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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:
2010-08-17 until 2010-08-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well performed GLP and OECD guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Manganese Oxalate, dihydrate
IUPAC Name:
Manganese Oxalate, dihydrate

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS: mice, CBA/CaOlaHsd
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 18.5 - 22.4 g
- Housing:single caging
- Diet: pelleted standard diet (Harlan Laboratories B.V., 5960 AD Horst, The Netherlands), ad libidum
- Water: tap water, (Gemeindewerke, 64380 Rossdorf, Germany), 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
- Bedding: granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-68 %
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.


Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
The highest test item concentration, which can be technically used was a 10 % (w/v) suspension in propylene glycol.
In the pre-test two mice were treated with test item concentrations of 5% and 10% (w/v).
The test item in the main study was assayed at 2.5, 5, and 10 % (w/v).
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
Compound solubility: The highest test item concentration, which can be technically used was a 10 % (w/v) suspension in propylene glycol. At higher concentrations an applicable formulation of the test item was not achieved.
In the pre-test, two mice were treated with test item concentrations of 5 and 10%.
At the tested concentrations the animals did not show any signs of local skin irritation or systemic toxicity.

MAIN STUDY:

TOPICAL APPLICATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 2.5, 5, and 10% (w/v) in propylene glycol. The application volume, 25 µL/ear/day, 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 relevant vehicle alone (control animals).

ADIMINISTRATION OF 3H-METHYL THYMIDINE
Five days after the first topical application, all mice were intraveneously injected into a tail vein with radio-labelled thymidine (3HTdR). Approximately five hours after treatment with 3HTdR all mice were sacrificed and the draining lymph nodes were excised and pooled per group (8 nodes per group). Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed with phosphate buffered saline and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a ß-scintillation counter.

INTERPRETATION OF RAW DATA:
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index S.I.). Before DPM/node 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.

OBSERVATIONS:
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:

Mortality / Viability:
Once daily (week day) from experimental start to necropsy.

Body weights:
In the pre-test: prior to the first application and prior to sacrifice.
In the main experiment: prior to the first application and prior to treatment with 3HTdR.

Clinical signs (local / systemic):
In the pre-test clinical signs were recorded within 1 hour and 24 ± 4 hours after each application as well as on day 7.
In the main experiment: clinical signs were recorded within 1 hour after each application, and 24 ± 4 hours after the first and second application as well as on the day of preparation. Especially the treatment sites were observed carefully.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Mean values and standard deviations were calculated for the body weights.

Results and discussion

Positive control results:
Experiment performed in May 2010 using concentrations of 5, 10, and 25 % alpha-hexyl cinnamic aldehyde in acetone:olive oil (4:1). These concentrations yielded S.I.´s of 2.04, 3.41, and 6.14, respectively.

The EC3 value calculated was 8.5 % (w/v).

The positive control substance alpha-hexyl cinnamic aldehyde was found to be a skin sensitizer under the described conditions, demonstrating the validity of the study.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
In this study Stimulation Indices of 1.00, 1.18, and 1.14 were determined with the test item at concentrations of 2.5, 5, and 10% in propylene glycol. A dose response was not observed. The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM per lymph node Control Group: 229.6 2.5% (w/v): 230.7 5.0% (w/v): 270.9 10% (w/v): 261.7

Any other information on results incl. tables

Calculation and results of individual data

Vehicle: propylene glycol

Test item concentration % (w/v)

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

15

---

---

---

---

---

BG II

20

---

---

---

---

---

1

1854

1837

8

229.6

1.00

2.5

2

1863

1846

8

230.7

1.00

5

3

2185

2168

8

270.9

1.18

10

4

2111

2094

8

261.7

1.14

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

1    =  Control Group

2-4 =  Test Group

S.I. =  Stimulation Index

a)   =  The mean value was taken from the figures BG I and BG II

b)    =  Since the lymph nodes of the animals of a dose group were pooled,

DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

The EC3 value could not be calculated, since all S.I.´s are below 3.

VIABILITY / MORTALITY

No deaths occurred during the study period.

CLINICAL SIGNS

No symptoms of local skin irritation at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS

The body weight of the animals, recorded prior to the first application (range: 18.5 - 22.4 g, mean +/- SD: 20.9 +/- 1.1) and prior to treatment (range: 19.1 - 23.2 g, mean +/- SD: 21.4 +/- 1.0) with 3HTdR, was within the range commonly recorded for animals of this strain and age.


Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item Manganese oxalate, dihydrate was not a skin sensitiser under the described conditions.
Executive summary:

In the study the test item Manganese oxalate, dihydrate suspended in propylene glycol was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay was performed using test item concentrations of 2.5, 5, and 10% (w/v).

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 1.00, 1.18, and 1.14 were determined with the test item at concentrations of 2.5, 5, and 10% in propylene glycol, respectively. A dose response was not observed.

The test item Manganese oxalate, dihydrate was not a skin sensitiser under the test conditions of this study.

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