Decyl dihydrogen phosphate, potassium salt

Endpoint summary

Description of key information

For the skin irritation/corrosion endpoint, the in vitro skin irritation study identified as key study was performed according to OECD 439 and under GLP with validity rating 1. The decylphosphoric acid, potassium salt (CAS 68427-32-7)was tested in the Reconstricted Human Epidermis test method, where tissues of the human skin model EpiSkin^™were treated with the test item for 15 minutes. After the treatment, the mean relative absorbance value decreased to 20.0%. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

In combination with the available data from thein vitro skincorrosion test (OECD 431) where the test substance, decyl dihydrogen phosphate, potassium salt (CAS# 68427-32-7), did not induce enough response to be classified as corrosive, the substance is considered to be classified asskin irritation Category 2.

 

The Eye irritation endpoint is covered by the only in vivo eye irritation study avaibalable for the test substance decyl dihydrogen phosphate, potassium salt (CAS# 68427-32-7. It has been identified as key study, and shows that after asingle application of the test substanceto the no-irrigated eye of three rabbits severe irritation is produced. As the changes in the eye were not reversible within 21 days in one animal, and the other 2 animals were sacrificed on day 7 because of the severity of the response, the test compound was considered a serious irritant to the rabbit eye. The test material requires classification in Category 1 according to OECD-GHS.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 05 May 2017. Experimental completion date: 09 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

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:

other: UN GHS (published 2003, last (6th) revision 2015).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

EpiSkin™ Kit Components Needed for the Assay
EpiSkin™ Kit Lot No.: 17-EKIN-023
1 Sealed 12-well plate Contains 12 inserts with EpiSkin™ tissues on agarose
1 12-well plate For MTT viability assay
1 bottle Assay Medium Basic medium for use in MTT assays
1 bottle EpiSkin™ Maintenance Medium Basic medium for incubations

Cell Culture
EpiSkin™ kits are purchased from SkinEthic Laboratories (69007 Lyon, France). The
EpiSkin™ tissue consists of NHEK, which have been cultured to form a multilayered, highly
differentiated model of the human epidermis. It consists of organized basal, spinous and
granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid
layers arranged in patterns analogous to those found in vivo. The EpiSkin™ tissues (surface
0.38 cm²) are cultured on specially prepared cell culture inserts.
EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose
gels in a 12-well plate and reached Envigo CRS GmbH on 07 June 2017. On the day of
experiment the pre-incubation phase of the EpiSkin™ tissues started.

Vehicle:

unchanged (no vehicle)

Details on test system:

Pre-warming of EpiSkin™ Tissues
Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates
containing the pre-warmed (37 ± 1.5 °C) maintenance medium. The EpiSkin™ tissues were
incubated for approximately 3 hours.

Treatment
The negative control, positive control and the test item were added into the insert atop the
concerning EpiSkin™ triplicate tissues for 15 minutes.
After the end of the treatment interval the inserts were immediately removed from the 12-
well plate. The tissues were gently rinsed with PBS to remove any residual test material.
Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting
paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues
were incubated for approximately 42 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

IL-1 α Immunoassay
Samples of all treatment groups were taken from the wells. The plates were shaken for
approximately 15 minutes to homogenise the released mediators in the medium before
sampling. At least 1.6 mL medium from each well was taken and was stored in the freezer at
–20 °C. Since the results derived from the MTT assay were not
unclear or borderline, the IL-1 α concentration in the medium was not determined and the
taken samples were discarded after report finalization

MTT Assay
A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.
After the 42 ± 1 hour incubation period was completed for all tissues the cell culture inserts
were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period
(37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was rinsed three times with PBS and the tissues
were plotted. Tissue samples were cut out of the inserts with a biopsy punch and transferred
into plastic vials. The tissue samples were immersed into extractant solution by gently
pipetting 0.5 mL extractant solution (isopropanol containing 0.04 N HCl) into each vial. The
tissue samples were completely covered by isopropanol. The formazan salt was extracted for
about 2.5 hours at room temperature with gentle agitation.
Per tissue sample 2 × 200 μL aliquots of the formazan extract were transferred into a 96-well
flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular
Devices, 85737 Ismaning, Germany, version 4.7.1) with 570 nm filter. Mean values were
calculated from the 2 wells per tissue sample.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 μL (26.3 μL/cm2) of undiluted test item
10 μL of both negative and positive control

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

incubated for approximately 42 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2

Number of replicates:

triplicate

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

20

Vehicle controls validity:

not applicable

Negative controls validity:

not valid

Remarks:

please see acceptance of results section

Positive controls validity:

not valid

Remarks:

please see acceptance of results section

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The optical pre-experiment (colour interference pre-experiment) to investigate the colour change potential of the test item in water did not lead to a change in colour.Optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent did not show blue colour.

The mean relative absorbance value of the test item, corresponding to the cell viability,decreased to 20.0% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin.

Concerning acceptance criteria:
•The mean OD of the three negative control exposed tissues is ≥ 0.6 till ≤ 1.5 (range: 1.384 to 1.468).
• The rel. standard deviations between tissues of the same treatment group was ≤ 18% (3.2% (negative control) and 13.1% (test item)). For the positive control, the acceptance criteria were not met (28.7%). Reason: extremely low absorbance values of 0.014, 0.014 and 0.022, where small differences cause high rel. standard deviations.

The OECD guideline 439 and the SkinEthic protocol request only the standard deviation being ≤ 18% as acceptance criteria between tissues of the same treatment group. If the standard deviation is considered (0.3%), the acceptance criteria are met.

This deviation from the acceptance criteria is not considered to have an impact on the outcome of the study.
• The mean relative tissue viability of the positive control was ≤ 40% (1.2%).
• The acceptance limit of the IC50 of the respective EpiSkin™ lot was between 1.5 and 3.0 mg/mL after 18 hours treatment with SLS (1.8 mg/mL).
• Historical Data for the negative control are not available. Reason: Envigo CRS GmbH changed the negative control for the test system “In vitro Skin Irritation Assay using RHE supplied by SkinEthic” from deionized water to PBS. The test is performed at Envigo CRS GmbH a long time prior to former a version of OECD 439 guideline, where deionized water or PBS are suggested as negative control. Originally, SkinEthic requested deionized water to be used as negative control in their protocol.

All of Envigo CRS GmbH’s historical data are based on deionized water. In the current SkinEthic protocol, PBS is suggested as negative control. Therefore, Envigo CRS GmbH decided to change the negative control to be in accordance with the SkinEthic protocol, but the number of performed assays using PBS as negative control is still too low to issue historical data.

Historical Data for the positive control are available (see annex 1). But the determined viability value of 1.2% is not within the historical data (viability range: 3.90% -32.73%). Since the historical data at Envigo CRS GmbH are updated at least once a year, after next update the present positive control viability of 1.2% will be within the historical range. Since neither the OECD guideline nor SkinEthic requests historical data for the positive control, the outcome of the study is not impacted by this.

The exceptions from the acceptance criteria did not have an impact on the outcome of the study.

Test Group	Absor-bance 570 nm Tissue Well 1	Absor-bance 570 nm Tissue Well 2	Mean Absor-bance 570 nm	Mean Absor-bance 570 nm*	Mean Absor-bance of 3 Tissues	Standard Deviation of Tissue Absorbance	Relative Absorbance [%] Tissue 1, 2 + 3**	Standard Deviation [%]	Rel. Standard Deviation [%]****	Rel. Absorbance [% of Negative Control]***
Blank	0.050	0.048	0.049	0.000
Negative Control	1.480	1.457	1.468	1.420	1.387	0.045	102.4	3.2	3.2	100.0
	1.469	1.438	1.453	1.405			101.3
	1.432	1.337	1.384	1.336			96.3
Positive Control	0.063	0.062	0.062	0.014	0.017	0.005	1.0	0.3	28.7	1.2
	0.061	0.065	0.063	0.014			1.0
	0.076	0.066	0.071	0.022			1.6
Test Item	0.305	0.290	0.298	0.249	0.277	0.036	18.0	2.6	13.1	20.0
	0.314	0.311	0.312	0.264			19.0
	0.368	0.366	0.367	0.318			22.9

*         Mean of two replicate wells after blank correction

**       relative absorbance per tissue [rounded values]: (100 x absorbance_tissue)/(mean absorbance _{negative control})

***     relative absorbance per treatment group [rounded values]: (100 x mean absorbance_{test item})/(mean absorbance_{negative control})

****      relative standard deviation per treatment group [rounded values]: (100 x standard deviation_{tissue absorbance})/(mean absorbance_tissue)

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, Decyl dihydrogen phosphate, potassium salt (CAS# 68427-32-7) is irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the irritation potential of Decyl dihydrogen phosphate, potassium salt (CAS# 68427-32-7) by means of the Human Skin Model Test according to OECD TG 439.

The test item did not reduce MTT (pre-test for direct MTT reduction), and it did not dye water, when mixed with it (pre-test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative control (PBS) or the positive control (5% sodium lauryl sulfate) for 15 minutes.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.6 till ≤ 1.5 thus showing the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control thus ensuring the validity of the test system.

After treatment with the test item the mean relative absorbance value decreased to 20.0%. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, Decyl dihydrogen phosphate, potassium salt (CAS# 68427-32-7) is irritant to skin according to UN GHS and EU CLP regulation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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 1974

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Limited reported study with only very basic data given; comparable to guideline/standard

Qualifier:

according to guideline

Guideline:

other: FDA, Federal Register (17 Sep 1964, 191.12)

GLP compliance:

no

Remarks:

pre-GLP

Species:

rabbit

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: no info
- Age at study initiation: no info
- Weight at study initiation: no info
- Housing: no info
- Diet (e.g. ad libitum): no info
- Water (e.g. ad libitum): no info
- Acclimation period: no info


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no info
- Humidity (%): no info
- Air changes (per hr): no info
- Photoperiod (hrs dark / hrs light): no info


IN-LIFE DATES: October 1974

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated eye served as control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL

VEHICLE: not used

Duration of treatment / exposure:

single application

Observation period (in vivo):

24, 48 and 72 h, day 4, day 7 and in one animal also on days 14 and 21

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no

SCORING SYSTEM:
CORNEA
Degree of Opacity (most dense area used)
No opacity 0
Scattered or diffuse areas, details of iris clearly visible 1
Easily discernible translucent areas, details of iris slightly obscured 2
Nacreous areas, no details of iris visible, size of pupil barely discernible 3
Opaque cornea, iris not discernible 4

IRIS
Normal 0
Folds above normal, congestion, swelling, circumcorneal injection
(any or all of these or combination of any thereof), iris still reacting to light
(sluggish reaction is positive) 1
No reaction to light, haemorrhage, gross destruction (any or all of these) 2

CONJUNCTIVAE
Redness (refers to palpebral and bulbar conjunctivae excluding cornea and iris)
Vessels normal 0
Vessels definitely injected above normal 1
More diffuse, deeper crimson red, indiviual vessels not easily discernible 2
Diffuse beefy red 3

Chemosis
No swelling 0
Any swelling above normal (includes nictitating membrane) 1
Obvious swelling with partial eversion of lids 2
Swelling with lids about half closed 3
Swelling with lids more than half closed 4

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: days 2/3/4

Score:

1.44

Max. score:

4

Reversibility:

not fully reversible within: 21 days (1/3 animals)

Remarks on result:

other: 2/3 animals sacrificed on day 7 because of the severity of the response

Irritation parameter:

iris score

Basis:

mean

Time point:

other: days 2/3/4

Score:

0.78

Max. score:

2

Reversibility:

fully reversible within: 4 days (1/3 animals)

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

other: days 2/3/4

Score:

2.11

Max. score:

3

Reversibility:

not fully reversible within: 21 days (1/3 animals)

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: days 2/3/4

Score:

2.56

Max. score:

4

Reversibility:

fully reversible within: 21 days (1/3 animals)

Irritant / corrosive response data:

Cornal opacities developed in all three animals, and an opacity was still present in one animal on day 21. The remaining two animals were sacrificed on day 7 due to the severity of the response. lritis was observed in all three animals. A diffuse crimson to beefy
red colouration of the conjuntivae and considerable swelling with the eyelids about half closed was observed in all three animals.

Other effects:

Not reported.

Summary of ocular lesions

Anim. No.	Effect	Days after application							Mean score cornea Days 2/3/4*	Mean score iritis Days 2/3/4*	Mean score redness Days 2/3/4*	Mean score chemosis Days 2/3/4*
		1	2	3	4	7	14	21
1	Cornea* Iris Redness Chemosis	1 1 2 2	1 1 2 2	1 1 2 3	2 0 2 2	4 0 1 1	3 0 1 1	2 0 1 0	1.33	0.67	2	2.33
2	Cornea* Iris Redness Chemosis	1 0 2 2	1 0 2 2	1 1 3 3	4 1 2 3	4 1 2 3	nm	nm	2	0.67	2.33	2.67
3	Cornea* Iris Redness Chemosis	1 0 1 2	1 1 2 2	1 1 2 3	1 1 2 3	4 1 2 3	nm	nm	1	1	2	2.67
Mean all anim.									1.44	0.78	2.11	2.56

nm = not measured as animals had been sacrificed; * score on days 2/3/4 was averaged as signs were generally more severe on day 4 than on day 1   

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Remarks:

Migrated information

Conclusions:

As the changes in the eye were not reversible within 21 days in one animal, and the other 2 animals were sacrificed on day 7 because of the severity of the response, the test compound was considered a serious irritant to the rabbit eye.

Executive summary:

A study was performed to assess the irritancy potential of the test material consisting of 45% decylphosphoric acid, potassium salt (CAS 68427-32-7) to the eye of the rabbit (strain not specified). A single application of the test material to the eye of three rabbits produced serious irritation. The treated eye of one animal was still not normal 21 days after application; the other 2 animals were sacrificed on day 7 due to the severity of the response. The test material requires classification in Category 1 according to OECD-GHS.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin
Two skin irritation/corrosion studies are available onthe substancedecyl dihydrogen phosphate, potassium salt (CAS# 68427-32-7). Both the studies are recently performed under GLP and according to current guidelines.

 

The decylphosphoric acid, potassium salt (CAS 68427-32-7)was tested inthe Reconstricted Human Epidermis test method, where tissues of the human skin model EpiSkin^™were treated with the test item, the negative control (PBS) or the positive control (5% sodium lauryl sulfate) for 15 minutes. After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.6 till ≤ 1.5 thus showing the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control thus ensuring the validity of the test system. After treatment with the test item the mean relative absorbance value decreased to 20.0%. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

Eye 

The in vivo eye irritation study identified as key study was performed according to the 1964 Federal Register of Food and Drug Administration in USA. This was before GLP was in place and the study is considered to be limitedly reported with only very basic data given. Anyhow it is given Klimish 2 rating as it is comparable to now available guideline/standard. Thereforeit is not considered justified from an animal welfare point of view to repeat any available studies performed under comparable guidelines. A single application ofthe substancetest substancedecyl dihydrogen phosphate, potassium salt (CAS# 68427-32-7)to the no-irrigated eye of three rabbits produced severe irritation. As the changes in the eye were not reversible within 21 days in one animal, and the other 2 animals were sacrificed on day 7 because of the severity of the response, the test compound was considered a serious irritant to the rabbit eye. The test material requires classification in Category 1 according to OECD-GHS.

Justification for classification or non-classification

Skin

The conclusion from the two availablein vitrostudies is that the substancedecyl dihydrogen phosphate, potassium salt (CAS# 68427-32-7)is not corrosive buthas to be classified for skin irritation as Category 2according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011), nor the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.The Signal Word “Warning” and Hazard Statement “H315: Causes skin irritation” are therefore required.

 

 

Eye

The available in vivo eye irritation study givesGHS Cat 1 for eye irritation as the changes in the eye were not reversible within 21 days in one animal, and the other 2 animals were sacrificed on day 7 because of the severity of the response, the test compound was considered a serious irritant to the rabbit eye. Based on this information, thedecyl dihydrogen phosphate, potassium salt (CAS# 68427-32-7)  is to be classified for eye irritation Category 1according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011), and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.