Manganese carbonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 January 2010 to 19 January 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. Use of data on MnO to address data requirements of MnCO3 is considered to be justified on the basis that manganese is in the same oxidation state in both substances, the anions in both substances are not regarded as toxic, and both substances are expected to have similar dermal absorption due to their poor water solubility and inorganic nature. Furthermore, both substances exhibited no dermal or eye irritation and both substances were found to exert no toxicity when dosed at the maximum permissible level in acute oral toxicity studies (thereby confirming the similarity of the toxicological profile of both substances, related to exposure to Mn2+).

Justification for type of information:

The data on the read-across substance is considered representative.

Cross-reference

Reason / purpose for cross-reference:

other: Target record

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

The data on the read-across substance is considered representative.

Reason / purpose for cross-reference:

read-across source

Parameter:

SI

Remarks on result:

other: Not sensitising

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Not sensitising

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)

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

Reference substance name:

1344-43-0

Cas Number:

1344-43-0

IUPAC Name:

1344-43-0

Constituent 2

Reference substance name:

Manganese oxide

EC Number:

215-695-8

EC Name:

Manganese oxide

Cas Number:

1344-43-0

Molecular formula:

MnO

IUPAC Name:

Oxomanganese

Constituent 3

Reference substance name:

Manganese oxide

IUPAC Name:

Manganese oxide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

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 substance 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 substance will be regarded as a sensitiser if at least one concentration of the test substance results in a threefold or greater increase in 3HTdR incorporation comapred to control values. Any test substance 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 substance 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 substance to the dorsal surface of each ear for three consecutive days (1, 2, and 3). The test substance formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further groups 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 teil 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 disintigrations 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)

Results

Results 1

Parameter:

SI

Value:

1.21

Test group / Remarks:

2.5 % (w/w) in propylene glycol

Remarks on result:

other: Negative

Results 2

Parameter:

SI

Value:

1.31

Test group / Remarks:

5 % (w/w) in propylene glycol

Remarks on result:

other: Negative

Results 3

Parameter:

SI

Value:

1.03

Test group / Remarks:

10 % (w/w) in propylene glycol

Remarks on result:

other: Negative

Results 4

Parameter:

other: DPM (Disintegrations Per Minute)

Value:

3 734.44

Test group / Remarks:

Vehicle

Remarks on result:

other: Negative

Results 5

Parameter:

other: DPM (Disintegrations Per Minute)

Value:

4 535.63

Test group / Remarks:

2.5 % (w/w) in propylene glycol

Remarks on result:

other: Negative

Results 6

Parameter:

other: DPM (Disintegrations Per Minute)

Value:

4 876.24

Test group / Remarks:

5 % (w/w) in propylene glycol

Remarks on result:

other: Negative

Results 7

Parameter:

other: DPM (Disintegrations Per Minute)

Value:

3 853.15

Test group / Remarks:

10 % (w/w) in propylene glycol

Remarks on result:

other: Negative

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/Node^a	Stimulation Index^b	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

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance was considerd to be a non-sensitiser under the conditions of the test.

Executive summary:

The skin sensitisation potential of the test substance was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 429 and EU Method B.42.

Under the conditions of the study the test substance was concluded to be a non sensitiser.