Manganese dichloride

Endpoint summary

Description of key information

In vitro assays for skin and eye irritation were followed up with in vivo studies which confirm that MgCl2 was non-irritating to skin, but caused serious damage to eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-12-03 to 2009-12-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to current guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd. Hillcrest, Belton, Loughborough, UK
- Age at study initiation: 12 to 20 weeks old
- Weight at study initiation: 2.32 to 2.72 kg
- Housing: housed individually in suspended cages
- Diet: 2030 Teklad Global Rabbit diet (supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK), available ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 17 to 23°C
- Humidity: 30-70%
- Air changes: at least 15 changes per hour
- Photoperiod (hrs dark / hrs light): lighting controlled by a time switch to give 12 h of continuous light (06:00 to 18:00) and 12 h of darkness

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

water

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.5 g of test material moistened sufficiently with 0.5 mL of distilled water to achieve a paste

Duration of treatment / exposure:

Animals were exposed to the test material for 4 hours.

Observation period:

Observations were made immediately following removal of the patches and approximately 1, 24, 48 and 72 hours later.

Number of animals:

3 animals

Details on study design:

TEST SITE
- Area of exposure: the test material was introduced under a 2.5 cm x 2.5 cm cotton gauze patch.
- Type of wrap if used: the trunk of each rabbit was wrapped in an elasticated corset.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): any residual test material removed by gentle swabbing with cotton wool soaked in distilled water.
- Time after start of exposure: four hours after application

SCORING SYSTEM: The test material was classified according to the Draize JH (1959) "Dermal Toxicity" In: Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. Association of the United States, Austin, Texas, p. 46-59.

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

mean

Remarks:

on 3 animals

Time point:

other: 24 and 72 h

Score:

4

Max. score:

6

Irritation parameter:

erythema score

Basis:

animal #1

Remarks:

68699

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

erythema score

Basis:

animal #2

Remarks:

68700

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

erythema score

Basis:

animal #3

Remarks:

68701

Time point:

24/48/72 h

Score:

1.3

Max. score:

2

Reversibility:

fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #1

Remarks:

68699

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

edema score

Basis:

animal #2

Remarks:

68700

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

The individual scores for erythema/eschar and oedema are given in Table 1.
Well-defined erythema and very slight oedema were noted at one treated skin site at the 24-hour observation with very slight erythema noted at the 48 and 72-hour observations. Slight desquamation was noted at this treated skin site at the 7-day observation.
No evidence of skin irritation was noted at two treated sites during the study.

Other effects:

not reported

Measurement of pH

The pH of the test material was determined prior to commencement of the study and found to be as follows:

 

Preparation
	immediately	after 10 minutes
10 % w/w aqueous preparation of the test material	7.1	7.2

Table 1: Individual Skin Reactions

 

Skin Reaction	Observation Time (Following Patch Removal)	Individual Scores – Rabbit Number and Sex			Total
		68699 Male	68700 Male	68701 Male
Erythema/Eschar Formation	Immediately	0	0	0	(0)
	1 Hour	0	0	0	(0)
	24 Hours	0	0	2	2
	48 Hours	0	0	1	(1)
	72 Hours	0	0	1	1
	7 Days	0	0	0D	(0)
Oedema Formation	Immediately	0	0	0	(0)
	1 Hour	0	0	0	(0)
	24 Hours	0	0	1	1
	48 Hours	0	0	0D	(0)
	72 Hours	0	0	0	0
	7 Days	0	0	0	(0)
Sum of 24 and 72-hour Readings (S): 4
Primary Irritation Index (S/6): 4/6 = 0.7
Classification: Mild Irritant

() = total values not included in calculating the primary irritation index

D = Slight desquamation

 

Table 2: Individual Bodyweights and Bodyweight Changes

 

Rabbit Number and Sex	Individual Bodyweight (kg)		Bodyweight Change (kg)
	Day 0	Day 7
68699 Male	2.40	2.53	0.13
68700 Male	2.72	2.85	0.13
68701 Male	2.32	2.51	0.18

Interpretation of results:

not irritating

Conclusions:

MnCl2 is not a dermal irritant.

Executive summary:

Introduction:

The study was performed to assess the irritancy potential of the test material to the skin of the New Zealand White rabbit. The method was designed to meet the requirements of the following:

- OECD Guidelines for the Testing of Chemicals No. 404 "Acute Dermal Irritation/Corrosion" (adopted 24 April 2002)

- Method B4 Acute Toxicity (Skin Irritation) of Commission Regulation (EC) No. 440/2008

Results:

A single 4-hour, semi-occluded application of the test material to the intact skin of three rabbits produced well-defined erythema, very slight oedema and slight desquamation at one treated skin site. No evidence of skin irritation was noted at two treated skin sites.

Conclusion:

The test material produced a primary irritation index of 0.7 and was classified as a mild irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-12-07 to 2010-02-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to the current guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Hillcrest, Belton, Loughborough, UK
- Age at study initiation: twelve to twenty weeks old
- Weight at study initiation: 2.32 or 2.65 kg
- Housing: individually housed in suspended cages
- Diet: 2030 Teklad Global Rabbit (Harlan Teklad, Blackthorn, Bicester, Oxon, UK), available ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23°C
- Humidity (%): 30-70%
- Air changes (per hr): at least 15 changes per hour
- Photoperiod: the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 100 mg of the test material was placed into the conjunctival sac

Duration of treatment / exposure:

Single application of the test material; up to 72 hours exposure.

Observation period (in vivo):

Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment. Additional observations were made on Days 7, 14 and 21 to assess the reversibility of the ocular effects.

Number of animals or in vitro replicates:

2 animals

Details on study design:

APPLICATION OF THE TEST MATERIAL:
Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.
Initially, a single rabbit was treated. 100 mg of the test material was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent the loss of the test material, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test material, an assessment of the initial pain reaction was made according to the six point scale (Appendix 1).
After consideration of the ocular responses produced in the first treated animal, a second animal was treated. In order to minimise pain on application of the test material, one drop of local anaesthetic (Tetracaine hydrochloride 0.5%, Chauvin Pharmaceuticals, Romford, Essex, UK) was instilled into both eyes of the second animal 1 to 2 minutes before treatment.

Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation given in Appendix 2 (from Draize JH (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC p.48 to 49).

Additional observations were made on Days 7, 14 and 21 to assess the reversibility of the ocular effects.

SCORING SYSTEM: modified Kay and Calandra classification system (Kay JH and Calandra JC (1962), J Soc Cosmet Chem. 13, 281-289) (see Appendix 3)
The numerical values corresponding to each animal, tissue and observation time were recorded. The data relating to the conjunctivae were designated by the letters A (redness), B (chemosis) and C (discharge), those relating to the iris designated by the letter D and those relating to the cornea by the letters E (degree of opacity) and F (area of cornea involved). For each tissue the score was calculated as follows:
Score for conjunctivae = (A+B+C) x 2
Score for iris = D x 5
Score for cornea = (E+F) x 5

Using the numerical data obtained a modified version of the system described by Kay and Calandra (1962) (Appendix 3) was used to classify the ocular irritancy potential of the test material. This was achieved by adding the scores for the cornea, iris and conjunctivae for each time point for each rabbit. The group means of the total scores for each observation were calculated. The highest of these group means (the maximum group mean score) together with the persistence of the reaction enabled classification of the eye irritancy potential of the test material. If evidence of irreversible ocular damage is noted, the test material will be classified as corrosive to the eye.

TOOL USED TO ASSESS SCORE: Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Irritation parameter:

maximum mean total score (MMTS)

Basis:

mean

Time point:

other: 48 h

Score:

53

Max. score:

110

Reversibility:

not reversible

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

68714

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 14 days

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

68794

Time point:

24/48/72 h

Score:

1.7

Max. score:

2

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

68714

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 14 days

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

68794

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 14 days

Irritation parameter:

conjunctivae score

Basis:

animal #1

Remarks:

68714

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

fully reversible within: 21 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Remarks:

68794

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

68714

Time point:

24/48/72 h

Score:

2.7

Max. score:

3

Reversibility:

fully reversible within: 21 days

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

68794

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

not fully reversible within: 21 days

Irritant / corrosive response data:

Individual and group mean scores for ocular irritation are given in Table 1 and Table2.
Scattered or diffuse corneal opacity was noted in the treated eyes at the 24-hour observation with translucent or scattered or diffuse corneal opacity noted at the 48 and 72-hours observations. Translucent corneal opacity was noted in the treated eyes at the 7-day observation. Scattered or diffuse corneal opacity was noted in one treated eye at the 14 and 21-day observations. Vascularisation with a localised ingrowth of vessels for approximately 3 mm and covering one or three quarters of the cornea was noted in the treated eyes at the 7-day observation.

Iridial inflammation was noted in the treated eyes at the 24, 48, 72-hour and 7-day observations.

Moderate conjunctival irritation was noted in the treated eyes one hour after treatment with severe conjunctival irritation noted at the 24 and 48-hour observation with moderate conjunctival irritation at the 7-day observation. Minimal or moderate conjunctival irritation was noted in the treated eyes at the 14-day observation with minimal conjunctival irritation at the 21-day observation. Haemorrhage scattered over the nictitating membrane and of the upper and lower conjunctival membrane were noted in the treated eyes at the 24, 48, 72-hour and 7-day observations. Alopecia around the treated eye was noted in one animal at the 7 and 14-day observations. Ectropion was noted in one treated eye at the 21-day observation.

Other effects:

not reported

Table 1: Individual Scores for Ocular Irritation

Rabbit Number and Sex	68714 Male							68794 Male
	IPR= 3							IPR= 0+
Time After Treatment	1 h	24 h	48 h	72 h	7 d	14 d	21 d	1 h	24 h	48h	72 h	7 d	14 d	21 d
CORNEA
E = Degree of Opacity	0	1	1	1	2V	0	0	0	1	2	2	2V.	1	1
F = Area of Cornea Involved	0	4	4	3	1	0	0	0	4	4	3	3	3	1
Score (E x F) x 5	0	20	20	15	10	0	0	0	20	40	30	30	15	5
IRIS
D	0	1	1	1	1	0	0	0	1	1	1	1	0	0
Score (D x 5)	0	5	5	5	5	0	0	0	5	5	5	5	0	0
CONJUNCTIVA
A = Redness	2	3H	3H	3H	2HP	1P*	0	2	3H	3H	3H	2HP AI	2PAI	1Ec
B = Chemosis	2	3	3	2	2	1	0	2	3	3	3	2	2	1
C = Discharge	2	3	3	2	2	1	1	2	3	3	3	2	2	2
Score (A + B + C) x 2	12	18	18	14	12	6	2	12	18	18	18	12	12	6
Total Score	12	43	43	34	27	6	2	12	43	63	53	47	27	11

 

IPR= Initial pain reaction + = one drop of local anaesthetic instilled into both eyes 1 to 2 minutes before treatment

V = vascularisation, with localised ingrowth of vessels for approx. 3 mm and covering ¼ of the cornea

V.= vascularisation, with localised ingrowth of vessels for approx. 3 mm and covering ¾ of the cornea

H = Haemorrhage scattered over the nictitating membrane and upper and lower conjunctival membrane

P = off white/pale area covering lower half of nictitating membrane and half of the lower conjunctival membrane

P* = off white/pale area covering lower half of nictitating membrane

Al = alopecia around the treated eye

Table 2: Individual Total Scores and Group Mean Scores for Ocular Irritation

 

Rabbit Number and Sex	Individual Total Scores At:
	1 h	24 h	48 h	72 h	7d	14 d	21 d
68714 Male	12	43	43	34	27	6	2
68794 Male	12	43	63	53	47	27	11
Group Total	24	86	106	87	74	33	13
Group Mean Score	12.0	43.0	53.0	43.5	37.0	16.5	6.5

 

Table 3: Individual Bodyweights and Bodyweight Changes

 

Rabbit Number and Sex	Individual Bodyweight (kg)		Bodyweight Change (kg)
	Day 0	Day 21
68714 Male	2.65	3.05	0.40
68794 Male	2.32	2.82	0.50

Interpretation of results:

irritating

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The hazard phrase H318: "Causes serious eye damage" is therefore required due to irreversible ocular damage (based on two rabbits).

Executive summary:

Introduction:

The study was performed to assess the irritancy potential of the test material following a single application to the eye of the New Zealand White rabbit. The method was designed to meet the requirements of the following: OECD Guidelines for the Testing of Chemicals No. 405 "Acute Eye Irritation/Corrosion" (adopted 24 April 2002) and Method B5 Acute Toxicity (Eye Irritation) of Commission Regulation (EC) No. 440/2008.

Result:

A single application of the test material to the non-irrigated eye of two rabbits produced translucent corneal opacity, iridial inflammation and severe conjunctival irritation. Other corneal effects noted were vascularisation with a localised ingrowth of vessels for approximately 3 mm, haemorrhage over the nictitating and conjunctival membranes and an off white/pale area covering the conjunctival membrane and/or nictitating membrane.

Conclusion:

The test material produced a maximum group mean score of 53.0 and was classified as a severe irritant (Class 6 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

In accordance with CLP Regulation (1272/2008), the hazard phrase H318: "Causes serious eye damage" is required due to irreversible ocular damage (based on two rabbits).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Additional information

Justification for selection of skin irritation / corrosion endpoint:
GLP Guideline compliant in vivo study.

Justification for selection of eye irritation endpoint:
GLP Guideline compliant in vivo study.

Effects on eye irritation: corrosive

Effect level: empty Endpoint conclusion: Adverse effect observed

Justification for classification or non-classification

MnCl2 is not irritating to skin, based on an in vivo study in rabbit (IUCLID section 7.3.1, Pooles A). MnCl2 is irritating to the eyes and should be classified as Eye Damage Cat. 1 on the basis of the severity of the effects seen (IUCLID section 7.3.2, Pooles A)