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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:
between 05 February 2009 and 18 February 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with GLP and agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Remarks:
Study conducted on read-across material
Justification for type of information:
A RAAF report will shortly be provided.
Cross-reference
Reason / purpose for cross-reference:
other: read-across target
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
A RAAF report will shortly be provided.
Reason / purpose for cross-reference:
read-across source
Type of study:
mouse local lymph node assay (LLNA)
Key result
Parameter:
SI
Value:
1.32
Test group / Remarks:
2.5 % concentration w/w
Key result
Parameter:
SI
Value:
1.13
Test group / Remarks:
5 % concentration w/w
Key result
Parameter:
SI
Value:
1.15
Test group / Remarks:
10 % concentration w/w

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of Signature: 15/10/2007; Date of Inspection: 21/08/2007
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of sodium hydrogen N-(1-oxooctadecyl)-L-glutamate and sodium hydrogen N-(1-oxohexadecyl)-L-glutamate
EC Number:
936-610-7
Molecular formula:
See remarks
IUPAC Name:
Reaction mass of sodium hydrogen N-(1-oxooctadecyl)-L-glutamate and sodium hydrogen N-(1-oxohexadecyl)-L-glutamate
Constituent 2
Reference substance name:
Reaction mass of sodium hydrogen N-(1-oxooctadecyl)-L-glutamate and sodium hydrogen N-(1-oxohexadecyl)-L-glutamate
IUPAC Name:
Reaction mass of sodium hydrogen N-(1-oxooctadecyl)-L-glutamate and sodium hydrogen N-(1-oxohexadecyl)-L-glutamate

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Limited, Bicester, Oxon, UK and female CBA/Ca (CBA/CaBkl) strain mice were supplied by B & K Universal Ltd, Hull, UK.
- Age at study initiation: 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 23 g
- Housing: The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet : Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK) was allowed throughout the study. ad libitum
- Water: Free access to mains tap water ( ad libitum)
- Acclimation period: acclimatisation period of at least five days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): in the range 19 to 25°C
- Humidity (%): in the range 30 to 70%,
- Air changes (per hr): The rate of air exchange was approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.


IN-LIFE DATES: From: Day 0 To: Day 6

Study design: in vivo (LLNA)

Vehicle:
other: 1% pluronic L92 in distilled water,
Concentration:
10%, 5% or 2.5% w/w in 1% pluronic L92 in distilled water.
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Not stated in report
- Irritation: None
- Lymph node proliferation response:
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 2004/73/EC
- Criteria used to consider a positive response:
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 sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:

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, 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, GE Healthcare UK Ltd) giving a total of 20 µCi to each mouse.
Positive control substance(s):
other: 2,4-Dinitrobenzenesulfonic acid, sodium salt
Statistics:
Not stated in report

Results and discussion

Positive control 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
1% pluronic L92 in distilled water Stimulation Index Result
10 13.71 Positive

Conclusion: 2,4 Dinitrobenzenesulfonic acid, sodium salt was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.32
Test group / Remarks:
2.5 % concentration w/w
Key result
Parameter:
SI
Value:
1.13
Test group / Remarks:
5 % concentration w/w
Key result
Parameter:
SI
Value:
1.15
Test group / Remarks:
10 % w/w concentration

Any other information on results incl. tables

 Preliminary Screening Test:

Table 1: Clinical observations, bodyweight and mortality data

Concentration (%w/w) in
1% pluronic L92in distilled water

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

17

17

0

0

0

0

0

0

0

0

0

0=      No signs of systemic toxicity

Based on this information the dose levels selected for the main test were 10%, 5% and 2.5% w/w in 1% pluronic L92 in distilled water.

Main Test:

Estimation of the Proliferative Response of Lymph Node Cells:

The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 1 of attachment 1.

A stimulation index of less than 3 was recorded for the three concentrations of the test material (10%, 5% and 2.5% w/win 1% pluronic L92 in distilled water).

Clinical Observations and Mortality Data:

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.


Bodyweight:

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period except for one animal treated with the test material at a concentration of 5% w/w in1 % pluronic L92 in distilled water which showed a slightly higher than expected bodyweight loss.

Table 2: Table to Show Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (% w/w) in 1 % pluronic L92 in distilled water

dpm

dpm/Node^a

Stimulation Index^b

Result

Vehicle

3441.01

430.13

N/A

N/A

2.5

4551.80

568.98

1.32

Negative

5

3886.80

485.85

1.13

Negative

10

3955.41

494.43

1.15

Negative

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Conclusions:
The test material 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 method was designed to meet the requirements of the following:

-   OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 24 April 2002)

-      Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 2004/73/EC.

Methods:

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 10% w/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 solution in 1% pluronic L92 in distilled water at concentrations of 10%, 5% or 2.5% w/w. A further group of four animals was treated with 1% pluronic L92 in distilled water 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
1% pluronic L92 in distilled water

Stimulation Index

Result

2.5

1.32

Negative

5

1.13

Negative

10

1.15

Negative

Conclusion:

The test material was considered to be a non‑sensitiser under the conditions of the test.