Registration Dossier

Diss Factsheets

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-01-05 to 2010-01-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted to current accepted guidelines.

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 (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Manganese oxide
EC Number:
215-695-8
EC Name:
Manganese oxide
Cas Number:
1344-43-0
Molecular formula:
MnO
IUPAC Name:
manganese oxide
Details on test material:
- Name of test material : MnO (Erachem)
- Substance type: Green powder
- Physical state: solid
- Impurities (identity and concentrations): Fe - 96 ppm, Ins ac -15 ppm, H20 @ 105°c - 0.01%
- Composition of test material, percentage of components: Mn -77.8 %, MnO2 - 0.46%
- Purity test date: 29/09/08
- Lot/batch No.: 104833-06
- Storage condition of test material: room temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Bicester. Oxon, UK
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 15 to 23 g
- Housing: Individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet : Free access to 2014 Teklad Global Rodent diet (Harlan Teklad, Bicester, Oxon, UK)
- Water : Free access to mains tap water
- Acclimation period: 5 days minimum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): 15 changes per hour
- Photoperiod (hrs dark / hrs light): Twelve hour cycle (06:00 to 18:00 continuous light).

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
2.5, 5 and 10 % w/w
No. of animals per dose:
4 animals per dose in the main test, with one animal in the preliminary test.
Details on study design:
RANGE FINDING TESTS:
- Irritation and toxicity : A preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the test material at a concentration of 10 % w/w in propylene glycol to the dorsal surface of each ear for three consecutive days. The mouse was observed daily on days 1, 2 and 3 and once a day on days 4, 5 and 6. Signs of toxicity or excessive local irritation were recorded. The bodyweight of the mouse was recorded on day 1 (prior to dosing) and 6.
- Compound solubility: The vehicle was chosen based on its ability to produce the highest concentration that was suitable for dosing. The concentration for dosing was selected on the basis that the dose produces a solution or fine homogenous suspension that can be administered via a micropipette.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporations will be classified as a non-sensitiser.


TREATMENT PREPARATION AND ADMINISTRATION: Groups of four mice were treated with the test material at concentrations of 10%, 5% or 2.5 w/w in propylene glycol. The mice were treated daily by application of 25 µL of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (1, 2, and 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone in the same manner.

Five days following the first topical application of the appropriate treatment all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmol) giving a total of 20 µCi to each mouse.

Five hours following administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.

A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labelled with the project number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 mL of 5 % Trichloroacetic acid (TCA).

After 18 hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase Trisafe). 3HTdR incorporation was measured by β-scintillation counting. The Poly Q vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes to reduce the risk of luminescence. After 20 minutes the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measure using the Beckman LS6500 scintillation system.
Positive control substance(s):
other: Phenylacetaldehyde (90%)
Statistics:
The stimulation Index was calculated by dividing the mean radioactive incorporation for each treatment group by the mean radioactive incorporation of the vehicle control.

Results and discussion

Positive control results:
The Stimulation Index for Phenylacetaldehyde (90%) was considered to be a positive sensitiser under the conditions of the test(see table 1).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.21
Test group / Remarks:
2.5 % (w/w) in propylene glycol
Parameter:
SI
Value:
1.31
Test group / Remarks:
5 % (w/w) in propylene glycol
Parameter:
SI
Value:
1.03
Test group / Remarks:
10 % (w/w) in propylene glycol
Parameter:
other: Disintegrations per minute (DPM).
Value:
4 535.63
Test group / Remarks:
2.5 % (w/w) in propylene glycol
Parameter:
other: Disintegrations per minute (DPM).
Value:
4 876.24
Test group / Remarks:
5 % (w/w) in propylene glycol
Parameter:
other: Disintegrations per minute (DPM).
Value:
3 853.15
Test group / Remarks:
10 % (w/w) in propylene glycol

Any other information on results incl. tables

Table 2. The stimulation index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

 

Concentration (% w/w) in propylene glycol

Stimulation Index

Result

2.5

1.21

Negative

5

1.31

Negative

10

1.03

Negative

 

Table 3. Clinical observations, Bodyweight and Mortality Data – Preliminary Screening Test.

 

Concentration (% w/w) propylene glycol

Animal Number

Bodyweight (g)

Day

1

2

3

4

5

6

Day

1

Day

6

Pre-Dose

Pre-Dose

Pre-Dose

Pre-Dose

Pre-Dose

Pre-Dose

10

S-1

20

20

0

0

0

0

0

0

0

0

0

 

0 = No signs of systemic toxicity

 

Table 4. Disintegrations per minute, Disintegrations per Minute/Node and Stimulation Index

 

Concentration (% w/w) in propylene glycol

dpm

Dpm/Nodea

Stimulation Indexb

Result

Vehicle

3734.44

466.81

na

na

2.5

4535.63

566.95

1.21

Negative

5

4876.24

609.53

1.31

Negative

10

3853.15

481.64

1.03

Negative

 

dpm = Disintegrations per minute

a = Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total) number of lymph nodes)

b = Stimulation Index of 3.0 or greater indicates a positive result

na = Not applicable

 

 

Table 5. Individual Bodyweights and Bodyweight changes

 

Concentration (% w/w) in propylene glycol

Animal Number

Bodyweight (g)

Bodyweight Change (g)

Day 1

Day 6

 

Vehicle

1-1

21

20

-1

1-2

20

19

1

1-3

23

21

-2

1-4

19

21

2

2.5

2-1

21

20

-1

2-2

18

17

-1

2-3

20

19

-1

2-4

22

21

-1

5

3-1

18

19

            1

3-2

19

20

1

3-3

19

18

-1

3-4

20

20

0

 

10

4-1

20

20

            0

4-2

20

20

0

4-3

22

21

-1

4-4

20

18

-2

 

 

 

 

 

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Conclusions:
The test material is not a sensitiser.
Executive summary:

This was chosen as Key study as it is the only available study which is of relevance and of sufficient quality for classification and labelling and for risk assessment.

 

The positive control, Phenylacetaldehyde (90%) in dimethyl formamide (vehicle) produced an increase in the stimulation index (SI) greater than 3 over the control groups.Also the quality criteria of the study have been fulfilled as the study was conducted to OCED 429, EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay) guidelines  and to GLP.

 Concentration of MnO at 2.5, 5 and 10 % w/w in propylene glycol (vehicle) did not produce a stimulation index greater then 1.31. Due to data showing a stimulation index of less than 3 in the study, this justifies the lack classification and labelling of this substance.

Categories Display