Sodium [[α,α'-(ethylenediimino)bis[2-hydroxybenzene-1-acetato]](4-)]ferrate(1-)

Skin sensitisation

Some information in this page has been claimed confidential.

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-04-13 to 2010-04-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2002

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Test material form:

other: Black red, fine grained and free-flowing solid

Details on test material:

- Name of test material (as cited in study report): Acetic acid, oxo-, sodium salt (FeNaEDDHA)
- Physical state: Dark red solid compound
- Analytical purity: 100 % (UVCB)
- Stability under test conditions: The test item was stable in all dose formulation samples for 2 hours at room temperature when protected from light.
- Storage condition of test material: At room temperature, protected from light

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 20.3 +/- 1.1 g
- Housing: single in Makrolon Type II, with wire mesh top cages
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 degree C
- Humidity (%): 45-65%
- Photoperiod (hrs dark / hrs light): 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

5, 10, and 25% (w/w) in dimethylformamide
Application volume: 25 µL

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which can be technically used, was a 25% (w/w) solution in dimethylformamide.
- Irritation:
To determine the highest non-irritant test item concentration that does at the same time not induce systemic toxicity, a pre-test was performed in two animals. Two mice were treated by topical (epidermal) application to the dorsal surface of each ear with test item at concentrations of 10% or 25% once daily each on three consecutive days. Clinical signs were recorded within 1 hour and 24 +/- 4 hours after each application as well as on day 6. Furthermore, prior to the first application of the test item and before sacrifice the ear thickness was determined using a micrometer (S0247 Kroeplin, 36381 Schlüchtern, Germany). Additionally, for both animals, the ears were punched after sacrifice at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm²) and were immediately pooled per animal and weighed using an analytical balance.
At the tested concentrations the animals did not show any signs of systemic toxicity. Also, the animals did not show relevant signs of local irritation as confirmed by the ear thickness and ear weight measurements.


MAIN STUDY
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 5, 10, and 25% (w/w) in dimethylformamide. The application volume, 25 µL, was spread over the entire dorsal surface of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the vehicle alone (control animals).
Five days after the first topical application, all mice were administered with 250 µL of 81.7 µCi/mL 3HTdR (corresponds to 20.4 µCi 3HTdR per mouse) by intravenous injection
via a tail vein.
For the determination of lymph node weights, after excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance. After excision of the lymph nodes, both ears (left and right) of mice were punched at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm²). For each animal both punches were immediately weighed per animal using an analytical balance. Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Sodium. The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared. The level of 3HTdR incorporation was then measured on β -scintillation counter. Similarly, background 3HTdR levels were also measured in two 1mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute
(DPM).
- Criteria used to consider a positive response:
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of lymph nodes of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/animal values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

In addition to the sensitising reactions the following observations were recorded during the test and observation period: Mortality / Viability, Body weights, Ear thickness, Ear weights, Lymph node weights, Clinical signs (local / systemic).

Positive control substance(s):

other: no positive control

Statistics:

The mean values and standard deviations were calculated in the body weight tables. The ANOVA (Dunnett-test) was conducted on the ear and lymph node weights to assess whether the difference is statistically significant between test item groups and negative control (vehicle) group. However, both biological and statistical significance were considered together.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Stimulation Indices (S.I.) of 1.6, 2.9, and 2.96 were determined with the test item at concentrations of 5, 10, and 25% in dimethylformamide, respectively.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: A statistically significant increase in DPM/animal and in lymph node weights was observed in all test item treated groups in comparison to the vehicle control group (p=0.008). A dose response was obtained.

Any other information on results incl. tables

Clinical signs, mortality and body weights:

During the course of the study, the animals did not show any signs of systemic toxicity and no cases of mortality were observed. The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age. Although reddening of the ears could not be assessed due to the intensive colour of the test item, other signs of local irritation (e.g. ear swelling) were also not seen. A statistically significant increase in ear weights was not observed in the test item treated groups in comparison to the vehicle control group.

Table 1: Calculation and results of individual data

Test item concentration % (w/w)	Group number	Animal number	DPM values measured	DPM-BG per animal (2 lymph nodes)^a)	S.I.^b)
—	BG I	—	16	—
—	BG II	—	15	—
0	1	1	488	473	—
0	1	2	1199	1187	—
0	1	3	569	554	—
0	1	4	716	701	—
0	1	5	916	901
5	2	6	887	872	1.1
5	2	7	1375	1360	1.8
5	2	8	1361	1346	1.8
5	2	9	1073	1058	1.4
5	2	10	1428	1413	1.9
10	3	11	3017	3002	3.9
10	3	12	2387	2372	3.1
10	3	13	1367	1352	1.8
10	3	14	3106	3091	4.1
10	3	15	1198	1183	1.6
25	4	16	2256	2241	2.9
25	4	17	2393	2378	3.1
25	4	18	2454	2439	3.2
25	4	19	1269	1254	1.6
25	4	20	3090	3075	4.0

BG = Background (1 mL 5% trichloroacetic acid) in duplicate;

1 = Vehicle Control Group for the Test Item (dimethylformamide);

2-4 = Test Groups S.I. = Stimulation Index;

a) = Values corrected for mean background value (BGI and BGII);

b) = Stimulation Indices relative to the mean of the control group (Group 1)

Table 2: Calculation of Stimulation Indices per dose group

Test item concentration	Mean DPM per Group (5 animals)^a)	SD^b)	S.I.
Vehicle for the Test Item (dimethylformamide)	762.1	286.3	1.0
5 % acetic acid, oxo-, sodium salt	1209.3	234.3^c)	1.6
10 % acetic acid, oxo-, sodium salt	2199.5	897.2^c)	2.9
25 % acetic acid, oxo-, sodium salt	2276.9	655.9^c)	3.0

Table 3: Calculation of the EC3 value

	Test item concentration%	S.I.
Test Group3	10(a)	2.9(b)
Test Group4	25(c)	3.0(d)
EC3=(a-c) [(3-d)/(b-d)]+c=25 %(w/w)

EC3 = Estimated concentration for a S.I. of 3.

Applicant's summary and conclusion

Interpretation of results:

ambiguous

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item FeNaEDDHA resulted to a SI of 2.96 under the conditions of this study for the highest concentration of 25% tested. The results can be considered ambiguous as both the conclusions non-sensiting and sensiting with EC3 value of 25% (w/w) are equally justified.

Executive summary:

In order to study a possible skin sensitization potential of FeNaEDDHA, three groups each of five female mice were treated daily with the test item at concentrations of 5, 10, and 25% (w/w) in dimethylformamide by topical application to the dorsum of each ear (left and right) once daily for three consecutive days. A control group of five mice was treated with the vehicle (dimethylformamide) only. 25% was the highest technically achievable concentration suitable for application which did at the same time not cause excessive skin irritation or systemic toxicity as confirmed by a pre-experiment. The test was performed according to the OECD Guideline 429 and the Commission Directive 2004/73/EC B.42. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in ab-scintillation counter.

Results of the formulation analysis showed that the measured concentrations of FeNaEDDHA were within the acceptable range set at 100 +/- 15% of the nominal concentration. The obtained results ranged between 100.8 – 104.5% of the nominal values. The test item was found to be stable in all dose formulation samples for 2 hours at room temperature when protected from light.

During the course of the study, the animals did not show any signs of systemic toxicity and no cases of mortality were observed. Although reddening of the ears could not be assessed due to the intensive colour of the test item, other signs of local irritation (e.g. ear swelling) were also not seen. A statistically significant increase in ear weights was not observed in the test item treated groups in comparison to the vehicle control group.

In this study Stimulation Indices of 1.6, 2.9, and 2.96 were determined with the test item at concentrations of 5, 10, and 25% in dimethylformamide, respectively. A statistically significant increase in DPM/animal and in lymph node weights was observed in all test item treated groups in comparison to the vehicle control group (p=0.008). A dose response was obtained.

The test item FeNaEDDHA resulted to a SI of 2.96 under the conditions of this study for the highest concentration of 25% tested. The results can be considered ambiguous as both the conclusions non-sensiting and sensiting with EC3 value of 25% (w/w) are equally justified.

Some remarks can be made to this study:

- With a maximum SI of 2.96 at the highest dose, it indicates a very weak sensitizing effect at the most. Also the notion that there is no difference in response between 10% and highest possible concentration of 25%.

- Notably the DPM of the non treated controls of the test group is much lower than that of the non-treated controls of the positive control group. In case these animals are treated exactly similarly (same size and age animals and same amount of radio-labelled thymidine injected and same timing of evaluations) one would expect similar levels. When considering the possibility that control group shows accidentally a too low activity, the study would be evaluated as negative.