Iron sinter

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 June 2010 - 7 July 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted according to official EC and OECD test guidelines, and in compliance with GLP.

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)

Version / remarks:

2002-04-24

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2008

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:

Reaction mass of diiron carbide, diiron phosphide and triiron phosphide

EC Number:

701-438-1

Molecular formula:

not applicable (multi constituent substance)

IUPAC Name:

Reaction mass of diiron carbide, diiron phosphide and triiron phosphide

Test material form:

solid

Details on test material:

- Name of test material (as cited in study report): Ferrophosphorus (Fe3P)
- Substance type: inorganic
- Physical state: solid
- Analytical purity: Iron: 82.7%
Phosphorus: 15.7%
- Lot/batch No.: 1068487
- Expiration date of the lot/batch: 15 February 2011
- Storage condition of test material: Room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Ltd.
- Age at study initiation: approx. 8 - 12 weeks
- Weight at study initiation: 16.8 - 20.8 g
- Housing: Animals were housed individually in polycarbonate cages with woodflake bedding. The mice were also given Nestlets and a plastic shelter for environmental enrichment. Each animal was assigned an alpha-numeric code and identified uniquely within the study by tail marking. Each cage label was colour-coded and was identified uniquely with the study number, dose level and animal mark.
- Diet (ad libitum): Standard rodent diet (Rat and Mouse No. 1 Maintenance Diet); ad libitum
- Water (ad libitum): Potable water taken from the public supply; ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23°C
- Humidity (%): 40 - 70%
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0, 25, 50 and 100% w/v

No. of animals per dose:

4 female mice

Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: The local lymph node assay
- Criteria used to consider a positive response: The test substance is regarded as a sensitizer if at least one concentration of the chemical results in a three-fold greater increase in 3HTdR incorporation compared to control values.

TREATMENT PREPARATION AND ADMINISTRATION: The mice were treated by daily application of 25 μl of each of one of these three concentrations, or control, to the dorsal surface of both ears for three consecutive days.

PRE-SCREEN TESTS:
A vehicle trial was conducted and the test substance showed that it formed a dark grey liquid suspension at 100% w/v in acetone:olive oil (4:1 v/v) which was satisfactory for dose administration.

TREATMENT PREPARATION:
The dose levels for the investigation were chosen based on the physical properties of the test substance e.g. solubility, viscosity. The test substance formulations were prepared on the day of dosing at the required concentrations.

MAIN STUDY.
Groups of mice were treated at one of three concentrations of the test substance. The mice were treated by daily application of 25 μL of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3). The test substance was spread over the entire dorsal surface of the ear using the tip of a pipette.

Five days following the first topical application of test substance (Day 6) all mice were injected via the tail vein with 250 μL of phosphate buffered saline containing 3H-methyl Thymidinea (3HTdR: 80 μCi/mL) giving a nominal 20 μCi to each mouse.

Five hours following the administration of 3HTdR on Day 6 all mice were humanely killed by carbon dioxide asphyxiation and the draining auricular lymph nodes excised and pooled for each experimental group. 1.0 mL of phosphate buffered saline was added to the pooled lymph nodes for each group.

A single cell suspension of lymph node cells (LNC) was prepared by gentle mechanical disaggregation through a stainless steel gauze (200 mesh size). The pooled LNC were then washed by adding 10 mL phosphate buffered saline, pelleted at 190 x g for 10 minutes and resuspended. The cells were washed twice again and resuspended in 3 mL trichloroacetic acid (TCA: 5%) following the final wash.

After overnight incubation with 5% TCA at 4°C, the precipitate was recovered by
centrifugation and resuspended in 1 mL 5% TCA and transferred to 10 mL Ultima gold scintillation fluid on Day 7. 3HTdR incorporation was measured by β-scintillation counting. The proliferative response of LNC was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio; Si value).

ANALYSIS OF RESULTS:
The test substance is regarded as a sensitizer if at least one concentration of the test substance results in a three-fold greater increase in 3HTdR incorporation compared to control values.

OBSERVATIONS:
- clinical signs: all animals were observed daily for signs of ill health or toxicity. The ears were also examined for signs of irritation.
- body weight: the weight of each mouse in the study was recorded on arrival (these data are not reported), Day 1 (first day of dosing) and prior to termination (Day 6).

Positive control substance(s):

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

Statistics:

No statistical analysis was performed on this study.

Results and discussion

Positive control results:

This positive control study is considered to be valid if the results from the hexyl cinnamic aldehyde (HCA) group have a three-fold greater increase in 3HTdR incorporation compared to control values.
In this assay the test/control ratios obtained for HCA at 10, 25 and 50% v/v were 2.3, 7.7 and 13.1. This indicates that Hexyl cinnamic aldehyde demonstrates the potential to induce skin sensitization (delayed contact hypersensitivity) and confirms the sensitivity.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.7

Test group / Remarks:

25 % w/v test substance

Results 2

Key result

Parameter:

SI

Value:

0.5

Test group / Remarks:

50 % w/v test substance

Results 3

Key result

Parameter:

SI

Value:

0.8

Test group / Remarks:

100 % w/v test substance

Cellular proliferation data / Observations:

CLINICAL OBSERVATIONS:
- There were no deaths and no signs of ill health or toxicity observed during this study, however the following observations were noted:

- Greasy fur was noted for all control and test animals post-dose from Day 1 for control animals and from Day 3 for all test animals. This sign had resolved completely in all animals by Day 5.

- Black dose residue on ears was noted for all test animals post-dose on Day 1. Wet fur was also noted for all test animals post-dose on Day 1. These signs had resolved completely in all animals by Day 4.

BODY WEIGHTS:
Body weight increases were recorded for all mice over the period of the study.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Ferrophosphorus (Fe3P) is not regarded as a potential skin sensitizer and therefore should not be classified and labelled for skin sensitisation according to Regulation (EC) No. 1272/2008 and its subsequent adaptations.