Manganese sulphide

Endpoint summary

Description of key information

Skin Irritation

Warren (2009)

Under the conditions of the study, MnS is considered to be an irritant and is classified according to EU criteria.

 

Eye Irritation

BCOP: Warren (2009)

Under the conditions of the study, the in vitro irritancy score for the test material was 4.8, indicating no prediction can be made.

 

Reconstituted Human Corneal model: Warren (2009)

Under the conditions of this study the test material was determined to be an irritant. 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Remarks:

other: EU method B.46

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-07-07 to 2009-07-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study follows GLP and internationally accepted guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: EU method B.46 (in vitro skin irritation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

EPISKIN™ Reconstituted Human Epidermis model

Source species:

human

Cell type:

non-transformed keratinocytes

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ± 2 mg


VEHICLE
Test material was used as supplied

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

The test was performed in triplicate

Details on study design:

APPLICATION OF TEST MATERIAL
- Area of exposure: The test material was applied topically to the reconstituted epidermis ensuring uniform coverage. The epidermis surface had been moistened with 5 µL of sterile distilled water to improve contact between the solid test material and the epidermis.


REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure period each tissue was rinsed with a Phosphate Buffered Saline solution containing Ca2+ and Mg2+ before incubating for approximately 42 hours at 37 °C in 5% CO2 air
- Time after start of exposure: 15 minutes


SCORING SYSTEM:
At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.
At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

>= 36.9 - <= 55.6

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Please refer to table 1 for tabulated results including control

Other effects / acceptance of results:

The relative mean viability of the test material treated tissues was 47.6% after a 15-minute exposure.

QUANTITATIVE MTT ASSESSMENT (percentage tissue viability):For the test material, the relative mean tissue viabilities were compared to the mean of the negative control treated tissues (n = 3). The relative mean viabilities were calculated in the following way:

% Relative mean viability = (mean OD540 of test material/mean OD540 of negative control) x 100

The test material was found not to directly reduce MTT.

Table 1: Mean OD₅₄₀ values and % viabilities^b for the negative control material, positive control material and test material

Material	OD540of tissues	Mean OD540of triplicate tissues	± SD of OD540	Relative individual tissue viability %	Relative mean % viability	± SD of % viability
Negative control material	0.826	0.756	0.061	109.3	100^a	8.15
	0.710			93.9
	0.733			97.0
Positive control material	0.069	0.074	0.012	9.1	9.7	1.55
	0.065			8.6
	0.087			11.5
Test material	0.420	0.360	0.073	55.6	47.6	9.65
	0.381			50.4
	0.279			36.9

^aThe mean viability of the negative control tissues is set at 100%;^bData are presented in the form of % viability (MTT reduction in the test material treated tissues relative to negative control tissues).

Table 2: Qualitative evaluation of tissue viability (MTT uptake visual evaluation)

Material	Tissue 1	Tissue 2	Tissue 3
Negative control Material	-	-	-
Positive Control Material	++	++	++
Test Material	-	-	-

MTT visual scoring scheme:

-         - = blue tissue (viable)

-         + = blue/white tissue (semi-viable)

-         ++ = tissue is completely white (dead)

Quality criteria

The quality criteria required for acceptance of results in the test were satisfied, i.e. positive control and negative control acceptance criteria.

Interpretation of results:

other: Classified according to EU criteria.

Conclusions:

Under the conditions of the study, MnS is considered to be an irritant and is classified according to EU criteria.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

29 May 2009 to 19 August 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study follows GLP and international test guideline.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 437

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: not applicable

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

PRE-TEST PROCEDURES
- Source of bovine eyes: eyes from adult cattle obtained from a local abattoir.
- Transportation to laboratory: eyes placed in Hanks' Balanced Salt Solution (HBSS) on ice packs.

PREPARATION CONDITIONS
- Incubation conditions: Immersion of eye corneas in HBSS and mounted onto BCOP holders.
- Incubation temperature (°C): 32 ± 1
- Incubation time (min): > 60
- Inclusion criteria: only corneas free of damage were used.
- Pre-treatment opacity reading: performed with calibrated opacitometer (MC2, Clermont-Ferrand, France)

Vehicle:

physiological saline

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Concentration : 20 % w/v


VEHICLE
- Concentration : 0.9% w/v sodium chloride solution

Duration of treatment / exposure:

240 min

Observation period (in vivo):

Not reported

Number of animals or in vitro replicates:

Each group (test material, positive control and negative control) was assigned three corneas.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: three times with fresh complete Hanks' Balanced Salt Solution (HBSS) containing phenol red and one time with complete HBSS.
- Time after start of exposure: 240 min

SCORING SYSTEM: Sina JF et al. (1995) Fund. Appl. Tox. 26: 20-31.

TOOL USED TO ASSESS SCORE:
- Opacity reading: calibrated opacitometer
- Permeability test: sodium fluorescein

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean at 240 minutes

Value:

4.8

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Other effects / acceptance of results:

- In Vitro Irritancy Score:
The mean values and mean permeability values (OD492) for each treatment group was used to calculate an in vitro score for each treatment group:
In Vitro Irritancy Score = mean opacity value + (15 x mean OD492 values)

The opacity and permeability values should be evaluated independently to determine whether a test substance induced irritation through only one of the two endpoints.

Evaluating Irritancy Score:

From 0 to 25 = mild irritant
From 25.1 to 55 = moderate irritant
From 55.1 and above = severe irritant

Corrected opacity values were calculated by subtracting the average change in opacity of the negative control from the treated and positive control groups. Corrected permeability OD492 values were calculated by subtracting the mean OD492 values from the negative control corneas from the OD492 of the treated and positive control corneas.

The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement (table 2) indicating a clear condition after exposure of test material saline solution for 240 minutes.

Table 1: Individual and Mean Corneal Opacity Measurements

Treatment	Cornea Number	Opacity				Permeability (OD)		In vitro Irritancy Score
		Pre-treatment (A)	Post-treatment (B)	(B)–()	Corrected value	-	Corrected value	-
Negative control	1	1	2	1	-	0.057	-	-
	2	3	3	0	-	0.040	-	-
	3	1	2	1	-	0.018	-	-
Mean	-	-	-	0.7*	-	0.038	-	1.2
Positive control	4	1	72	71	70.3	2.430	2.392	-
	5	3	66	63	62.3	2.000	1.962	-
	6	2	70	68	67.3	3.110	3.072	-
Mean**	-	-	-	-	66.7	-	2.475	103.8
Test material	7	2	8	6	5.3	0.059	0.021	-
	8	3	9	6	5.3	0.035	0.0	-
	9	1	5	4	3.3	0.026	0.0	-
Mean**	-	-	-	-	4.7	-	0.007	4.8

* = Mean of the post-treatment - pre-treatment values

** = Mean corrected value

OD = Optical density

Table 2: Corneal Epithelium Condition

Treatment	Cornea Number	Observation
		Immediately after Rinsing
Negative control	1	Clear
	2	Clear
	3	Clear
Positive control	4	Cloudy
	5	Cloudy
	6	Cloudy
Test material	7	Clear
	8	Clear
	9	Clear

Table 3: Summary of Results 

Treatment	In Vitro Irritancy Score
Test material	4.8
Negative control	1.2
Positive control	103.8

 

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of the study, the in vitro irritancy score for the test material was 4.8 indicating no prediction can be made.

Executive summary:

The eye irritation potential of the test material was assessed according to OECD Test Guideline 437 and in compliance with GLP using bovine eyes.

Under the conditions of the study, the in vitro irritancy score for the test material was 4.8 indicating no prediction can be made.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation

Warren (2009)

The skin irritation potential of the test material was assessed according to EU Method B.46 and in compliance with GLP using the EPISKIN™ Reconstituted Human Epidermis model. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The relative mean viability of the test material treated tissues was 47.6 % after a 15-minute exposure. 

Under the conditions of the study the test material was considered to be a skin irritant.

 

Eye Irritation

BCOP: Warren (2009)

The eye irritation potential of the test material was assessed according to OECD Test Guideline 437 and in compliance with GLP using bovine eyes. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Under the conditions of the study, the in vitro irritancy score for the test material was 4.8, indicating no prediction can be made.

 

Reconstituted Human Corneal model: Warren (2009)

The eye irritation potential of the test material was assessed using the SkinEthic Reconstituted Human Corneal model (HCE SkinEthic Laboratories, Nice, France) after a treatment period of 10 minutes. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The relative mean viability of the test material treated tissues after a 10 minute exposure was 46.4 %.

Under the conditions of this study the test material was determined to be an irritant. 

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the material does require classification with respect to skin irritation (Category 2) and eye irritation (Category 2).