemic toxicity, a pre-test was performed in two animals and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25 and 50% (v/v) once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible reddening of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer (S0247 Kroeplin, 36381 Schlüchtern, Germany). Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if reddening of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6. The measured ear weight was also considered in this evaluation.
On day 3 and 4, both animals showed an erythema of the ear skin (Score 1). Additionally, on day 4, the animal treated with 50% test item concentration showed reduced spontaneous activity. Furthermore, regarding the ear weight, the threshold value of 25% for excessive local skin irritation mentioned in OECD guideline 429 was exceeded in this animal (value was compared to the mean value of historical control data for the ear weight, 25.32 mg).
The test item in the main study was thus assayed at test item concentrations of 5, 10, and 25% (v/v).


MAIN STUDY:

TOPICAL APPLICATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 5, 10, and 25% (v/v) in acetone:olive oil (4+1 v/v). The application volume, 25 µL was spread over the entire dorsal surface (~ 8 mm) of each ear once daily for three consecutive days. A futher group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

ADMINISTRATION OF 3H-METHYL THYMIDINE AND DETERMINATION OF INCORPORATED 3H-METHYL THYMIDINE

Five days after the first topical application, all mice were intraveneously injected into a tail vein with radio-labelled thymidine (3HTdR). Approximately five hours after treatment with 3HTdR all mice were sacrificed and the draining lymph nodes were excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed with phosphate buffered saline and incubated with trichloroacetíc acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a ?-scintillation counter.

INTERPRETATION OF RAW DATA

The proliferation response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitiliser in the LLNA if the following criteria are fulfilled:
-First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
-Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

OBSERVATIONS

In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:

Mortality / Viability: At least once daily from experimental start to necropsy.

Body weights: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with 3HTdR.

Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6), the ear thickness will be determined using a micrometer (S0247 Kroeplin, 36381 Schlüchtern, Germany).

Ear weights: In the pre-test and main experiment after sacrifice; biopsy punches will be taken from each ear.

Clinical signs (local / systemic): Clinical signs (local irritation at the application site or systemic toxicity) will be recorded at least once daily. Especially the treatment sites will be observed carefully.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables and for the DPM values (group mean DPM ± standard deviation).
A statistical analysis was conducted to assess whether the difference was statistically significant between test item groups and negative control group. For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons was performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test was used for identification of possible outliers (performed with Microsoft Excel 2003).
Furthermore, the ANOVA (Dunnett-test) was conducted on the ear weights to assess whether the difference was statistically significant between test item groups and negative control (vehicle) group.

Results and discussion

Positive control results:
Experiment performed in August 2011 using concentrations of 5, 10, and 25 % alpha-hexyl cinnamic aldehyde in acetone:olive oil (4:1). These concentrations yielded S.I.´s of 1.35, 2.18, and 8.08, respectively.
The EC3 value calculated was 12.1 % (w/v).
The positive control substance alpha-hexyl cinnamic aldehyde was found to be a skin sensitizer under the described conditions, demonstrating the validity of the study.

In vivo (LLNA)

Results
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see depicted table below

Any other information on results incl. tables

Calculation and results of individual data; Vehicle: acetone/olive oil (4:1 v/v)

Test item concentration

DPM values measured

DPM-BG per animal
(2 lymph nodes)a)

S.I.b)

% (v/v)

Group No.

Animal No.

---

BG I

---

27

---

---

---

BG II

---

22

---

---

0

1

1

745

721

---

0

1

2

966

942

---

0

1

3

583

559

---

0

1

4

643

619

---

0

1

5

797

773

---

5

2

6

881

857

1.2

5

2

7

780

756

1.0

5

2

8

96

72

0.1

5

2

9

81

57

0.1

5

2

10

225

201

0.3

10

3

11

1442

1418

2.0

10

3

12

833

809

1.1

10

3

13

1839

1815

2.5

10

3

14

1620

1596

2.2

10

3

15

1018

994

1.4

25

4

16

1460

1436

2.0

25

4

17

1590

1566

2.2

25

4

18

1723

1699

2.4

25

4

19

1205

1181

1.6

25

4

20

2219

2195

3.0

BG   =   Background (1 ml 5% trichloroacetic acid) in duplicate

1      =   Control Group

2-4  =   Test Group

S.I.   =   Stimulation Index

a)     =   values corrected for mean background value (BGI and BGII).b)          =     Stimulation Indices relative to the mean of the control group (Group 1)

Table 2: Calculation of Stimulation Indices per Dose Group

Test item concentration

Group Calculation

DPM per
lymph nodea)

SD

S.I.

Vehicle

722.3

148.5

1.00

5 % 1,1,3,3-
tetramethylbutyl peroxyneodecanoate

388.1

387.2

0.54

10 % 1,1,3,3-tetramethylbutyl peroxyneodecanoate

1325.9

417.7

1.84

25 % 1,1,3,3-tetramethylbutyl peroxyneodecanoate

1614.9

376.2

2.24

a)      Mean DPM/animal was determined by dividing the sum of the measured values from
lymph nodes of all animals within a group by the number of animals in that group

 

The EC3 value could not be calculated, since all obtained SI´s were below the threshold value of 3.

 

VIABILITY / MORTALITY

No deaths occurred during the study period.

CLINICAL SIGNS

No symtoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

EAR WEIGHT

The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant increase in ear weights was not observed in any treated group in comparison to the vehicle control group.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In this study Stimulation Indices (S.I.) of 0.54, 1.84, and 2.24 were determined with the test item at concentrations of 5, 10, and 25% (v/v) in acetone:olive oil (4+1 v/v), respectively. A dose response was observed. Although a statistically significant increase in DPM value was observed in the mid and the high dose group in comparison to the vehicle control group, this was not considered as biologically relevant as the S.I. determined for these concentrations did not exceed the threshold value of 3 for a positive response.
The EC3 value could not be calculated, since all obtained SI´s were below the threshold value of 3. The test item was thus not a skin sensitizer under the test conditions of this study.
Executive summary:

In order to study a possible skin sensitization potential of 1,1,3,3-tetramethylbutyl peroxyneodecanoate, three groups each of five female mice were treated once daily with the test item at concentrations of 5, 10, and 25% (v/v) in acetone:olive oil (4+1 v/v) by topical application to the dorsum of each ear for three consecutive days. A control group of five mice was treated with the vehicle (acetone:olive oil (4+1 v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled for each animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of3H-methyl thymidine measured in ab-scintillation counter.

The animals did not show any signs of local skin irritation or systemic toxicity during the course of the study and cases of mortality were also not observed. A statistically significant increase in ear weights was not observed in any dose group in comparison to the vehicle control group.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3-fold or greater increase in incorporation of3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices (S.I.) of 0.54, 1.84, and 2.24 were determined with the test item at concentrations of 5, 10, and 25% (v/v) in acetone:olive oil (4+1 v/v), respectively. A dose response was observed. Although a statistically significant increase in DPM value was observed in the mid and the high dose group in comparison to the vehicle control group, this was not considered as biologically relevant as the S.I. determined for these concentrations did not exceed the threshold value of 3 for a positive response.

The EC3 value could not be calculated, since all obtained SI´s were below the threshold value of 3.