Slags, ferromanganese-manufg.

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:

16 November 2009 to 1 December 2009

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. Since the study was conducted with silicomanganese slag, rather than with the registered substance itself, it has been assigned a reliability score of 2 in line with the criteria of Klimisch (1997). Use of data generated with silicomanganese slag, to address information requirements of ferromanganese slag, is considered to be justified on the basis of the similar compositions of the two substances. Both substances are UVCB substances, containing metallic oxides; each substance is obtained as a by-product in the manufacture of SiMn and FeMn alloy, respectively.

Cross-reference

Reason / purpose for cross-reference:

other: Target substance

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.

Reason / purpose for cross-reference:

read-across source

Parameter:

SI

Value:

0.85

Test group / Remarks:

2.5 % w/w in dimethyl formamide

Remarks on result:

other: Negative

Parameter:

SI

Value:

1.09

Test group / Remarks:

5.0 % w/w in dimethyl formamide

Remarks on result:

other: Negative

Parameter:

SI

Value:

1.14

Test group / Remarks:

10.0 % w/w in dimethyl formamide

Remarks on result:

other: Negative

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

Details on test material:

- Name of test material : SiMn slag
- Substance type: light grey granules
- Physical state: Solid
- Lot/batch No.: CDSNC-08072009
- 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 - 12 weeks
- 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 - 25 °C
- Humidity (%): 30 - 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:

dimethylformamide

Concentration:

10, 5 and 2.5 % 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 dimethyl formamide 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 % in dimethyl formamide. 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 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 disintegrations per minute was then measure 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.

For positive results an EC3 value is calculated to determine the concentration of test material expected to cause a 3 fold increase in 3HRdR incorporation.

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:

other: disintegrations per minute (DPM)

Remarks on result:

other: Please refer to table 1 under section "Any other information on results incl. tables" for tabulated data .

Results 2

Key result

Parameter:

SI

Value:

0.85

Test group / Remarks:

2.5 % w/w in dimethyl formamide

Remarks on result:

other: Negative

Results 3

Key result

Parameter:

SI

Value:

1.09

Test group / Remarks:

5.0 % w/w in dimethyl formamide

Remarks on result:

other: Negative

Results 4

Key result

Parameter:

SI

Value:

1.14

Test group / Remarks:

10.0 % w/w in dimethyl formamide

Remarks on result:

other: Negative

Any other information on results incl. tables

For full tabulated results please refer to the attached document, Appendix I.

 

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

Concentration (% w/w) in dimethyl formamide	dpm	dpm/Node^a	Stimulation Index^b	Results
Vehicle	9777.48	1222.19	na	na
2.5	8323.41	1040.43	0.85	Negative
5	10621.27	1327.66	1.09	Negative
10	11119.31	1389.91	1.14	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 observed. In the main test, no deaths occurred and no signs of systemic toxicity were noted in the test or control animals.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test material was considered to be a non-sensitiser under the conditions of the study.

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.

Since the study was conducted with silicomanganese slag, rather than with the registered substance itself, it has been assigned a reliability score of 2 in line with the criteria of Klimisch (1997). Use of data generated with silicomanganese slag, to address information requirements of ferromanganese slag, is considered to be justified on the basis of the similar compositions of the two substances. Both substances are UVCB substances, containing metallic oxides; each substance is obtained as a by-product in the manufacture of SiMn and FeMn alloy, respectively.