Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
23 October 2012 to 06 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as an unpublished report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Solid
Details on test material:
- Physical state: Pale Yellow Solid
- Purity: Not applicable - UVCB
- Substance identity: Aluminum, benzoate C16-18-fatty acids complexes
- Batch number: 11074091 + Benzoic acid
- Carbon Content: 65.1%
- Analysis code: A118
- Date recieved: 07 June 2012
- Expiration date: 01 July 2013
- Storage of test material: Room temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan UK Limited, Oxon, UK.

- Age at study initiation: At the start of the study the animals were eight to twelve weeks old.

- Weight at study initiation: At the start of the study the animals were in the weight range of 15 to 23g.

- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.

- Diet (e.g. ad libitum): ad libitum (2014 Teklad Global Rodent diet supplied by Harlan UK Limited, Oxon, UK)

- Water (e.g. ad libitum): ad libitum.

- Acclimation period: At least five days.


ENVIRONMENTAL CONDITIONS

- Temperature (°C): The temperature was controlled to remain within the target ranges of 19 to 25 ºC.

- Humidity (%): The humidity was controlled to remain within the target ranges of 30 to 70%.

- Air changes (per hr): The rate of air exchange was approximately fifteen changes per hour.

- Photoperiod (hrs dark / hrs light): The lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

IN-LIFE DATES: From: Day 1 To: Day 6

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
Each group was exposed to the test material at concentrations of 10%, 5% or 2.5% w/w in propylene glycol.
No. of animals per dose:
Groups of four mice were treated for each test item concentration plus vehicle control
Details on study design:
RANGE FINDING TESTS: Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the test item at a maximum attainable concentration (as a suspension) of 10% w/w in propylene glycol, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale included as Appendix 4. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

- Lymph node proliferation response: No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.
Based on this information the dose levels selected for the main test were 10%, 5% and 2.5% w/w in propylene glycol.


MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT

- Name of test method: Local Lymph Node Assay in the Mouse. The assay has undergone extensive inter-laboratory validation and has been shown to reliably detect test materials that are moderate to strong sensitisers.

- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node(dpm/node) and as the ratio of 3HTdR incorporation in lymph node cells of test nodes relative to that recorded for the control nodes (stimulation Index). The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitier".

TREATMENT PREPARATION AND ADMINISTRATION: For the purpose of the study, the test material was used at concentrations of 10%, 5% or 2.5% w/w in propylene glycol. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest
suitable concentration. The mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface
of each ear for three consecutive days (Days 1, 2 and 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone in the same manner

3H-Methyl Thymidine Administration: Five days following the first topical application of the test material (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20 µCi to each mouse.
Positive control substance(s):
other: Phenylacetaldehyde
Statistics:
None provided.

Results and discussion

Positive control results:
One group of five animals was treated with 50 µL (25 µl per ear) of phenylacetaldehyde (>90%) as a solution in propylene glycol at a concentration of 2.5% v/v. A further control group of five animals was treated with propylene glycol alone.

The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration % v/v in propylene glycol Stimulation Index (SI) Result
2.5 6.48 Positive

Phenylacetaldehyde (>90%) was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: A stimulation index of less than 3 was recorded for the undiluted test material and the test material at concentrations of 10%, 5% and 2.5% w/w in propylene glycol. The stimulation index (SI) results are given in Table 4.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegrations per minute (dpm) per lymph node and the stimulation index (SI) are given in Table 2.

Any other information on results incl. tables

Table 1              Clinical Observations, Bodyweight and Mortality Data – Preliminary Screening Test

Concentration (% w/w) in propylene glycol

Animal Number

Bodyweight (g)

Day

1

2

3

4

5

6

Day 1

Day 6

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Pre-Dose

Post Dose

10

S-1

21

20

0

0

0

0

0

0

0

0

0

0=      No signs of systemic toxicity

Table 2              Local Skin Irritation – Preliminary Screening Test

Concentration
(%
w/w) in
propylene glycol

Animal Number

Local Skin Irritation

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

left

right

left

right

left

right

left

right

left

right

left

right

10

S-1

0

0

0

0

0

0

0

0

0

0

0

0

Table 3              Measurement of Ear Thicknessand Mean Ear Thickness Changes – Preliminary Screening Test

Concentration
(%
w/w) in
propylene glycol

Animal Number

Ear Thickness Measurement (mm)

Day 1

Day 3

Day 6

pre‑dose

post dose

left

right

left

right

left

right

10

S-1

0.245

0.240

0.230

0.240

0.235

0.245

overall mean (mm)

0.243

0.235

0.240

overall mean ear thickness change (%)

na

-3.093

-1.031

na=     Not applicable

Table 4              Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration
(%w/w) in
propylene glycol

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

5012.31

626.54

na

na

2.5

5528.98

691.12

1.10

Negative

5

4181.55

522.69

0.83

Negative

10

5039.56

629.95

1.01

Negative

dpm= Disintegrations per minut

a=      Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

b=      Stimulation Index of 3.0 or greater indicates a positive result

na =    Not applicable

Table 5              Individual Clinical Observations and Mortality Data

Concentration
(% w/w) propylene glycol

Animal Number

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Vehicle

1-1

0

0

0

0

0

0

0

0

0

1-2

0

0

0

0

0

0

0

0

0

1-3

0

0

0

0

0

0

0

0

0

1-4

0

0

0

0

0

0

0

0

0

2.5

2-1

0

0

0

0

0

0

0

0

0

2-2

0

0

0

0

0

0

0

0

0

2-3

0

0

0

0

0

0

0

0

0

2-4

0

0

0

0

0

0

0

0

0

5

3-1

0

0

0

0

0

0

0

0

0

3-2

0

0

0

0

0

0

0

0

0

3-3

0

0

0

0

0

0

0

0

0

3-4

0

0

0

0

0

0

0

0

0

10

4-1

0

0

0

0

0

0

0

0

0

4-2

0

0

0

0

0

0

0

0

0

4-3

0

0

0

0

0

0

0

0

0

4-4

0

0

0

0

0

0

0

0

0

0=      No signs of systemic toxicity

Table 6              Individual Bodyweights and Bodyweight Changes

Concentration
(% w/w) in
propylene glycol

Animal Number

Bodyweight (g)

Bodyweight Change (g)

Day 1

Day 6

Vehicle

1-1

22

21

-1

1-2

20

22

2

1-3

22

20

-2

1-4

17

18

1

2.5

2-1

18

19

1

2-2

20

20

0

2-3

18

19

1

2-4

21

22

1

5

3-1

19

18

-1

3-2

19

19

0

3-3

18

19

1

3-4

20

18

-2

10

4-1

20

22

2

4-2

20

20

0

4-3

18

21

3

4-4

19

19

0

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: other: Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitiser".
Conclusions:
The test material, aluminum, benzoate C16-18-fatty acids complexes was considered to be a non-sensitiser under the conditions of the test.
Executive summary:

Introduction

A study was performed to assess the skin sensitisation potential of the test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The skin sensitisation potential was assessed in a proprietary, GLP-compliant experimental study (Harlan 2013) following OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay"(adopted 22 July 2010) and Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008.The study is considered reliable and relevant for use for this endpoint.

Methods. 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 10w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test material as a suspension in propylene glycol at concentrations of10%, 5% or 2.5w/w. A further group of four animals was treated with propylene glycol alone.

Results. 

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%w/w) in propylene glycol

Stimulation Index

Result

2.5

1.10

Negative

5

0.83

Negative

10

1.01

Negative

Conclusion. 

The test material, aluminum, benzoate C16-18-fatty acids complexes was considered to be a non-sensitiser under the conditions of the test.