Registration Dossier

Administrative data

Description of key information

Local Lymph Node Assay (LLNA): sensitising (EU B.42, GLP compliant).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24.10.2012 - 6.11.2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: 16.50 – 19.66 g
- Housing: Maximum six in macrolon cages with sterilized softwood shavings
- Diet (e.g. ad libitum): Pelleted standard diet for experimental animals ad libitum
- Water (e.g. ad libitum): Drinking tap water ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.4 – 21.6 °C
- Relative humidity (%): 30-70 %
- Air changes (per hr): Approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

STUDY TIME SCHEDULE
Animal arrival/ start of acclimatization: 16.10.2012
Pilot experiment: 24.10.12 - 29.10.2012
Main study:
First day of administration: 31.10.2012
End of treatment period: 02.11.2012
Application of radionuclide and necropsy: 05.11.2012
Vehicle:
other: DAE 433 - mixture of 40 % dimethylacetamide, 30 % acetone and 30 % ethanol
Concentration:
Concentrations in formulation:
50 % (w/v) - 500 mg/mL
5 % (w/v) - 50 mg/mL
0.5 % (w/v) - 5 mg/mL
No. of animals per dose:
5 females
Details on study design:
PILOT EXPERIMENT
The pilot experiment was conducted under the conditions identical to the main study, except assessment of lymph node proliferation. The appropriate dilution of the test substance (50 %, 5 %, 0.5 % w/v) was applied to three animals in volume 25 µl to the dorsum of each ear once a day morning for 3 consecutive days. Both ears of each mouse was observed for erythema and scored and subsequently thickness was measured using digital thickness gauge

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Animals were subjected to a clinical examination (health check) shortly after arrival. No clinical changes were recorded.
Study animals were randomly allocated to the dose groups manually and assigned animal numbers.

TREATMENT PREPARATION AND ADMINISTRATION:
Dosage volume: 25 μL / ear / animal
Preparation for administration: The suspensions were prepared before the start of application by mixing on magnetic stirrer and then were still mixed during application.
Frequency of preparation: On each day immediately before administration.

IN VIVO EXAMINATION
- mortality
- clinical observation
- body weight

POST MORTEM INVESTIGATIONS
- ears weights
- incorporation of 3H-methyl thymidine

DATA ANALYSIS
Mean values and standard deviations of ears weight and incorporation of 3H-methyl thymidine were computed for the test item groups and for the positive as well as the vehicle control group. Stimulation index (for incorporation of 3H-methyl thymidine) was calculated by dividing mean values from the test substance groups and the positive control group by the corresponding mean value of the vehicle control group. The index for the vehicle control group was set at 1 by definition.
Positive control substance(s):
other: Dinitrochlorobenzene (DNCB)
Statistics:
For statistical calculations the software Statgraphic ® Centurion (version XV, USA) was used. Statistical evaluation of measured parameters was performed at first by applying the non-parametric Kruskal-Wallis test for the comparison of the measured effect in all treatment groups with the vehicle control group, as global test, and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) for all two-group comparisons.
Positive control results:
The positive control substance DNCB produced positive LLNA response at an exposure level expected to give an increase in the Stimulation Index SI ≥ 3 over the negative control group, which was in congruence with the expected mode of action of a contact allergen. The positive control also elicited a reaction pattern with statistically significant increase in ear weight. These results demonstrate that the method performed in conditions of our laboratory has sufficient reliability.
Key result
Parameter:
SI
Value:
8.35
Test group / Remarks:
50 %
Remarks on result:
other: see Remark
Remarks:
The test substance at the highest dose level caused a dose dependent increase in radioisotope incorporation into the DNA of dividing lymphocytes.
Key result
Parameter:
SI
Value:
3.27
Test group / Remarks:
5 %
Remarks on result:
other:
Remarks:
The test substance at the highest dose level caused a dose dependent increase in radioisotope incorporation into the DNA of dividing lymphocytes
Key result
Parameter:
SI
Value:
1.64
Test group / Remarks:
0.5 %
Remarks on result:
other:
Remarks:
0.5% is below the threshold
Parameter:
other: DPM
Value:
943.17
Test group / Remarks:
50 %
Parameter:
other: DPM
Value:
369.29
Test group / Remarks:
5 %
Parameter:
other: DPM
Value:
184.83
Test group / Remarks:
0.5 %

Summary table

Group

Radioisotope incorporation

Ear weight

Mean DPM

SI

Mean (mg)

NC

112.89

1.00

24.10

PC

890.38

 7.89+

 34.84*

50%

943.17

 8.35+

 46.34*

5%

369.29

 3.27+

 26.08*

0.5%

184.83

1.64

25.04

Figures with asterisk * = values statistically significant on probability level < 0.05 (Mann-Whitney test)

Figures with cross + = values ≥ 3 

DISCUSSION

The animals exposed to the test substance showed no clinical symptoms of intoxication throughout the experiment. The skin reaction could not be evaluated because the test substance caused marked colouration of skin.

 

The test substance Solvent Red 19E showed a tendency to increase ear weight with relation to the dose. Statistically significant increase of ear weight was recorded in animals at the highest and the middle dose level (50%, 5% test substance concentrations). The test substance showed a tendency to form residues. Residues of the test substance on the ears could cause this weight increase. With respect to the results of the study No. 188/12/4 – Histopathological examination it could not be excluded possibility of irritating effect.

Interpretation of results:
sensitising
Conclusions:
The test material elicited a sensitising response in LLNA assay and therefore could be a contact allergen in mice.
Executive summary:

The test material was assessed for skin sensitisation potential according to the Method B.42 – Skin Sensitisation: Local Lymph Node Assay, Council Regulation (EC) No.640/2012, and OECD Test Guideline 429.

In this study the contact allergenic potential was evaluated after topical application to female BALB/c mice. Five mice per group were exposed on the dorsum of both ears once a day by test substance and control substances during 3 consecutive days. Primary proliferation of lymphocytes in the lymph node draining the site of application was evaluated using radioactive labelling of proliferating cells. The ratio of the proliferation in treated groups to that in vehicular controls, termed the Stimulation Index, was determined.

The evaluation of ear weight was performed for elimination of false positive findings with certain skin irritants.

Concentrations of test substance in vehicle (DAE 433): 50%, 5%, 0.5% (w/v) The animals exposed to the test substance at all concentrations showed no pathological and no other negative clinical symptoms of intoxication throughout the experiment. The test substance caused marked colouration of skin on dorsum of both ears of animals so the assessment of erythema and other skin reactions could not be evaluated.

There was no significant difference in body weight increment of all groups in comparison to the vehicle control.

The positive control substance DNCB (concentration 0.5 % (w/v) elicited a reaction pattern with statistically significant increase in Stimulation Index of cell proliferation and of ear weight, which was in congruence with his expected mode of action as a contact allergen.

The test substance showed a tendency to increased ear weight in the highest and the middle of concentrations tested. Residues of the test substance on the ears could cause this weight increase.

Comparison of Stimulation Indexes between all treated groups and control vehicle group revealed that the test substance caused a significant and dose dependent increase in radioisotope incorporation into the DNA of dividing lymphocytes. The Stimulation Index of the highest treated group (50% w/v) was 8.35 and of the middle treated group 3.27.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The test material was assessed for skin sensitisation potential according to the Method B.42 – Skin Sensitisation: Local Lymph Node Assay, Council Regulation (EC) No.640/2012, and OECD Test Guideline 429.

In this study the contact allergenic potential was evaluated after topical application to female BALB/c mice. Five mice per group were exposed on the dorsum of both ears once a day by test substance and control substances during 3 consecutive days. Primary proliferation of lymphocytes in the lymph node draining the site of application was evaluated using radioactive labelling of proliferating cells. The ratio of the proliferation in treated groups to that in vehicular controls, termed the Stimulation Index, was determined.

The evaluation of ear weight was performed for elimination of false positive findings with certain skin irritants.

Concentrations of test substance in vehicle (DAE 433): 50, 5, 0.5 % (w/v) The animals exposed to the test substance at all concentrations showed no pathological and no other negative clinical symptoms of intoxication throughout the experiment. The test substance caused marked colouration of skin on dorsum of both ears of animals so the assessment of erythema and other skin reactions could not be evaluated.

There was no significant difference in body weight increment of all groups in comparison to the vehicle control.

The positive control substance DNCB (concentration 0.5 % (w/v) elicited a reaction pattern with statistically significant increase in Stimulation Index of cell proliferation and of ear weight, which was in congruence with his expected mode of action as a contact allergen.

The test substance showed a tendency to increased ear weight in the highest and the middle of concentrations tested. Residues of the test substance on the ears could cause this weight increase.

Comparison of Stimulation Indexes between all treated groups and control vehicle group revealed that the test substance caused a significant and dose dependent increase in radioisotope incorporation into the DNA of dividing lymphocytes. The Stimulation Index of the highest treated group (50 % w/v) was 8.35 and of the middle treated group 3.27.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does require classification with respect to skin sensitisation, Category 1B (H317; May cause an allergic skin reaction).