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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. The key study (Warren N, 2013) 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 an ex vivo study has been performed prior to conducting an in vivo study. This study is not sufficient for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP). However, the study does support the conclusion that magnesium bis(dihydrogenorthophosphate) is not corrosive to the eyes and the data can therefore be used to support the conclusions made in the in vivo study. The in vivo study (Bradshaw J, 2013) 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 the regulatory requirement as a key study for this endpoint.

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 06 November 2012 and 12 November 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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: 10 July 2012, Date of Signature: 30 November 2012
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:
Triplicate tissues were treated
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:
% tissue viability
Value:
86.2
Other effects / acceptance of results:
The relative mean viability of the test material treated tissues was 86.2% after a 15-minute exposure.

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 82.6% 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 5.4% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 2.2%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was 0.697 and the standard deviation value of the percentage viability was 8.2%. The negative control acceptance criterion was therefore satisfied. The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 3.4%. 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¤

0.728

0.697

0.057

104.4

100*

8.2

0.631

90.5

0.732

105.0

Positive Control Material¤

0.032

0.038

0.015

4.6

5.4

2.2

0.055

7.9

0.026

3.7

Test Material

0.588

0.601

0.024

84.4

86.2

3.4

0.628

90.1

0.586

84.1


SD=    Standard deviation

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

¤ = Control group shared with Harlan Laboratories Ltd, Project numbers 41205113, 41205115, 41205120, 41205125, 41205128 and 41205133


Interpretation of results:
GHS criteria not met
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). Magnesium bis(dihydrogenorthophosphate) 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
Remarks:
In vivo. Study was performed prior to the update of the REACH Regulation and prior to the validation of in vitro test methods.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 02 January 2013 and 21 January 2013.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of GLP inspection: 10 July 2012 Date of Signature on GLP certificate: 30 November 2012
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK.

- Age at study initiation: Twelve to twenty weeks old

- Weight at study initiation: 2.70 or 2.83 kg

- Housing: The animals were individually housed in suspended cages. The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

- Diet (e.g. ad libitum): ad libitum (2930 Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK)

- Water (e.g. ad libitum): ad libitum.

- Acclimation period: At least five days


ENVIRONMENTAL CONDITIONS

- Temperature (°C): 17 to 23°C

- Humidity (%): 30 to 70%

- Air changes (per hr): At least fifteen changes per hour

- Photoperiod (hrs dark / hrs light): Twelve hours continuous light (06:00 to 18:00) and twelve hours darkness


IN-LIFE DATES:
From: day 1 To:day 7
Vehicle:
unchanged (no vehicle)
Controls:
other: The left eye remained untreated and was used for control purposes.
Amount / concentration applied:
TEST MATERIAL

- Amount(s) applied (volume or weight with unit): 100 mg of the test item 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 loss of the test item, and then released. The left eye remained untreated and was used for control purposes.

- Concentration (if solution): Undiluted and used as supplied

VEHICLE

- Amount(s) applied (volume or weight with unit):
Not applicable

Duration of treatment / exposure:
72 hours
Observation period (in vivo):
Approximately 1 hour and 24, 48 and 72 hours following treatment. An additional observation was made on Day 7 to assess the reversibility of the ocular
effects.
Number of animals or in vitro replicates:
2 animals were tested in total. (After consideration of the ocular responses produced in the first treated animal, one additional animal was treated. )

Details on study design:
REMOVAL OF TEST SUBSTANCE

- Washing (if done):
Not applicable

- Time after start of exposure:
Not applicable


SCORING SYSTEM:
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 J H (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).


TOOL USED TO ASSESS SCORE:

Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
Irritation parameter:
cornea opacity score
Basis:
animal: 72826 Male
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: No effects observed
Remarks on result:
other: Initial pain reaction = 2
Irritation parameter:
cornea opacity score
Basis:
animal: 72873 Male
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: No effects observed
Remarks on result:
other: Inital pain reaction = 2
Irritation parameter:
iris score
Basis:
animal: 72826 Male
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: No effect observed
Irritation parameter:
iris score
Basis:
animal: 72873 Male
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: No effect observed
Irritation parameter:
conjunctivae score
Basis:
animal: 72826 Male
Time point:
24/48/72 h
Score:
1.66
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Basis:
animal: 72873 Male
Time point:
24/48/72 h
Score:
1.66
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal: 72826 Male
Time point:
24/48/72 h
Score:
1.33
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal: 72873 Male
Time point:
24/48/72 h
Score:
1.33
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritant / corrosive response data:
Individual and group mean scores for ocular irritation are given in Table 1 and Table 2.
No corneal effects were noted during the study.
Iridial inflammation was noted in one treated eye one hour after treatment.
Moderate conjunctival irritation was noted in both treated eyes one hour after treatment
and at the 24 and 48-Hour observations with minimal conjunctival irritation noted at the
72-Hour observation.
Both treated eyes appeared normal at the 7-Day observation.
Other effects:
Bodyweight
Both animals showed expected gain in bodyweight during the study.

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. 

If evidence of irreversible ocular damage is noted, the test material will be classified as corrosive to the eye.

 

Table1               Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex

72826 Male

72873 Male

IPR= 2

IPR = 2

Time After Treatment

1
Hour

24
Hours

48
Hours

72
Hours

7

Days

1
Hour

24
Hours

48
Hours

72
Hours

7

days

CORNEA

 

 

E = Degree of Opacity

0

0

0

0

0

0

0

0

0

0

F = Area of Cornea Involved

0

0

0

0

0

0

0

0

0

0

Score (E x F) x 5

0

0

0

0

0

0

0

0

0

0

IRIS

 

 

D

1

0

0

0

0

0

0

0

0

0

Score (D x 5)

5

0

0

0

0

0

0

0

0

0

CONJUNCTIVAE

 

 

A = Redness

2

2

2

1

0

2

2

1

1

0

B = Chemosis

2

2

1

1

0

2

2

1

1

0

C = Discharge

3

2

1

0

0

2

1

1

0

0

Score (A + B + C) x 2

14

12

8

4

0

12

10

8

4

0

Total Score

19

12

8

4

0

12

10

8

4

0

 

IPR=  Initial pain reaction

 

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

Rabbit Number

and Sex

Individual Total Scores At:

1 Hour

24 Hours

48 Hours

72 Hours

7 Days

72826 Male

19

12

8

4

0

72873 Male

12

10

8

4

0

Group Total

31

22

16

8

0

Group Mean Score

15.5

11.0

8.0

4.0

0.0

 

Interpretation of results:
GHS criteria not met
Conclusions:
The test material did not meet the criteria for classification as irritant according to Regulation (EC) No. 1272/2008 (EU CLP). This study is conducted according to the appropriate guidelines (OECD 405) 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. Study is sufficient for classification and labelling purposes, in accordance with Regulation (EC) No. 1272/2008 (EU CLP).
Executive summary:

Introduction

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit. The method was designed to be compatible with the following:

 OECD Guidelines for the Testing of Chemicals No. 405 “Acute Eye Irritation/Corrosion” (adopted 24 April 2002)

 Method B5 Acute Toxicity (Eye Irritation) of Commission Regulation (EC) No. 440/2008

Result

A single application of the test item to the non-irrigated eye of two rabbits produced iridial inflammation and moderate conjunctival irritation. Both treated eyes appeared normal at the 7-Day observation.

Conclusion

The test item produced a maximum group mean score of 15.5 and was classified as a moderate irritant (Class 5 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

The test item does not meet the criteria for classification according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

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

Eye irritation: No classification is proposed in accordance with 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 magnesium bis(dihydrogenorthophosphate) is irritating to the respiratory tract.