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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:
10 January 2018 to 16 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
OECD Guidelines for Testing of Chemicals No.429. Skin Sensitisation: Local Lymph Node Assay (Adopted 22 July 2010)
Deviations:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Commission Regulation (EC) No 440/2008 of 30 May 2008, B.42., “Skin Sensitisation: Local Lymph Node Assay” (Official Journal L 142, 31/05/2008) amended by Commission Regulation (EU) No 640/2012 of 6 July 2012)
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
No further details specified in the study report.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
Species and strain: CBA/CaOlaHsd mice
Source: Envigo, San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy
Hygienic level: SPF at arrival; standard housing conditions during the study
Justification of strain: On the basis of OECD Guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals: 4 animals / group
Sex: Female, nulliparous, non-pregnant
Age of animals at starting: 8 weeks old (age-matched, within one week)
Body weight range at starting: 17.1 – 18.8 grams
(The weight variation in animals in the study did not exceed ± 20 % of the mean weight.)
Acclimatization time: 21 days
Note: In the Preliminary Experiment, mice of 8 weeks of age (21.2-21.9 grams) were used.

Husbandry
Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Group caging / mice were provided with glass tunnel-tubes
Cage type: Type II. polypropylene / polycarbonate
Bedding: Bedding was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 18.2 – 26.7 °C
Relative humidity: 23 - 88 %
Ventilation: 15-20 air exchanges/hour
The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases.

Food and feeding
Animals received ssniff® SM Rat/Mouse – Breeding and Maintenance, 15 mm, autoclavable “Complete feed for Rats and Mice – Breeding and Maintenance" (Batch number: 262 21592, Expiry date: 31 January 2018, and Batch number: 382 24962, Expiry date: 30 April 2018, produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), and Gel diet Transport (Batch Number: 60172230020101, Expiry date:11 August 2018, Scientific Animal Food & Engineering, Route de Saint Bris, 89290 Augy, France) ad libitum. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Water supply
Animals received tap water from the municipal supply from 500 mL bottles, ad libitum.
Water quality control analysis was performed once every three months and microbiological assessment was performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József Attila u. 36., Hungary).

Bedding
Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. Lignocel 3/4-S Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH + Co.KG (D-73494 Rosenberg, Germany) was available to animals during the study. Certified nest building material was also provided for animals (ARBOCEL crinklets natural produced by J. Rettenmaier & Söhne GmbH + Co.KG).

Identification and randomisation
A unique number written on the tail with a permanent marker identified each animal. The animal number was assigned on the basis of Citoxlab Hungary Ltd.’s master file. The cages were marked with identity cards with information including study code, cage number, and dose group, sex and individual animal number. The animals were randomised and allocated to the experimental groups. The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
The highest concentration was 49.4% (undiluted test substance as received).
The formulation at 25% (w/v), 12.5% (w/v), 5% (w/v) and 1% (w/v) using acetone:olive oil 4:1 (v/v) as vehicle were suitable for the test.
No. of animals per dose:
four animals
Details on study design:
Dose Selection and Justification of Dose Selection
The Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/CaOlaHsd mice using two concentrations (2 animals/dose) of the test item, 49.4% (corresponding to the test substance as received) and 24.7% w/v (corresponding to 50% of the test substance as received) in acetone: Olive oil 4:1 (v/v) mixture.
The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed.

The maximum concentration of test item in an acceptable solvent was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum available concentration was 49.4% (corresponding to the test substance as received).

In the Preliminary Irritation / Toxicity Test, all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.

During the Preliminary Irritation / Toxicity Test, no mortality or signs of systemic toxicity were observed. There were no indications of any irritancy at the site of application at Day 1, 2, 4, 5 and 6. Very slight erythema (barely perceptible) was observed on Day 3 in both treatment group. No test item precipitate was observed on the ears of the animals in each experiment.

A slight decrease was observed in body weight (<5% reduction) in 1 out of the 2 animals in the group treated with concentration of 24.7% in the preliminary study. However, this fact was considered to have no toxicological significance and differences arise from individual variability. Average body weight loss of the groups were within 1%.

Ear thickness of the animals was measured by using a thickness gauge on Days 1, 3 and 6, and by ear punch weight determination after the euthanasia of the experimental animals on Day 6.
The ear thickness values and ear punch weights were within the acceptable range.

The draining auricular lymph nodes of the animals were visually examined: they were normal in the 24.7% (w/v) group and larger than normal in the 49.4% group (subjective judgement by analogy with observations of former experiments).

Based on these results, 49.4 % (corresponding to undiluted test substance as received) concentration was selected as top dose for the main test.

Topical application
During the study, animals were topically dosed with 25 µL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

OBSERVATIONS
Clinical Observations
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6.
Individual records were maintained.

Measurement of Body Weight
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

PROLIFERATION ASSAY
Injection of Tritiated Thymidine (3HTdR)
On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (phosphate buffered saline) containing approximately 20 µCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses).
The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels.
Once removed, the nodes of each mouse were collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing. The nodes of each animal were processed individually.

Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C.
After centrifugation, supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for the lymph nodes of each individual animal.

Determination of Incorporated 3HTdR
After the final washing step, supernatants were removed. Pellets were gently agitated, resuspended and 3 mL of 5 % (w/v) TCA solution was added to the tubes for precipitation of macromolecules.
After overnight (approximately 18 hours) incubation at 2-8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4 °C), and supernatants were removed. Pellets were resuspended in 1 mL of 5 % (w/v) TCA solution and dispersed by using an ultrasonic bath.
Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a ß-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).
The ß-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5 % (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not specified

Results and discussion

Positive control results:
The result of the positive control substance a-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25 % in the relevant vehicle (acetone:olive oil 4:1 (v/v)) using CBA/CaOlaHsd mice.
No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historic positive control data (stimulation index value of 11.3) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.
Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were in within the historical control range. Each treated and control group included 4 animals.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
2.2
Test group / Remarks:
49.4% 2,2-di-(tertamylperoxy) butane
Key result
Parameter:
SI
Value:
1.3
Test group / Remarks:
25% 2,2-di-(tertamylperoxy) butane
Key result
Parameter:
SI
Value:
1.6
Test group / Remarks:
12.5% 2,2-di-(tertamylperoxy) butane
Key result
Parameter:
SI
Value:
1.8
Test group / Remarks:
5% 2,2-di-(tertamylperoxy) butane
Key result
Parameter:
SI
Value:
1.4
Test group / Remarks:
1% 2,2-di-(tertamylperoxy) butane
Cellular proliferation data / Observations:
CLINICAL OBSERVATION
No mortality or signs of systemic toxicity were observed during the study. No test item precipitate was observed on the ears of the experimental animals. There were no indications of any irritancy at the site of application.

BODY WEIGHT MEASUREMENT
No treatment related effects were observed on the mean body weight changes.

PROLIFERATION ASSAY
The appearance of the lymph nodes was normal in the negative control group 1%, 5%, 12.5%, 25% (w/v) and 49.4% (undiluted) dose groups. Larger than normal lymph nodes were observed only in the positive control group.
The stimulation index values induced by the test item were 2.2, 1.3, 1.6, 1.8 and 1.4 at concentrations of 49.4% (undiluted test substance), 25% (w/v), 12.5% (w/v), 5% (w/v) and 1% (w/v) respectively.

INTERPRETATION OF OBSERVATIONS
The test substance was formulated in acetone:olive oil 4:1 (v/v). Since there were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay. Although there was some observational evidence of enlarged lymph nodes in the Preliminary Irritation / Toxicity Test, the proliferation data takes precedence. The resulting stimulation indices observed under these exaggerated test conditions were considered to be good evidence that 2,2-DI-(TERTAMYLPEROXY)BUTANE is a non-sensitizer.

Any other information on results incl. tables

Individual Body Weights for all Animal with Group Means

Animal Number

Test Group Name

Initial Body Weight (g)

Terminal Body Weight* (g)

Change# (%)

5238

5245

5250

5243

 

Negative (vehicle) control acetone:olive oil 4.1 (v/v)

 

 

Mean

18.2

17.8

17.2

17.1

17.6

19.2

18.5

17.9

17.1

18.2

5.5

3.9

4.1

0.0

3.4

5252

5253

5257

5236

 

49.4%

2,2-di-(tert-amylperoxy)butane

 

 

Mean

18.8

17.7

17.4

17.5

17.9

19.0

18.9

17.4

17.9

18.3

1.1

6.8

0.0

2.3

2.5

5260

5244

5247

5256

 

25%

2,2-di-(tert-amylperoxy)butane

 

 

Mean

18.8

17.8

17.6

17.7

18.0

20.4

17.7

18.4

17.9

18.6

8.5

-0.6

4.5

1.1

3.4

5259

5237

5251

5262

 

12.5%

2,2-di-(tert-amylperoxy)butane

 

 

Mean

18.0

18.3

17.6

17.5

17.9

19.0

19.0

18.6

19.2

19.0

5.6

3.8

5.7

9.7

6.2

5263

5240

5254

5246

 

5%

2,2-di-(tert-amylperoxy)butane

 

 

Mean

17.8

18.0

17.1

17.5

17.6

18.8

18.7

17.3

17.7

18.1

5.6

3.9

1.2

1.1

3.0

5242

5248

5258

5261

 

1%

2,2-di-(tert-amylperoxy)butane

 

 

Mean

18.2

17.7

17.8

17.1

17.7

18.3

18.4

18.9

19.7

18.8

0.5

4.0

6.2

15.2

6.5

5239

5241

5255

5249

 

Positive Control 25% HCA in acetone:olive oil 4:1 (v/v)

 

 

Mean

18.8

17.2

17.1

17.1

17.6

19.4

18.2

18.1

18.6

18.6

3.2

5.8

5.8

8.8

5.9

Notes:

*: Terminal body weight measured in Day 6.

#: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

DPM, DPN and Stimulation Index Values for all Groups

Test Group Name

Identity Number

Measured DPM

DPM

Number of lymph nodes

DPN

Group DPN

Stimulation Index

Background (5% (w/v) TCA)

-

39

33

-

-

-

-

-

Negative vehicle control acetone:olive oil 4:1 (v/v)

1

2

3

4

559

415

1205

1630

523.0

379.0

1169.0

1594.0

2

2

2

2

261.5

189.5

584.5

797.0

458.1

1.0

49.4%

2,2-di-)tert-amylperoxy)butane

5

6

7

8

1925

1599

2697

1813

1889.0

1563.0

2661.0

1777.0

2

2

2

2

944.5

781.5

1330.5

888.5

986.3

2.2

25%

2,2-di-)tert-amylperoxy)butane

9

10

11

12

1516

1785

248

1392

1480.0

1749.0

212.0

1356.0

2

2

2

2

740.0

874.5

106.0

678.0

599.6

1.3

12.5%

2,2-di-)tert-amylperoxy)butane

13

14

15

16

751

1251

1695

2205

715.0

1215.0

1659.0

2169.0

2

2

2

2

357.5

607.5

829.5

1084.5

719.8

1.6

5%

2,2-di-)tert-amylperoxy)butane

17

18

19

20

607

1839

3533

867

571.0

1803.0

3497.0

831.0

2

2

2

2

285.5

901.5

1748.5

415.5

837.8

1.8

1%

2,2-di-)tert-amylperoxy)butane

21

22

23

24

1496

1124

1063

1423

1460.0

1088.0

1027.0

1387.0

2

2

2

2

730.0

544.0

513.5

693.5

620.3

1.4

Positive control 25% HCA in acetone:olive oil 4:1 (v/v)

25

26

27

28

11859

12126

10358

7114

11823.0

12090.0

10322.0

7078.0

2

2

2

2

5911.5

6045.0

5161.0

3539.0

5164.1

11.3

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, under the conditions of the present assay, the test item 2,2-DI-(TERTAMYLPEROXY)BUTANE (content of LUPEROX 520M50 ) was shown to have no skin sensitization potential (non-sensitizer) in the Local Lymph Node Assay.
Executive summary:

The aim of this study was to determine the skin sensitisation potential of 2,2-DI-(TERT-AMYLPEROXY)BUTANE following dermal exposure in mice. The study was performed with vertebrate animals in order to provide a proper classification as the test item was incompatible with in vitro tests (insoluble for in vitro test). Based on the results of the Preliminary Compatibility Test, acetone:olive oil 4:1 (v/v) was used as vehicle for the test substance in the study. The test item of this study was 2,2-DI-(TERT-AMYLPEROXY)BUTANE, the organic peroxide contained at the concentration of 49.4% in the test substance (LUPEROX 520M50) supplied. The highest concentration was 49.4% (undiluted test substance as received). The formulation at 25% (w/v), 12.5% (w/v), 5% (w/v) and 1% (w/v) using acetone:olive oil 4:1 (v/v) as vehicle were suitable for the test. As acetone:olive oil 4:1 (v/v) is one of the vehicles recommended by the relevant OECD guideline, it was selected for vehicle of the study. The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose) at 49.4% (undiluted test substance) and 24.7% (w/v) in acetone:olive oil 4:1 (v/v). Based on the observations recorded in the preliminary test, the 49.4% dose was selected as top dose for the main test. 

In the main assay, twenty-eight female CBA/CaOlaHsd mice were allocated to seven groups of four animals each:

- five groups received 2,2-DI-(TERT-AMYLPEROXY)BUTANE at 49.4%, 25% (w/v), 12.5% (w/v), 5% (w/v) and 1% (w/v) concentrations from the test item,

- the negative control group received the vehicle (acetone:olive oil 4:1 (v/v)),

- the positive control group received 25 % (w/v) HCA (dissolved in acetone:olive oil 4:1 (v/v)).

The test item solutions were applied on the dorsal surface of ears of experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

No mortality or signs of systemic toxicity were observed during the study. No test item precipitate was observed on the ears of the experimental animals. There were no indications of any irritancy at the site of application. No treatment related effects were observed on the mean body weight changes in the main study. The stimulation index values were 2.2, 1.3, 1.6, 1.8 and 1.4 at concentrations of 49.4% (undiluted test substance), 25% (w/v), 12.5% (w/v), 5% (w/v) and 1% (w/v) respectively. The result of the positive control substance a-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historic positive control data was noted for the positive control chemical, this result confirmed the validity of the assay.

In conclusion, the test item 2,2-DI-(TERTAMYLPEROXY)BUTANE (organic peroxide content of LUPEROX 520M50 ) was shown to have no skin sensitization potential (non-sensitizer) in the Local Lymph Node Assay.