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Description of key information

Key value for chemical safety assessment

Skin sensitisation

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

- 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

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A study evaluating the potential of the test item Mexoryl SBO to induce contact sensitization using the murine Local Lymph Node Assay (LLNA) is available. The study was conducted according to OECD TG 429 and was performed under GLP.

Five groups of four female mice received the test item at the concentration of 10, 25, 50, 75 or 100% in dimethylformamide (DMF), one negative control group of four animals receiving the vehicle (DMF) and one positive control group of four animals received 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. 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. 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.The stimulation indexes were all below 1.41 for the test item. Treatment with 25 % HCA (v/v) in DMF led to 88.66 % cell viability and a SI of 4.97. Under the experimental conditions of this study, the test item Mexoryl SBO did not induce contact sensitization in the murine Local Lymph Node Assay.

Another murine Local Lymph Node Assay (LLNA) was performed using the test item. The study was conducted under GLP and according to OECD TG 429 (Pelcot 2008). A preliminary test was performed to confirm concentrations for the main test. The experimental design of the main test, group allocation and dose levels were the same as in the above mentioned study.No mortality and no clinical signs were observed during the study.No cutaneous reactions and no noteworthy increase in ear thickness were observed in the animals of the treated groups. All validity criteria of the study were respected. No noteworthy lymphoproliferation was noted at any of the tested concentrations of both batches.The stimulation indexes were all below 1.57 for both batches of the test item. Treatment with 25% HCA (v/v) in DMF resulted in a SI of 5.54. Based on the results of this study the batches Nos. 1 and 2 of the test item Mexoryl SBO did not induce contact sensitization in the murine Local Lymph Node Assay

Migrated from Short description of key information:

not sensitising, mouse, OECD 429

Justification for selection of skin sensitisation endpoint:

two GLP compliant and guideline conform skin sensitisation studies

Respiratory sensitisation

Endpoint conclusion
Additional information:

No data on respiratory sensitisation is available.

Migrated from Short description of key information:

No data available

Justification for classification or non-classification

Mexoryl SBO was determined to be not sensitising to the skin in a LLNA test. The substance has therefore not to be classified as sensitising to the skin according to Regulation (EC) No 1272/2008 (CLP).

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