Iron sulphide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to GLP and valid testing guidelines, therefore it is considered relevant, adequate and reliable for classification.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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:

Iron sulphide

EC Number:

215-268-6

EC Name:

Iron sulphide

Cas Number:

1317-37-9

Molecular formula:

FeS

IUPAC Name:

sulfanylideneiron

Test material form:

other: powder

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760118
- Age at start of dosing: 8 to 10 weeks
- Weight at start of dosing: 17.06 – 20.30 g
- Housing: Maximum six in macrolon cages (35x20x15 cm) with sterilized softwood shavings. Cleaning and disinfection of animal room was regularly performed.
- Diet (e.g. ad libitum): Pelleted standard diet for experimental animals ad libitum (ST 1 BERGMAN, manufacturer: Ing. Miroslav Mrkvička – Výroba krmných směsí, Mlýn Kocanda No. 19, 252 42 Jesenice u Prahy).
- Water (e.g. ad libitum): Drinking tap water ad libitum.
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2 – 21.9 °C, permanently monitored
- Humidity (%): 43.4 – 49.0 %, permanently monitored
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: January 2, 2013 To: January 29, 2013

Study design: in vivo (LLNA)

Vehicle:

other: DAE 433 - mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol

Concentration:

80%, 8%, 0.8% w/v

No. of animals per dose:

5 (main experiment)
1 (pilot experiment)

Details on study design:

RANGE FINDING TESTS:
The pilot experiment was conducted under the conditions identical to the main study, except assessment of lymph node proliferation. The appropriate dilution of the test substance (80%, 8%, 0.8% w/v) was applied to three animals in volume 25 µL to the dorsum of each ear once a day morning for 3 consecutive days. The suspensions were prepared before the start of application by mixing on magnetic stirrer and then were still mixed during application. The application was performed very slowly by micropipette. The route of administration was the same as in the main study.
Both ears of each mouse was were observed for erythema and scored and subsequently thickness was measured using digital thickness gauge. Body weight was recorded before application and prior to termination (Day 6).
According to the results of pilot experiment the doses used were confirmed also for main experiment.
- Compound solubility: The highest concentration 80% (w/v) was maximum technically practicable concentration.
- Irritation: No erythema and no relevant differences in thickness of ears were observed during the pilot experiment.
- Lymph node proliferation response: Not performed

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
Cell proliferation
The response towards the test substance is considered positive, if the stimulation index (SI)
is ≥ 3, and the response increases in dose-related manner (dose-response relationship).
The response is considered negative, if the stimulation index (SI) is < 3 without the dose–response relationship.
The response is considered ambiguous if the stimulation index is < 3, but the response increases in dose-related manner (dose–response relationship), and eventually statistical significance is observed.

Ear weight – irritation effect
If after treatment with the test substance a statistically significant increase of ear weight together with clear concentration dependence of the effect is recorded, the inflammatory effect will be considered as irritation induced by the test substance.
Positive result in cell proliferation reveals that the test substance could be a contact allergen. When positive irritation effect in animals is demonstrated simultaneously, the possibility cannot be ruled out, that the evaluation based on cell proliferation could be a false positive.

TREATMENT PREPARATION AND ADMINISTRATION:
The test substance was administered in the form of suspension in DAE 433.
The volume of the application form was constant at all groups of animals - 25 µL of the appropriate dilution to the dorsum of each ear once a day morning for 3 consecutive days. The application was performed very slowly by micropipette. Losses caused by draining from the ear were minimized.
This route of administration is listed in the guideline and it is similar to expected exposure conditions at the workplace. The application form of test substance was prepared immediately before administration.
Day 1:
Open application of 25μL (in the morning, by pipette) of appropriate suspensions of the test substance, the vehicle alone or the positive control to the dorsum of each ear.

Days 2 and 3:
The application procedure repeated as carried out on day 1.

Days 4 and 5:
No treatment.

Day 6:
The weight of animals was recorded. Injection 250 μL of phosphate-buffered saline (PBS) containing 7.26 x105 Bq of 3H-methyl thymidine into all test and control mice via the tail vein.
Five hours later, the animals were killed.

Positive control substance(s):

other: Dinitrochlorobenzene (DNCB) CAS No: 97-00-7 (0.5% (w/v) solution in the vehicle DAE 433)

Statistics:

For statistical calculations the software Statgraphic ® Centurion (version XV, USA) was used. Statistical evaluation of measured parameters was performed at first by applying the non-parametric Kruskal-Wallis test for the comparison of the measured effect in all treatment groups with the vehicle control group, as global test, and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) for all two-group comparisons.

Results and discussion

Positive control results:

The positive control substance DNCB produced positive LLNA response (SI=9.67) at an exposure level expected to give an increase in the Stimulation Index SI ≥ 3 over the negative control group, which was in congruence with the expected mode of action of a contact allergen. The positive control also elicited a reaction pattern with statistically significant increase in ear weight. The negative control group did not reveal any changes. These results demonstrate that the method performed in conditions of our laboratory has sufficient reliability.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

negative (vehicle) control group

Results 2

Key result

Parameter:

SI

Value:

1.04

Test group / Remarks:

80% test group

Results 3

Key result

Parameter:

SI

Value:

1.17

Test group / Remarks:

8% test group

Results 4

Key result

Parameter:

SI

Value:

1.13

Test group / Remarks:

0.8% test group

Results 5

Key result

Parameter:

SI

Value:

9.67

Test group / Remarks:

positive control group

Results 6

Key result

Parameter:

other: mean DPM

Remarks:

disintegrations per minute

Value:

250

Test group / Remarks:

negative (vehicle) control group

Results 7

Key result

Parameter:

other: mean DPM

Remarks:

disintegrations per minute

Value:

261.02

Test group / Remarks:

80% test group

Results 8

Key result

Parameter:

other: mean DPM

Remarks:

disintegrations per minute

Value:

293.22

Test group / Remarks:

8% test group

Results 9

Key result

Parameter:

other: mean DPM

Remarks:

disintegrations per minute

Value:

282.64

Test group / Remarks:

0.8% test group

Results 10

Key result

Parameter:

other: mean DPM

Remarks:

disintegrations per minute

Value:

2 418

Test group / Remarks:

positive control group

Any other information on results incl. tables

Table 1. Summary table

Group	Radioisotope incorporation		Ear weight
	Mean DPM	SI	Mean (mg)
Negative control (vehicle)	250.00	1.00	22.62
Positive Control	2418.00	9.67+	34.92*
80%	261.02	1.04	23.64
8%	293.22	1.17	22.90
0.8%	282.64	1.13	23.50

Figures with asterisk * = values statistically significant on probability level < 0.05 (Mann-Whitney test)
Figures with cross + = values ≥ 3 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

At the given experimental conditions the test substance Tribotecc® - Ferrostar provided a negative result in LLNA.

Executive summary:

The test substance, Tribotecc® - Ferrostar, was tested for the assessment of skin sensitisation potential with the murine local lymph node assay (LLNA) with radionuclides was used according to the OECD TG 429 or Method B.42 – Skin Sensitisation: Local Lymph Node Assay.

Tribotecc® - Ferrostar was evaluated after topical application to female BALB/c mice. Five mice per group were exposed on the dorsum of both ears once a day by test substance and control substances during 3 consecutive days. Primary proliferation of lymphocytes in the lymph nodes draining the site of application was evaluated using radioactive labelling of proliferating cells. The ratio of the proliferation in treated groups to that in vehicular controls, termed the Stimulation Index (SI), was determined. The evaluation of ear weights was performed for elimination of false positive findings with certain skin irritants. Concentrations of test substance in vehicle (DAE 433) were: 80%, 8%, 0.8% (w/v). The animals exposed to the test substance at all concentrations showed no pathological and no other negative clinical symptoms of intoxication throughout the experiment. There was no significant difference in body weight increment of the lowest and middle dose levels in comparison to the vehicle control. In highest dose level body weight increment was decreased.

The positive control substance DNCB elicited a reaction pattern with statistically significant increase in ear weight and Stimulation Index of lymphocytes proliferation equal to 9.67, which was in congruence with his expected mode of action as a contact allergen. The test substance did not show a tendency to increased ear weight in any of concentrations tested. The result of skin irritation effect was considered as negative – it means the test substance did not cause irritation of skin. Comparison of Stimulation Indexes between all treated groups and negative vehicle control group (1.13, 1.17 and 1.04 at the 0.8, 8 and 80% dosed groups, respectively) revealed that the test substance Tribotecc® - Ferrostar did not cause significant increase of lymphocytes proliferation – it means that the test substance is not a contact allergen.

In conclusion, at the given experimental conditions the test substance Tribotecc® - Ferrostar provided a negative result in LLNA.