Reaction mass of copper oxide and manganese dioxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-11-16 to 2009-12-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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.

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:

Manganese dioxide

EC Number:

215-202-6

EC Name:

Manganese dioxide

Cas Number:

1313-13-9

Molecular formula:

MnO2

IUPAC Name:

Manganese dioxide

Test material form:

solid: particulate/powder

Details on test material:

Sponsor's identification : MnO2
Purity : 97.6%
Batch number : 103721-02
Description : black powder
Date received : 8 September 2008
Storage conditions : 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:

acetone/olive oil (4:1 v/v)

Concentration:

50, 25 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 50 % w/w in acetone/olive oil 4:1 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. No signs of toxicity or excessive local irritation were recorded. The bodyweight of the mouse was recorded on day 1 (prior to dosing) and 6.
- Substance 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 50 %, 25 % or 10 % in acetone/olive oil 4:1. 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 measured using the Beckman LS6500 scintillation system.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

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 α-Hexylcinnamaldehyde, Tech. 85 % was 5.16 at 15 % in dimethyl formamide and was considered to be a positive sensitiser under the conditions of the test.

The Stimulation Index expressed as the mean radioactive incorporation for the positive control group divided by the mean radioactive incorporation of the vehicle control group is listed in table 1.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Please refer to table 1 under section Remarks on results including tables and figures for tabulated data .

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Please refer to table 1 under section Remarks on results including tables and figures for tabulated data .

Any other information on results incl. tables

Table 1: Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index.

Concentration (% w/w) in acetone/olive oil 4:1	dpm	dpm/Node^a	Stimulation Index^b	Results
Vehicle	7167.27	895.91	na	na
10	16164.34	2020.54	2.26	Negative
25	16208.91	2026.11	2.26	Negative
50	13352.70	1669.09	1.86	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

 

During the preliminary screening test no signs of systemic toxicity were noted. Black staining of the fur and ears was noted post dose on Days 2 and 3. During the main test, no deaths or signs of systemic toxicity were recorded. Black staining of the fur was noted in all animals treated with the test material. Bodyweight changes of the test animals between Day 1 and 6 were comparable to those observed in the control group animals over the same period.

Table 2: Summary of Positive Control Data

Start Date	Finish Date	Test Material	Concentration	Vehicle	Stimulation Index^a	Classification^b
16/04/09	22/04/09	α-Hexylcinamaldehyde, 85 %	15 % v/v	Butanone	4.08	Positive
17/04/09	23/04/09	α-Hexylcinamaldehyde 85 %	15 % v/v	Dimethyl sulphoxide	4.60	Positive
25/06/09	01/07/09	α-Hexylcinamaldehyde 85 %	15 % v/v	Ethanol/distilled water 7:3	10.68	Positive
25/06/09	01/07/09	α-Hexylcinamaldehyde 85 %	15 % v/v	Acetone	10.72	Positive
25/06/09	01/07/09	α-Hexylcinamaldehyde 85 %	5, 10, 25 % v/v	1 % pluronic L92 in distilled water	1.30, 2.37, 8.14	Positive
08/07/09	14/07/09	α-Hexylcinamaldehyde 85 %	15 % v/v	Acetone/olive oil 4:1	3.70	Positive
11/09/09	17/09/09	α-Hexylcinamaldehyde 85 %	25 % v/v	1 % pluronic L92 in distilled water	5.11	Positive
01/10/09	07/10/09	α-Hexylcinamaldehyde 85 %	2.5 % v/v	Propylene glycol	4.20	Positive
05/11/09	11/11/09	Phenylacetaldehyde (90 %)	15 % v/v	Dimethyl formamide	5.16	Positive
11/11/09	17/11/09	α-Hexylcinamaldehyde 85 %	50 % v/v	Cottonseed oil	6.40	Positive
11/11/09	17/11/09	α-Hexylcinamaldehyde 85 %	15 % v/v	Butanone	7.45	Positive
11/11/09	17/11/09	α-Hexylcinamaldehyde 85 %	15 % v/v	Dimethyl sulphoxide	6.07	Positive
05/11/09	11/11/09	α-Hexylcinamaldehyde 85 %	15 % v/v	Acetone/olive oil 4:1	3.12	Positive
a = Ratio of test to control lymphocyte proliferation b = Stimulation index greater than 3.0 indicates a positive result.

 

Table 3: Preliminary Screening Test (Bodyweight and Mortality Data)

Concentration (% w/w) inacetone/olive oil 4:1	Animal Number	Bodyweight (g)		Day
				1		2		3		4	5	6
		Day 1	Day 6	Pre-Dose	Post-Dose	Pre-Dose	Post-Dose	Pre-Dose	Post-Dose
50	S-1	19	19	0	0	0	0Fs	0	0Fs	0	0	0

Fs = Black staining of the fur and ears

 

Table 4: Individual Clinical Observations and Mortality Data

Concentration (% w/w) in	Animal Number	Day 1		Day 2		Day 3		Day 4	Day 5	Day 6
		Pre-Dose	Post-Dose	Pre-Dose	Post-Dose	Pre-Dose	Post-Dose
Vehicle	1-1	0	0	0	0	0	0	0	0	0
	1-2	0	0	0	0	0	0	0	0	0
	1-3	0	0	0	0	0	0	0	0	0
	1-4	0	0	0	0	0	0	0	0	0
10	2-1	0	0	0	0Fs	0	0Fs	0	0	0
	2-2	0	0	0	0Fs	0	0Fs	0	0	0
	2-3	0	0	0	0Fs	0	0Fs	0	0	0
	2-4	0	0	0	0Fs	0	0Fs	0	0	0
25	3-1	0	0	0	0Fs	0	0Fs	0	0	0
	3-2	0	0	0	0Fs	0	0Fs	0	0	0
	3-3	0	0	0	0Fs	0	0Fs	0	0	0
	3-4	0	0	0	0Fs	0	0Fs	0	0	0
50	4-1	0	0Fs	0	0Fs	0	0Fs	0	0	0
	4-2	0	0Fs	0	0Fs	0	0Fs	0	0	0
	4-3	0	0Fs	0	0Fs	0	0Fs	0	0	0
	4-4	0	0Fs	0	0Fs	0	0Fs	0	0	0
0 = No signs of systemic toxicity

 

Table 5: Individual Bodyweights and Bodyweight Changes

Concentration (% w/w) in	Animal Number	Bodyweight (g)		Bodyweight Change (g)
		Day 1	Day 6
Vehicle	1-1	17	17	0
	1-2	19	19	0
	1-3	20	19	-1
	1-4	20	20	0
10	2-1	20	20	0
	2-2	16	16	0
	2-3	19	19	0
	2-4	19	20	1
25	3-1	20	20	0
	3-2	18	18	0
	3-3	19	18	-1
	3-4	17	18	1
50	4-1	19	18	-1
	4-2	18	19	1
	4-3	17	18	1
	4-4	17	18	1

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

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

Executive summary:

The skin sensitisation potential of the test material 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 material was concluded to be a non sensitiser.