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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:
from August 8, 2007 to September 20, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was carried out in accordance with internationally valid GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
During the study the deviations from the guideline limit for temperature (20±3ºC) in animal room were recorded. The temperature was higher (up to 25,6ºC). These temperature fluctuations did not affect the welfare of animals and had no impact on the study.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Humic acids, sodium salts
- Molecular formula: not known - UVCB substance
- Molecular weight: not known - UVCB substance
- Substance type: technical product
- Physical state: solid
- Lot/batch No.: 9. 5. 2007/R
- Expiration date of the lot/batch: 05/2022
- Stability under test conditions: stable
- Storage condition of test material: dry conditions

In vivo test system

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeding farm BioTest s.r.o., Konárovice, Czech Republic
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: 19 to 24 g
- Housing: Animals in groups of 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: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Room temperature 22 +/- 3ºC, permanently monitored
- Humidity (%): Relative humidity 30 – 70 %, permanently monitored
- Photoperiod (hrs dark / hrs light): Light: 12 hours light/dark cycle

Study design: in vivo (LLNA)

Vehicle:
other: DAE 433 - mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol
Concentration:
30% (w/v)
3% (w/v)
0.3% (w/v)

No. of animals per dose:
Pilot experiment: 3 females
Exposed groups: 18 females (6 animals in 3 groups)
Positive control group: 6 females
Negative control group: 6 females
Details on study design:
RANGE FINDING TESTS:
- Irritation: Method LLNA contains also determination of irritation effect of the test substance (weight comparison of circular target from ears) for elimination of false positive result.
- Lymph node proliferation response: The proliferation – cell concentration in lymph node is measured by cell counter and provides a simple means of obtaining an objective, quantitative measurement in test groups is compared to that in vehicle treated controls.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
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 – cell concentration in lymph node is measured by cell counter and provides a simple means of obtaining an objective, quantitative measurement in test groups is compared to that in vehicle treated controls. Method LLNA contains also determination of irritation effect of the test substance (weight comparison of circular target from ears) for elimination of false positive result.
The results of the LLNA were evaluated according to the following criteria.
The following thresholds were determined by Ulrich (2007) from analysis of historical data:
Ear weight index: 1.05
LN weight: 1.2
LN cell count:1.3

1. Values which exceed these thresholds were considered positive
- when a statistically significant increase in one of the parameters occurs and a clear concentration-dependence can be derived
- or with no statistical significance, but a clear concentration-dependence.

2. Values which are below these thresholds were considered positive
- when a statistical significant occurs in one of the parameters together with a clear concentration-dependence.

3. Values were considered negative:
- in case of being below the thresholds and without a statistical significance
- in case of being below the thresholds, with a statistical significance, but without a clear concentration-dependence
- in case of being above the thresholds, without statistical significance and without a clear concentration-dependence.

TREATMENT PREPARATION AND ADMINISTRATION:
The volume of the application form was constant at all groups of animals - 25 microliter 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 guideline and it is similar to expected exposure conditions at the workplace. The application form of test substance was prepared immediately before administration.
Positive control substance(s):
other: Dinitrochlorbenzene, 0.5% (w/v) solution. A dose level for this positive control was determined by Ulrich (2007). The vehicle and dosage volume were the same as in treated groups.
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.

Results and discussion

Positive control results:
In the positive control group, the weight of ear target was statistically significantly increased against negative control group – test LLNA was efficient.See Table.

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: Index = Mean Cell Count (Treated Group)/(Control group) Indexes (LN weight, LN cell count and ear weight) - see Table

Any other information on results incl. tables

Summary of results

Group

LN weight

LN cell count

Ear weight

Mean  (mg)

Index

Mean (x106/mL)

Index

Mean (mg)

Index

NC

4.92

1.00

7.13

1.00

23.72

1.00

PC

16.43*

3.34+

28.02*

3.93+

31.77*

1.34+

0.3%

5.97

1.21+

6.93

0.97

23.73

1.00

3%

6.88*

1.40+

9.58

1.34+

22.98

0.97

30%

8.10*

1.65+

11.97*

1.68+

23.38

0.99

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

Figures with cross = values exceeding thresholds

NC = Negative control group

PC = Positive control group

Index = Mean Cell Count (Treated Group)/(Control Group)

Applicant's summary and conclusion

Conclusions:
The positive control substance DNCB elicited a reaction pattern with statistically significant increase in ear weight and LN hyperplasia, which was in congruence with the expected mode of action of a contact allergen. In the positive control groups (with DNCB) the LN weight, LN cell count and ear weight were statistically significantly increased from negative control values.


The animals exposed to test substance showed no pathological skin reactions and no other negative clinical symptoms of intoxication throughout the experiment.

The test substance did not show a tendency to increase ear weight in anyone of dose levels tested. These results confirmed existing dermal irritation data (acute dermal irritation study was performed in test facility with negative results, see study No. 27/07/4 - endpoint Skin irritatoin/corrosion.001).

Comparison of values between treated groups and control group revealed that the test substance caused statistically significantly increased LN cell count in the highest dose level and LN weight in middle and the highest dose level. Index of LN weight was exceeded in all dose levels. Index of LN cell count exceeded the threshold in middle and the highest dose levels. Test substance caused a dose-dependent increase in LN weight and cell counts. According to criteria given in chapter "Details on study design - LLNA" the values of both parameters were considered positive.
The calculation for the LN hyperplasia threshold concentration (by linear regression) was performed with the respective LN cell count threshold index. Calculated threshold concentration is 12.7% (w/v). According to three scenarios the test substance could be assigned as a weak sensitizer and no further action is needed.

Executive summary:

The test substance was tested for the assessment of skin sensitisation potential.   

The Local Lymph Node Assay (LLNA) was used. This method was performed with endpoint different from that described in original guideline (non Radioactive measuring of cell proliferation). This testing was conducted according to EU method B.42 with modifications as described in publications of Ulrich P, Streich J, Suter W, 2001; Ehling et al., 2005; Ehling et al., 2005A.

This study was conducted to evaluate the contact allergenic potential after topical application to female BALB/c mice. Six mice per group were exposed by test and control substances on the dorsum of both ears once a day during 3 consecutive days. Lymph nodes were taken 24 hours after the last application. Concentrations: positive control DNCB (dinitrochlorobenzene): 0.5% (w/v) and Humic acids, sodium salts: 30%, 3%, 0.3% (w/v). Endpoints: ear weight, auricular (ear-draining) lymph node weights and cell counts = lymph node (LN) hyperplasia.

The animals exposed to test substance showed no pathological skin reactions and no other negative clinical symptoms of intoxication throughout the experiment. There was no difference in body weight increment of all groups in comparison to the vehicle control. There were no clinical observations attributable to treatment with test substance.

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

                         

The test substance did not show a tendency to increase ear weight in anyone of dose levels tested. These results confirmed existing dermal irritation data (acute dermal irritation study was performed in test facility with negative results - see endpoint Skin irritation/corrosion.001).

Comparison of values between treated groups and control group revealed that the test substance Humic acids, sodium salts caused statistically significantly increased LN cell count in the highest dose level and LN weight in middle and the highest dose level. Index of LN weight was exceeded in all dose levels. Index of LN cell count exceeded the threshold in middle and the highest dose levels. Test substance caused a dose-dependent increase in LN weight and cell counts. Hence, at the given test conditions the test substance Humic acids, sodium salts, provided a weak positive response in LLNA test.