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Description of key information

Skin irritation/corrosion: Two studies are available for this endpoint; 
- An in vitro GLP study conducted according to OECD guideline 431 is available and indicates that the test material has no corrosive potential.
- The key study is an in vitro GLP study conducted according to EU Method B.46 and is valid for the assessment of skin irritation potential. The study demonstrated that the test material was not irritating to SKINETHIC(TM) Reconsituted Human Epidermal Models. This result is a sufficient to consider the test material not to be classified for skin irritation according to Regulation (EC) No 1272/2008 (EU CLP).
Eye irritation/corrosion: Two studies are available for this endpoint;
- In accordance with the testing strategy detailed in Annex VIII, column 1 of Regulation (EC) No. 1907/2006 an in vitro study has been performed prior to conducting an in vivo study. 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).
- The key study (Bradshaw 2010) is conducted under the conditions of GLP and according to OECD guideline 405 (in vivo). Under the conditions of this test, and in accordance with Regulation (EC) 1272/2008, disodium fluorophosphate was not considered to be classified for eye irritation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation / corrosion
Remarks:
other: VALIDATED IN VITRO METHOD
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 19 January 2010 and 25 January 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
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:
other: EU Guideline Testing of Chemicals B46
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Draft Test Guideline (version 4)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspectection: 15-09-2009 Date of Signature: 26-11-2009
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):
10 ± 2 mg of the test material was applied to the epidermis surface pre-moistened with 5 µl of sterile distilled water.

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

VEHICLE
No vehicle used
Duration of treatment / exposure:
15 Minutes & 42 hour post exposure incubation
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
TEST SITE
- Area of exposure:
10 ± 2 mg of the test material was applied to the epidermis surface pre-moistened with 5 µl of sterile distilled water.

- % 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 PBS 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.

- 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 (I) R38

Mean tissue viability is >50% : Non-Irritant (NI)
Irritation parameter:
other: Viability of cells
Basis:
mean
Remarks:
Viability of cells (%)
Time point:
other: Day 6
Score:
103.4
Max. score:
100
Reversibility:
other: Not applicable
Remarks on result:
other: See relative mean viability below.
Irritant / corrosive response data:
The relative mean viability of the test material treated tissues was 103.4% after a 15-minute exposure.
Other effects:
No

RESULTS

Direct MTT Reduction

The MTT solution containing the test material did not turn blue/purple 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 103.4% after a 15 minute exposure.

The qualitative evaluation of tissue viability is given in Table 2.

Following the 15-minute exposure the test material treated tissues appeared blue/white which was considered indicative of viable tissue.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was ≤40% relative to the negative control treated tissues and the standard deviation value of the percentage viability was ≤20%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was ≥0.6 and the SD value of the percentage viability was ≤20%. The negative control 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

0.787

0.825

0.054

95.4

100*

6.6

0.887

107.5

0.801

97.1

Positive Control Material

0.037

0.033

0.005

4.5

4.0

0.6

0.034

4.1

0.027

3.3

Test Material

0.882

0.853

0.025

106.9

103.4

3.1

0.834

101.1

0.844

102.3

SD=    Standard deviation

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


Table2 : 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)

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).
Executive summary:
Introduction: The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKINTMreconstituted human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 -[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 hour post-exposure incubation period is also determined for test materials which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.

Methods:

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for approximately 42 hours. 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.

Data are presented in the form of percentage viability (MTT reduction in the test material treated tissues relative to negative control tissues).

Results: 

The relative mean viability of the test material treated tissues was 103.4% after a 15-minute exposure.

Quality criteria: 

The quality criteria required for acceptance of results in the test were satisfied.

Conclusion:  The test material was considered to be Non-Irritant (NI).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation, other
Remarks:
In vivo study performed in accordance with REACH at the time of first submission (2010).
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 27 April 2010 and 08 May 2010.
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: Date of Signature on GLP certificate: 26/11/2009
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source:
Harlan Laboratories UK Ltd, Oxon, UK

- Age at study initiation:
Twelve to twenty weeks old

- Weight at study initiation:
2.0 to 3.5 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 (2030 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 3
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):
A volume of 0.1 ml 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.

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

VEHICLE

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

- Concentration (if solution):
Not applicable

- Lot/batch no. (if required):
Not applicable

- Purity:
Not reported
Duration of treatment / exposure:
72 hours
Observation period (in vivo):
Approximately 1 hour and 24, 48 and 72 hours following treatment
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
Remarks:
Degree of opacity
Basis:
animal: 69147 Male
Time point:
other: Mean 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
not specified
Remarks:
No effects observed
Irritation parameter:
cornea opacity score
Remarks:
Degree of opacity
Basis:
animal: 69180 Male
Time point:
other: Mean 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
not specified
Remarks:
No effects observed
Irritation parameter:
iris score
Basis:
animal: 69147 Male
Time point:
other: Mean 24, 48 and 72 hours
Score:
0
Max. score:
2
Reversibility:
not specified
Remarks:
No effects observed
Irritation parameter:
iris score
Basis:
animal: 69180 Male
Time point:
other: Mean 24, 48 and 72 hours
Score:
0
Max. score:
2
Reversibility:
not specified
Remarks:
No effects observed
Irritation parameter:
other: redness
Basis:
animal: 69147 Male
Time point:
other: Mean 24, 48 and 72 hours
Score:
0.66
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
other: redness
Basis:
animal: 69180 Male
Time point:
other: Mean 24, 48 and 72 hours
Score:
0.66
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
chemosis score
Basis:
animal: 69147 Male
Time point:
other: Mean 24, 48 and 72
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
animal: 69180 Male
Time point:
other: Mean 24, 48 and 72 hours
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritant / corrosive response data:
Ocular Reactions
Individual and group mean scores for ocular irritation are given in Table 1 and Table 2.
No corneal effects were noted during the study.
No iridial inflammation effects were noted during the study.
Moderate conjunctival irritation was noted in all treated eyes one hour after treatment with minimal conjunctival irritation noted at the 24 and 48-Hour observations.
Both treated eyes appeared normal at the 72-Hour observation.
Other effects:
Body weight
All animals showed expected gain in bodyweight during the study.
Individual bodyweights and bodyweight changes are given in Table 3.

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.

Table1              Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex

69147Male

69180Male

IPR= 2

IPR = 2

Time After Treatment

1
Hour

24
Hours

48
Hours

72
Hours

1
Hour

24
Hours

48
Hours

72
Hours

CORNEA

 

 

 

 

 

 

 

 

E = Degree of Opacity

0

0

0

0

0

0

0

0

F = Area of Cornea Involved

0

0

0

0

0

0

0

0

Score (E x F) x 5

0

0

0

0

0

0

0

0

IRIS

 

 

 

 

 

 

 

 

D

0

0

0

0

0

0

0

0

Score (D x 5)

0

0

0

0

0

0

0

0

CONJUNCTIVAE

 

 

 

 

 

 

 

 

A = Redness

2

1

1

0

2

1

1

0

B = Chemosis

2

1

0

0

2

1

0

0

C = Discharge

2

1

0

0

2

1

0

0

Score (A + B + C) x 2

12

6

2

0

12

6

2

0

Total Score

12

6

2

0

12

6

2

0

 

IPR= Initial pain reaction


Table 2              Individual Total Scores and Group Mean Scoresfor Ocular Irritation

Rabbit Number

and Sex

Individual Total Scores At:

1 Hour

24 Hours

48 Hours

72 Hours

69147Male

12

6

2

0

69180Male

12

6

2

0

Group Total

24

12

4

0

Group Mean Score

12.0

6.0

2.0

0.0

 


Table 3              Individual Bodyweights and Bodyweight Changes

Rabbit Number
and Sex

Individual Bodyweight (kg)

Bodyweight Change (kg)

Day 0

Day 3

69147Male

2.60

2.71

0.11

69180Male

2.15

2.24

0.09


Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test material produced a maximum group mean score of 12.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.
The test material is not classified as an eye irritant according to the EU labelling regulations Commision Directive 2001/59/EC. In addition the substance is not classified as irritating to the eyes in accordance with Regulation (EC) No 1272/2008 (EU CLP).

This study has been selected as the key study 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).
Executive summary:

Introduction. 

The study was performed to assess the irritancy potential of the test material 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)

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 moderate conjunctival irritation. Both treated eyes appeared normal at the 72‑Hour observation.

Conclusion. 

The test material produced a maximum group mean score of 12.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for selection of skin irritation / corrosion endpoint:
One key study exists. This study is acceptable for classification in accordance with Regulation (EC) No.1272/2008 (EU CLP).

Justification for selection of eye irritation endpoint:
One key study exists. This study is acceptable for classification in accordance with Regulation (EC) No.1272/2008 (EU CLP).

Justification for classification or non-classification

Skin irritation/corrosion: Disodium fluorophosphate is not considered to be classified for skin irritation/corrosion in accordance with Regulation (EC) No. 1272/2008 (EU CLP).The key study is considered to be adequate and reliable for the purposes of classification and therefore further testing is not considered to be scientifically justified.

Eye irritation/corrosion: Disodium fluorophosphate is not considered to be classified for eye irritation/corrosion in accordance with Regulation (EC) No. 1272/2008 (EU CLP). The key study is considered to be adequate and reliable for the purposes of classification and therefore further testing is not considered to be scientifically justified.

There are no data (workplace or study data) to suggest that disodium fluorophosphate is irritating to the respiratory tract.