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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation, other
Remarks:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-12-03 to 2009-05-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline compliantGLP compliant

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:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
as at 2002
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
as at 2004
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Reaction mass of (3-hydroxy-2-pentylcyclopentyl)acetic acid and sodium (3-hydroxy-2-pentylcyclopentyl)acetate
EC Number:
932-898-3
IUPAC Name:
Reaction mass of (3-hydroxy-2-pentylcyclopentyl)acetic acid and sodium (3-hydroxy-2-pentylcyclopentyl)acetate
Details on test material:
- Name of test material (as cited in study report): R0034833C
- Physical state: slightly yellow liquid
- Analytical purity: 28 %
- Composition of test material, percentage of components:
NaCI content: 4.1%
Water content: 44.4%
Dipropylene glycol content: 20.1%
Active content: 28%*
Majority diastereoisomers ratio: 59/41
Other diastereoisomers and isomers content: 10%
- Impurities content:
ER2423 content Detected: < 0.1%
ER2425 content Detected: < 0.1%
Inter-molecular esters content: 0.39%*
Intra-molecular esters content: 2.29%*
ER2410 content: 0.02%*
Ruthenium content: 0.5 ppm
Methanol content: Not Detected, < 1000 ppm

Results consistent with ID LIMS 2%13385 (*) and Aulnay report AILC/FB/08.0103/J22611

- Purity test date: 2008-11-18
- Lot/batch No.: R0034833C 023 D 001 awes 105 Ours a 45°C
- Expiration date of the lot/batch: June 2009
- Stability under test conditions: not reported
- Storage condition of test material: at +4°C and protected from light

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS- Strain: CBA/J mouse (nulliparous and non-pregnant females)- Source: Janvier, Le Genest-Saint-Isle, France- Age at study initiation: approximately 9 weeks- Weight at study initiation: 21.8 ± 1.1 g- Housing: individually in disposable crystal polystyrene cages (22.00 cm x 8.50 cm x 8.00 cm) containing autoclaved sawdust (SICSA, Alfortville, France).- Diet (e.g. ad libitum): ad libitum, SSNIFF R/M-H pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany)- Water (e.g. ad libitum): ad libitum, tap water (filtered using a 0.22 micron filter)- Acclimation period: at least 5 dENVIRONMENTAL CONDITIONS- Temperature (°C): 22 ± 2- Humidity (%): 30 to 70- Air changes (per hr): approximately 12, (filtered, non-recycled air)- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Remarks:
batch No. 00937HH (Aldrich, Saint-Quentin-Fallavier, France)
Concentration:
0, 10, 25, 50, 75, 100 % (v/v)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:- no preliminary test performed, at the request of the Sponsor- Compound solubility: soluble (solubility assays performed in a previous study (CIT/Ref. No. 34489 TSS))- Irritation: irritant potential of the test item (through ear thickness measurement) was assessed in a previous study (CIT/Ref. No. 34489 TSS)- Lymph node proliferation response: not reportedMAIN STUDYANIMAL ASSIGNMENT AND TREATMENT- Name of test method: pooled approach- Criteria used to consider a positive response:Validity criteria:The study is considered valid if all the following criteria are met: - the incorporation of 3H-TdR expressed as disintegrations/mn (dpm) in the vehicle control group should be at least 2 fold higher than that of the control blank, and should fall within CIT historical data range, - the cell viability in the vehicle group should be higher than 70%, - the SI for the positive control should be higher than the threshold positive value of 3.(Historical data see Table ??????????)Decision criteriaPositive: - SI for a dose group is ≥ 3 together with consideration of a dose-response relationship - other relevant criteria: cellularity, radioactivity levels and ear thickness to be taken into account for the interpretation of results. - assessment of the biological significance of the proliferative results in the control groups: comparison with appropriate historical control dataTREATMENT PREPARATION AND ADMINISTRATION:- substance dissolved in DMF at 0, 10, 25, 50, 75, 100 % (v/v) and using α-hexylcinnamaldehyde (HCA) at the concentration of 25% in DMF as positive control substance- test item, vehicle (DMF) or reference item applied over the ears (25 μL per ear) for 3 consecutive days (days 1, 2 and 3)- after 2 days of resting iv injection of 250 μL of 0.9% NaCl containing 20 μCi of 3H-TdR (specific activity of 25 Ci/mmol, batch No. B509A (Amersham, Les Ulis, France)) into the tail vein of animals- sacrifice of animals 5h later, excision of draining lymph nodes and pooling per dose group - suspensions washed with 15 mL of 0.9% NaCl and pellets re-suspended in 0.9% NaCl for numeration of lymphocytes (cellularity) and determination of their viability by exclusion of trypan blue. - cell suspension centrifuged and pellets precipitated with 3 mL of 5% (w/v) trichloroacetic acid (TCA ; batch No. 047K0780, Sigma, Saint-Quentin- Fallavier, France) in purified water at +4°C overnight. - centrifugation followed by precipitation of pellets with 1 mL of 5% TCA and addition of 3 mL of Ultima GoldxR scintillation fluid (Packard)- β-scintillation counting for measurement of 3H-TdR incorporation expressed as disintegrations/mn (dpm) per group and as dpm/node- assessment of irritant potential of the test item by measurement of ear thickness on days 1, 2, 3 and 6
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Stimulation Indices (SI) were calculated according to the following formula:SI = dpm of treated group / dpm of control groupCellularity and viability of enumerated cells were also determined in each group.Cellularity index (CI) was calculated as follows: CI = Number of viable cells (x10^6)/number of nodes in the treated group / Number of viable cells (x10^6)/number of nodes in the vehicle control group

Results and discussion

Positive control results:
Treatment with 25 % HCA (v/v) in DMF led to 88.66 % cell viability and a SI of 4.97.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: - SI = 0.62 - 1.41 in treatment groups- see Table 3 for details
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: - 59.61 - 136.34 dpm/node in treatment groups as compared to 96.72 dpm/node in controls - see Table 3 for details

Any other information on results incl. tables

- Table 3: Summary of study results

Groups

Treatment and concentrations

Cell count

 

Viability (%)

Amount of cells (x 10^6 cells)

Cellularity index

Number of nodes per group

viable

dead

1

Vehicle

70

7

90.91

7.00

 

8

2

Test item 10%

97

18

84.35

9.70

1.39

8

3

Test item 25%

76

11

87.36

7.60

1.09

8

4

Test item 50%

76

24

76.00

7.60

1.09

8

5

Test item 75%

83

11

88.30

8.30

1.19

8

6

Test item 100%

148

9

94.27

14.80

2.11

8

7

HCA 25%

219

28

88.66

21.90

3.13

8

Groups

Treatment and concentrations

dpm per group

Dpm per node

Stimulation index (SI)

Increase in ear thickness (% between day 1 and day 6)

Irritation level

EC3 value

1

Vehicle

773.74

96.72

 

7.84

 

NA

2

Test item 10%

1090.75

136.34

1.41

7.29

I

3

Test item 25%

886.81

110.85

1.15

7.07

I

4

Test item 50%

874.72

109.34

1.13

5.10

I

5

Test item 75%

476.90

59.61

0.62

9.00

I

6

Test item 100%

862.70

107.84

1.11

3.06

I

7

HCA 25%

3848.22

481.03

4.97

 

 

- Table 4: Historical data: Skin sensitization potential in mice (CBA1J strain) using the Local Lymph Node Assay (LLNA) Vehicle control Dimethylformamlde from July 2004 to May 2007

 

Amount of cells ( x106 cells)

Dpm/node

increase in ear thickness (%)

minimum

2.10

31.76

-4.95

maximum

16.40

121.35

6.45

Mean

7.42

69.34

1.29

SD

2,94

20.09

2.84

Nb

40

SD = standard deviation

Nb number of values

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The aim of this study was to evaluate the potential of the test item R0034833C (batch No. R0034833C 023 D 001 après 105 jours à 45°C) to induce contact sensitization using the murine Local Lymph Node Assay (LLNA) according to OECD TG 429 and GLP.Under the experimental conditions of this study, the test item R0034833C (batch No. R0034833C 023 D 001 après 105 jours à 45°C) did not induce contact sensitization in the murine Local Lymph Node Assay.
Executive summary:

The aim of this study was to evaluate the potential of the test item Mexoryl SBO (termed R0034833C in the study report, batch No. R0034833C 023 D 001 après 105 jours à 45°C) to induce contact sensitization using the murine Local Lymph Node Assay (LLNA) according to OECD TG 429 and GLP.

Twenty-eight female CBA/J mice were allocated to seven groups:

• five treated groups of four animals receiving the test item R0034833C at the concentration of 10, 25, 50, 75 or 100% in dimethylformamide (=DMF),

• one negative control group of four animals receiving the vehicle (= DMF),

• one positive control group of four animals receiving the reference item, α-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25% in DMF.

During the induction phase, the test item, vehicle or reference item was applied over the ears (25 μL per ear) for 3 consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate stimulation indices (SI). The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.

Neither mortality nor clinical signs were observed during the study. A dryness of the skin of the ears was noted on day 6 in 1/4 animals treated at the concentration of 75%. No noteworthy increase in ear thickness was observed in the animals of the treated groups.

As all acceptance criteria were met, this experiment was therefore considered valid. No noteworthy lymphoproliferation was noted at any of the tested concentrations.

The results are presented in the following table:

Treatment

Concentration

Irritation level (%)

Stimulation Index (SI)

Test item

10

non-irritant

1.41

Test item

25

non-irritant

1.15

Test item

50

non-irritant

1.13

Test item

75

non-irritant

0.62

Test item

100

non-irritant

1.11

HCA

25

-

4.97

Under the experimental conditions of this study, the test item R0034833C (batch No. R0034833C 023 D 001 après 105 jours à 45°C) did not induce contact sensitization in the murine Local Lymph Node Assay.

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