Registration Dossier

Administrative data

Description of key information

Skin irritation: In accordance with the testing strategy detailed in Annex VIII, column 1 of Regulation (EC) No. 1907/2006 the assessment of the endpoint ‘skin irritation or skin corrosion’  has been performed following the consecutive steps detailed in the Regulation. As such an in vitro skin corrosion study has been performed. This study is not considered as the key study because it is not sufficient for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP) and is therefore submitted as supporting data.  The key study (Warren N, 2012) is conducted according to an appropriate validated in vitro guideline and under the conditions of GLP and therefore the study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint. In addition, the data is considered to be adequate and reliable for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP). 
Eye irritation: In accordance with the testing strategy detailed in Annex VIII, column 1 of Regulation (EC) No. 1907/2006 (REACH) an ex vivo study has been performed prior to conducting an in vivo study. In addition to this an in vivo study was performed. In this instance the in vivo data are considered to be more reliable when comparing against a real-world situation and as such this study (Lowe C, 2013) is used for the purpose of classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP) and as a key study for REACH.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 12 June 2012 and 18 June 2012
Reliability:
1 (reliable without restriction)
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. This study is conducted according to an appropriate validated in vitro guideline and under the conditions of GLP and therefore the study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint. In addition, the data is considered to be adequate and reliable for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP).
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspectection: 19-21 July 2011 Date of Signature: 31 August 2011
Species:
other: reconstituted human epidermis model
Strain:
other: reconstituted human epidermis model
Details on test animals or test system and environmental conditions:
Not applicable
Type of coverage:
other: Topical
Preparation of test site:
other: Not applicable
Vehicle:
other: No vehicle used
Controls:
no
Amount / concentration applied:
TEST MATERIAL

- The test material was applied neat.

- Amount(s) applied (volume or weight with unit):
Approximately 10 mg of the test item was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 μl of sterile distilled water to improve contact between the solid test item and the epidermis.

- Concentration (if solution):
The test material was used as supplied.

VEHICLE
No vehicle used
Duration of treatment / exposure:
15 minute exposure & 42 hour post-exposure incubation
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
TEST SITE
- Area of exposure:
Approximately 10 mg of the test item was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 μl of sterile distilled water to improve contact between the solid test item and the epidermis.

- % coverage:
The test material was applied topically to the corresponding tissues ensuring uniform covering.

- Type of wrap if used:
None used

REMOVAL OF TEST SUBSTANCE
- Washing (if done):
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test material. The rinsed tissues were transferred to the second column of 3 wells containing 2 ml of maintenance medium in each well. The rinsed tissues were incubated at 37ºC, 5% CO2 in air for 42 hours.

- Time after start of exposure:
15 minutes post-exposure

SCORING SYSTEM:
Quantitative MTT Assessment (percentage tissue viability)
For the test material the relative mean tissue viabilities obtained after the 15 minute treatment followed by the 42 hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:

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

Classification of irritation potential is based upon relative tissue viability following the 15 minute exposure period followed by the 42 hour post-exposure incubation period according to the following:

Mean tissue viability is ≤50% : Irritant: H315, Category 2

Mean tissue viability is >50% : Non-Irritant (NI)
Irritation / corrosion parameter:
other: other: Viability of cells
Value:
107.3
Remarks on result:
other:
Remarks:
Basis: mean Viability of cells (%). Time point: Day 6. Max. score: 100.0. Reversibility: other: Not applicable. Remarks: See relative mean viability below.. (migrated information)
Irritant / corrosive response data:
The relative mean viability of the test material treated tissues was 107.3% after a 15-minute exposure.
Other effects:
No

RESULTS

Direct MTT Reduction

The MTT solution containing the test material did not turn blue which indicated that the test material did not directly reduce MTT.

Test Material, Positive Control Material and Negative Control Material

The individual and mean OD540 values, standard deviations and tissue viabilities for the test material, negative control material and positive control material are given in Table 1. The mean viabilities and standard deviations of the test material and positive control, relative to the negative control are also given in Table 1.

The relative mean viability of the test material treated tissues was 128.1% after a 15 minute exposure.

The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 8.6% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.0%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was 0.659 and the standard deviation value of the percentage viability was 6.2%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 8.7%. The test item acceptance criterion was therefore satisfied.

Table1 : Mean OD540 Values and Percentage Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material

OD540of tissues

Mean OD540of triplicate tissues

±SDof OD540

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Material¤

0.666

0.659

0.041

101.1

100*

6.2

0.696

105.6

0.615

93.3

Positive Control Material¤

0.059

0.056

0.006

9.0

8.6

1.0

0.061

9.3

0.049

7.4

Test Material

0.721

0.707

0.057

109.4

107.3

8.7

0.644

97.7

0.756

114.7


SD=    Standard deviation

*=     The mean viability of the negative control tissues is set at 100%

¤ = The control groups served as common controls with Harlan Laboratories Ltd, Project numbers 41200853, 41200860, 41200861, 41200871, 41200880, 41200884 and 41201543.


Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test material was considered to be Non-Irritant (NI).
This study is conducted according to an appropriate validated in vitro guideline and under the conditions of GLP and therefore the study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint. In addition, the data is considered to be adequate and reliable for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP).Manganese bis(dihydrogen phosphate) is not considered to be classified in accordance with Regulation (EC) No. 1272/2008 (EU CLP).
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:
October 1 – 11, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no deviations from standard test guidelines and no minor methodological deficiencies. This study is conducted according to an appropriate guideline and under the conditions of GLP and therefore the study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement (Regulation (EC) No. 1907/2006; REACH) as a key study for this endpoint. In addition, this study is considered to be acceptable for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP).
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: Received from Robinson Services, Inc. on September 26, 2012.
- Age at study initiation: Young adult.
- Sex: Female, nulliparous and non-pregnant.
- Housing: The animals were singly housed in suspended stainless steel caging with mesh floors, which conform to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011). Litter paper was placed beneath the cage and was changed at least three times per week.
- Diet: Harlan Teklad Global High Fiber Rabbit Diet® #2031. A designated amount of the diet was available to each rabbit (approximately 150 grams/day).
- Water: Filtered tap water was supplied ad libitum by an automatic water dispensing system.
- Acclimation period: 5

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23ºC
- Humidity (%): 43-84% Humidity was above the targeted upper limit for 1 day during the study. A portable dehumidifier was used to lower the humidity levels during this time.
- Air changes (per hr): 12. Airflow measurements are evaluated regularly and the records are kept on file at Product Safety Labs.
- Photoperiod (hrs dark / hrs light): 12 hours light/dark

Contaminants: There were no known contaminants reasonably expected to be found in the food or water at levels which would have interfered with the results of this study. Analyses of the food and water are conducted regularly and the records are kept on file at Product Safety Labs.
Vehicle:
unchanged (no vehicle)
Controls:
other: untreated eye served as control
Amount / concentration applied:
Instillation
Prior to instillation, 2-3 drops of ocular anesthetic (Tetracaine Hydrochloride Ophthalmic Solution, 0.5%) were placed into both the treated and control eye of each animal. One-tenth of a milliliter (0.10 grams) of the test substance was then instilled into the conjunctival sac of the right eye of each rabbit by pulling the lower lid away from the eyeball. The upper and lower lids were then gently held together for about one second before releasing to minimize loss of the test substance. The other eye of each rabbit remained untreated with the test substance and served as a control. The rabbits were then returned to their designated cages.
Duration of treatment / exposure:
10 days
Observation period (in vivo):
10 days post-instillation
Number of animals or in vitro replicates:
3
Details on study design:
Prior to test initiation, both eyes of a group of animals were examined using a white light source and a fluorescein dye procedure. One drop of 2% ophthalmic fluorescein sodium was instilled into both eyes of each rabbit. The eyes were rinsed with physiological saline (0.9% NaCl) approximately 30 seconds after instillation of the fluorescein and then evaluated for corneal damage using an ultraviolet light source. Prior to test substance instillation, the eyes were reexamined and scored for abnormalities according to the "Scale for Scoring Ocular Lesions". Three healthy animals without pre-existing ocular irritation were selected for test.


TOOL USED TO ASSESS SCORE: Ocular irritation was evaluated using a high-intensity white light (Mag Lite) in accordance with Draize et al at 1, 24, 48, and 72 hours and at 4, 7, and 10 days post-instillation. Individual scores were recorded for each animal. The fluorescein dye evaluation procedure was used in the treated eye at 24 hours and as needed at subsequent scoring intervals to evaluate the extent of corneal damage or to verify reversal of effects. In addition to observations of the cornea, iris and conjunctivae, any other observed lesions were noted. The average score for all rabbits at each scoring period was calculated to aid in data interpretation.
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
other: Mean 24, 48 and 72 hours
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
other: Mean 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
other: Mean 24, 48 and 72 hours
Score:
0.66
Max. score:
4
Reversibility:
not fully reversible within: 72 hours
Irritation parameter:
iris score
Basis:
other: All animals tested
Time point:
other: Mean 24, 48 and 72 hours
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
other: all animals tested
Time point:
other: Mean 24, 48 and 72 hours
Score:
3
Max. score:
3
Reversibility:
fully reversible within: 10 days
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
other: Mean 24, 48 and 72 hours
Score:
2.33
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
other: Mean 24, 48 and 72 hours
Score:
3
Max. score:
4
Reversibility:
not fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
other: Mean 24, 48 and 72 hours
Score:
3.66
Max. score:
4
Reversibility:
fully reversible within: 10 days
Irritant / corrosive response data:
Individual eye irritation scores are presented in Table 1.

There was no iritis observed in any treated eye during this study. Within 24 hours of test substance instillation, two treated eyes exhibited corneal opacity and all three treated eyes exhibited "positive‟ conjunctivitis. The overall incidence and severity of irritation decreased gradually thereafter. An area of blanching on the conjunctivae was noted for one animal from 24 hours through Day 7. All animals were free of ocular irritation by Day 10 (study termination).
Other effects:
All animals appeared active and healthy during the study. Apart from the eye irritation noted below, there were no other signs of gross toxicity, adverse pharmacologic effects, or abnormal behavior.

Interpretation of Results

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 x F) x 5

Using the numerical data obtained a modified version of the system (Modified Kay and Calandra Interpretation of Eye Irritation Test was used to classify the ocular irritancy potential of the test material. This was achieved by adding together 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 reactions 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.

 

Table 1             Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit 3401 Female

Rabbit 3402 Female

Rabbit 3403 Female

Hours

Days

Hours

Days

Hours

Days

Time After Treatment

1

24

48

72

4

7

10

1

24

48

72

4

7

10

1

24

48

72

4

7

10

CORNEA

 

 

 

 

 

 

 

 

 

E = Degree of Opacity

0

11

01

0

0

0

0

0

01

0

0

0

0

0

1

11

1

01

0

0

0

F = Area of Cornea Involved

4

1

4

4

4

4

4

4

4

4

4

4

4

4

1

1

1

4

4

4

4

Score (E x F) x 5

0

5

0

0

0

0

0

0

0

0

0

0

0

0

5

5

5

0

0

0

0

IRIS

 

 

 

 

 

 

 

 

 

D

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Score (D x 5)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

CONJUNCTIVAE

 

 

 

 

 

 

 

 

 

A = Redness

2

3

3

3

2

0

0

3

3

3

3

3

0

0

3

32

32

32

32

12

0

B = Chemosis

3

3

2

2

1

0

0

4

4

3

2

2

0

0

3

4

4

3

2

0

0

C = Discharge

2

2

2

1

1

0

0

2

3

2

2

1

0

0

2

3

3

2

1

0

0

Score (A + B + C) x 2

14

16

14

12

8

0

0

18

20

16

14

12

0

0

16

20

20

16

12

2

0

Total Score

14

21

14

12

8

0

0

18

20

16

14

12

0

0

21

25

25

16

12

2

0

 

12% ophthalmic fluorescein sodium used to evaluate the extent or verify the absence of corneal opacity.

2Area of blanching present on conjunctivae.

Interpretation of results:
Category II
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In accordance with Regulation EC No. 1272/2008 on the Classification, Labeling, and Packaging of Substances and Mixtures, and based on the results of this study, Manganese bis(dihydrogen phosphate) classification: Category 2 (irritating to eye).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for selection of skin irritation / corrosion endpoint:
This study has been selected as the key study in accordance with Regulation (EC) No. 1907/2006 (REACH) because the results are sufficient in order to derive a reliable conclusion on classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP). No effects are noted.

Justification for selection of eye irritation endpoint:
This study is considered to be sufficient for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP) and therefore is also considered to be adequate and reliable for use as a key study in accordance with REACH.

Effects on eye irritation: irritating

Justification for classification or non-classification

Skin irritation: No classification is proposed in accordance with Regulation (EC) No. 1272/2008 (EU CLP).

Eye irritation: Manganese bis(dihydrogen phosphate) was found to meet the classification of irritating to the eyes (Category 2) according to Regulation (EC) No. 1272/2008 (EU CLP).

The above conclusions are based on reliable (Klimisch 1) and adequate data and as such no further testing is anticipated. There are no workplace data to suggest that manganese bis(dihydrogen phosphate) is irritating to the respiratory tract.