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Description of key information

The Local Lymph Node Assay (LLNA) with radionuclides was used. The testing was conducted according to the EU Method B.42, Skin sensitization: Local Lymph Node Assay, Council Regulation (EC) No. 440/2008, published in O.J. L142, 2008. GLP study.

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:
27.07 - 10.08.2009
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)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
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, RČH CZ 21760118
- Age at study initiation: 8 to 10 weeks (at start of dosing)
- Weight at study initiation: 19.5 to 22.4 g (at start of dosing)
- Housing: Animals in groups of maximum six in macrolon cages with sterilized softwood shavings
- Diet: Pelleted standard diet for experimental animals ad libitum. Microbiological control and content of nutrients will be performed according SOP No. 72
- Water: Drinking tap water ad libitum. Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Health Ministry
- Acclimation period: 5 days
- Animal rooms: Monitored conditions, microbiologically defined background, according to SOP No.40
- Prophylactic arrangement: Cleaning and disinfection of animal room was regularly performed as it is described in appropriate SOP No.10
- Health examination: All animals were examined during the acclimatisation period
- Cage identification: By cage number, study number and group specific colour.
- Animal identification: By felt tip marking (from 1 to 5 per each group and group specific colour)
- Random selection: According to SOP No.42

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Room temperature 22 + 3°C, permanently monitored
- Humidity (%): Relative humidity 30 – 70 %, permanently monitored
- Air changes (per hr): approximately 15/air changes per hour
- Photoperiod (hrs dark / hrs light): Light: 12 hours light/dark cycle: 6am-6pm/6pm-6am


STUDY TIME SCHEDULE
Animal arrival/ start of acclimatization: 22.07.2009
Pilot experiment: 27.- 30.07 2009

MAIN STUDY
First day of administration: 05.08.2009
End of treatment period: 07.08.2009
Application of radionuclide and necropsy: 10.08.2009
Experimental completion date: 17.08.2009
Date of final report elaboration: 11.09.2009
Vehicle:
other: DAE 433 - mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol
Concentration:
30% (w/v) 300 mg/mL
3% (w/v) 30 mg/mL
0.3% (w/v) 3 mg/mL
No. of animals per dose:
Pilot experiment – 3 females
Exposed groups - 15 females (5 animals in three groups)
Positive control group – 5 females
Negative control group –5 females
Reserve group – 2 females
Details on study design:
PRINCIPLE OF THE TEST METHOD
The basic principle of the Local Lymph Node Assay (LLNA) is that sensitizers induce a primary proliferation of lymphocytes in the lymph node draining the site of chemicals application. The proliferation is proportional to the dose applied (and to the potency of the allergen) and provides a simple means of obtaining an objective, quantitative measurement of sensitisation. The LLNA assesses this proliferation as a dose-response relationship in which the proliferation in test group is compared to that in vehicle treated controls, termed the Stimulation Index. This index served for the classification of the substance in respect of its sensitizing properties. The method is based on the use of radioactive labelling to measure cell proliferation.

PILOT EXPERIMENT
The highest concentration 30% (maximum technically practicable concentration) was administered to three animals to assess a possible systemic toxicity. The route of administration was same as in the main study. During pilot experiment no clinical symptoms of systemic toxicity and no macroscopic changes (after necropsy) were found out in all three animals.

MAIN STUDY
Animal check-in and allocation
Animals were subjected to a clinical examination (health check) shortly after arrival. Study animals were randomly allocated to the dose groups manually and assigned

Application
The volume of the application form was constant at all groups of animals - 25 l of the appropriate dilution to the dorsum of each ear once a day morning for 3 consecutive days. The application was performed very slowly by micropipette. Losses caused by draining from the ear must be minimized.
This route of administration is listed in the guideline and it is similar to expected exposure conditions at the workplace. The application form of test substance was prepared immediately before administration.
Dosage volume: 25μL / ear / animal

Preparation for administration:
All suspensions were prepared by suspending an appropriate amount of Semi Dry Absorption (SDA) Product in the vehicle to obtain a concentration of 30%, 3% or 0.3% (w/v). The suspensions were prepared before the start of application by mixing on magnetic stirrer and then were still mixed during application.

Frequency of preparation: Each day of administration

In vivo examination
Mortality
During working hours the animals were checked for general health whenever other activities were performed – at least twice daily during the application period.

Clinical observations
Clinical signs were assessed using a defined scoring system described in SOP M/8 – at least twice daily during the dosing period. Efforts were made to characterize onset and duration of signs observed.

Body weight
Individual body weights were measured using an electronic balance. Weighing was performed on the first day of treatment before dosing and on the day of necropsy before application of radionuclide.

Necropsy
Third day after last administration (five hours after application of radionuclide), all test animal were sacrificed by injection of veterinary preparation T 61 (0.5 mL ip.)

T 61* inj. a.u.v.
Manufacturer: Intervet International GmbH, Feldstrasse 1A, 85716 Unterschleissheim, Germany
Composition: Embutramidum 200mg, Mebenzonii iodidum 50 mg, Tetracaini hydrochloridum 5 mg in 1 mL of water for injections

POST MORTEM INVESTIGATION
Ears weights
Immediately after death, the both ears were cut off and circular pieces from the apical area of each ear with a diameter of 8 mm (=0.5 cm2) were excised using a disposable punch and weighed together on analytical scales.

Incorporation of 3H-methyl thymidine
Both of the lymph nodes were prepared by gentle mechanical disaggregation through 100 m-mesh nylon gauze with pooling of 1 mL PSB (Phosphate Buffered Saline). Cells were washed twice an excess of PBS and precipited with 5% trichloroacetic acid (TCA) at 4 oC for 18h. Pellets were re-suspended in 1 mL TCA and transferred to scintillation vials containing 10 mL of scintillation fluid. Incorporation of 3H-methyl thymidine were measured by β-scintillation counting (Beckmann LS 6500) as disintegrations per minute (DPM).


DATA ANALYSIS
Mean values and standard deviation of ears weight and incorporation of 3H-methyl thymidine were computed for the test substance groups and for the positive as well as the vehicle control group. Results (incorporation of 3H-methyl thymidine) for each treatment group were expressed as the mean SI. The Stimulation Index was the ration of the mean dpm/mouse within each test substance treatment group against the mean dpm/mouse for the vehicle treated 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. At first the global comparison of all three values of the concentration groups with vehicle control was performed by applying the non-parametric Kruskal-Wallis test, and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) was applied to all two-group comparisons.
Positive control results:
All animals in the positive control group showed these symptoms: hyperaemia of skin and clonospasm.
In the positive control group, the SI was ≥ 3 (12.31) – test LLNA was efficient.
In the positive control group the weight of ear target was statistically significantly increased against negative control group – the test design used is efficient in the detection of irritation effect.
Key result
Parameter:
SI
Value:
< 3
Remarks on result:
other: The SI for the test groups treated by the test substance was not increased with dependence on the dose level. At the all dose levels the SI was belong threshold.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See table No. 6

Table No. 6. Summary of results

Group

Radioisotope incorporation

Ear weight

Mean DPM

SI

Mean (mg)

NC

711.51

1.00

23.62

PC

8756.00

12.31+

34.38*

30%

683.34

0.96

24.98

3%

709.13

1.00

23.64

0.3%

1480.39

2.08

25.14

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

+ Figures with cross = values ≥ 3

DPM disintegrations per minute

NC – Negative control group

PC – Positive control group

The SI for the test groups treated by the test substance was not increased with dependence on the dose level. At the all dose levels the SI was belong threshold.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the given test conditions the test substance Semi Dry Absorption (SDA) Product elicited negative response in LLNA test.
Executive summary:

The test substance, Semi Dry Absorption (SDA) Product, was tested for the assessment of skin sensitisation potential with the murine local lymph node assay. This study is a part of the test substance health hazard evaluation.

The Local Lymph Node Assay (LLNA) with radionuclides was used. The testing was conducted according to the EU Method B.42, Skin sensitization: Local Lymph Node Assay, Council Regulation (EC) No. 440/2008, published in O.J. L142, 2008.

In this study the contact allergenic potential of Semi Dry Absorption (SDA) Product was evaluated after topical application to female BALB/c mice. Five mice per group were exposed by test and control substances on the dorsum of both ears once a day during 3 consecutive days. Primary proliferation of lymphocytes in the lymph node draining the site of application was evaluated on the base on using of radioactive labelling. The ratio of the proliferation in treated groups to that in vehicular controls, termed the Stimulation Index, was determined. Statistical evaluation of ear weight was performed for elimination of potential false positive findings with certain skin irritants.

Concentrations: positive control DNCB (dinitrochlorobenzene): 0.5% (w/v) and Fluidized Bed Combustion (FBC) Fly Ash: 30%, 3%, 0.3% (w/v) in DAE 433.

  The animals exposed to the test substance at the all dose levels showed no pathological skin reactions and no other negative clinical symptoms of intoxication throughout the experiment. There was no significant difference in body weight increment of treated groups in comparison to the vehicle control.

The positive control substance DNCB elicited a reaction pattern with statistically significant increase in ear weight and Stimulation Index of cell proliferation 12.31, which was in congruence with his expected mode of action as a contact allergen.

Ear weight of animals in all dose level was not statistically significantly increased. The result of skin irritation effect was not considered as positive.

Comparison of Stimulation Indexes between treated groups and control group revealed that the test substance Semi Dry Absorption (SDA) Product did not cause significant increase in radioisotope incorporation into the DNA of dividing lymphocytes.

The test substance Semi Dry Absorption (SDA) Product had a negative result in LLNA test. Negative results in cell proliferation and no clinical symptoms of systemic toxicity revealed that the test substance Semi Dry Absorption (SDA) Product is not a contact allergen in mice.

Based on the negative results of LLNA study of the test substance Semi Dry Absorption (SDA) Product, it could be suggested, that the test substance does not have to be classified as the substance, which may cause sensitisation by skin contact.

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

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

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 sensitivity. The animals exposed to test substance showed no clinical symptoms of intoxication throughout the experiment. Ear weight of animals in all dose level was not statistically significantly increased. The result of skin irritation effect was considered as negative – it means the test substance did not cause irritation of skin.

Comparison of Stimulation Indexes between treated groups and control group revealed that the test substance, Semi Dry Absorption (SDA) Product, did not cause significant increase in radioisotope incorporation into the DNA of dividing lymphocytes.

Based on the negative results of LLNA study of the test substance Semi Dry Absorption (SDA) Product, it could be suggested, that the test substance does not have to be classified as the substance, which may cause sensitisation by skin contact.