Reaction mass of hexadecyl dihydrogen phosphate and dihexadecyl hydrogen phosphate

Endpoint summary

Description of key information

Reaction mass of dihexadecyl hydrogen phosphate and hexadecyl dihydrogen phosphate  was tested for its skin irritant properties using the three-dimensional human skin model Episkin-SM. The study was performed according to OECD Guideline 439. 10 mg of the test item and 5 µL distilled water were applied topically for 15 minutes. After 42 h post-incubation cytotoxic effects were determined via MTT reduction assay. The mean relative tissue viability (% negative control) was > 50%. The test item is therefore classified as "non-irritant" (EU CLP and UN GHS: No Category). 
Reaction mass of dihexadecyl hydrogen phosphate and hexadecyl dihydrogen phosphate  was tested for its eye irritant properties in a bovine corneal opacity and permeability assay. The study was performed according to OECD Guideline 437. According to the mean in vitro score of 130.9 the test item is classified as very severe irritant.

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:

Jan - Feb 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

other: EU Method B.46 (Skin Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Species:

other: EpiSkin reconstructed human epidermis model (SkinEthic Laboratories)

Details on test animals or test system and environmental conditions:

organotypic reconstructed three-dimensional model of the human epidermis

Type of coverage:

other: direct application

Amount / concentration applied:

10 mg + 5 µL aqua dest.
The amount of aqua dest. was increased to 50 µL to allow for evenly spreading of the test item to match size of the tissue.

Duration of treatment / exposure:

15 minutes

Observation period:

42 +/- 1 h

Details on study design:

3 replicate tissues are dosed with the test item, the negative control (PBS) and the positive control (5% SDS), respectively. After 15 minutes treatment period at room temperature the test item and the controls are rinsed off with PBS and the tissues are post-incubated for 42 +/- 1 h. Then the tissues are stained via MTT for 3 hours. Isopropanol extracts are measured photometrically at 550 nm.

Irritation / corrosion parameter:

other: other: mean relative tissue viability

Value:

> 50

Remarks on result:

other:

Remarks:

Basis: other: mean tissue viability of the negative control tissues. Time point: 15 min treatment 42 h post-incubation. Remarks: non-irritant; EU CLP and UN GHS: No Category. (migrated information)

Irritation / corrosion parameter:

other: other: mean relative tissue viability

Value:

<= 50

Remarks on result:

other:

Remarks:

Basis: other: mean tissue viability of the negative control tissues. Time point: 15 min treatment 42 h post-incubation. Remarks: irritant; EU CLP and UN GHS Category 2. (migrated information)

Irritant / corrosive response data:

If mean tissue viability is > 50% relative to the mean negative control, the test item is classified as non-irritant (EU CLP and UN GHS: No Category).
If mean tissue viability is <= 50% relative to the mean negative control, the test item is classified as irritant (EU CLP and UN GHS: Category 2).

Other effects:

The test item showed no direct MTT reducing capability and no colouring potential.

 

Name	NC			PC			TI
Tissue	1	2	3	1	2	3	1	2	3
absolute OD550	0.913	0.905	0.854	0.061	0.058	0.061	1.076	1.059	1.121
	0.929	0.892	0.908	0.060	0.061	0.064	1.077	1.070	1.151
blank-corrected OD550	0.868	0.860	0.809	0.016	0.013	0.015	1.031	1.014	1.076
	0.884	0.847	0.863	0.014	0.015	0.018	1.032	1.025	1.106
mean OD550of the duplicates (blank-corrected)	0.876	0.853	0.836	0.015	0.014	0.017	1.031	1.019	1.091
total mean OD550of 3 replicate tissues (blank-corrected)	0.855^*			0.015			1.047
SD OD550	0.03			0.00			0.04
relative tissue viabilities [%]	102.5	99.8	97.7	1.8	1.6	2.0	120.6	119.2	127.6
mean tissue viability [%]	100.0			1.8^**			122.5
SD tissue viability [%]^***	2.4			0.2			4.5
CV [% viability]	2.4			9.1			3.7

*       Corrected mean OD₅₅₀of the negative control corresponds to 100% absolute tissue viability.

^**      Mean relative tissue viability of the three positive control tissues is <= 40%

^***     Standard deviation (SD) obtained from the three concurrently tested tissues is < 18%

Interpretation of results:

other: non-irritant; EU CLP and UN GHS: No Category

Remarks:

Criteria used for interpretation of results: other: OECD 439

Conclusions:

The test item is classified as "non-irritant" (No Category).

Executive summary:

In the in vitro skin irritation test using the EpiSkin human epidermis model 10 mg test item + 50 µl A. dest. were applied topically for 15 minutes. After 42 h post-incubation cytotoxic effects were determined via MTT reduction assay.

The mean relative tissue viability (% negative control) was > 50%.

The test item is therefore classified as "non-irritant" (EU CLP and UN GHS: No Category).

This study is classified as acceptable:

OD550 of the blank is < 0.1 (0.045).

Mean OD550 of the three negative control tissues is >= 0.6 and <= 1.5 (0.855).

Mean relative tissue viability of the three positive contol tissues is <= 40% (1.8%).

Standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is < 18% (0.2% - 4.5%).

This study satisfies the requirement for Test Guideline OECD 439 for in vitro skin irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-02-27 to 2012-06-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants), adopted: 7 September 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und dLebensmittelsicherheit, München, Germany)

Vehicle:

physiological saline

Amount / concentration applied:

The test item was diluted with physiological saline 0.9% NaCl to gain a 20% concentration.

Duration of treatment / exposure:

750 microL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method).
After 4 hours ± 5 minutes incubation at 32 +/- 1 °C either the test substance or the control substance was removed.

Details on study design:

Test System

Preparation of the Corneas:
The assay uses isolated corneas obtained as a by-product from an abattoir from freshly slaughtered animals
(from Attenberger Fleisch GmbH & Co. KG).
On the test day, fresh eyes were collected from the slautherhouse and were transported in HBSS containing Pen/Strep
on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera.
The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders
(MC2, Clermont, France) with the endothelial side against the O-ring of the posterior chamber, they had been visually
examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top
of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red)
containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were
incubated for one hour at 32 +/- 1 °C in a water bath.

Calibration of the Opacitometer:
The opacitometer had been switched on 15 min before the calibration procedure was started. Empty cornea holders were
placed into the opacitometer and the readout was adjusted to zero using the “BAL”-turning knob. For calibration the polyester
foil no. 1 was introduced into the test chamber and the readout was adjusted to 75 using the “CAL”-turning knob.
To test the linearity of the measurement, two additional calibration foils, polyester foil no. 2 and polyester foil no. 3,
were measured. For these, the opacitometer was supposed to display 150 and 225, respectively (± 3%).
If this had not been the case, the calibration procedure would have had to be repeated. The calibration procedure
was performed before each test and was documented in the raw data.

Treatment of the Corneas:
After the equilibration period, the medium was removed from both chambers and replaced with fresh Complete RPMI.
An initial opacity measurement was performed on each of the corneas using an opacitometer (MC2, Clermont, France).
Three corneas with opacity readings approximately equivalent to the median opacity of all corneas were selected
as negative-control corneas. The opacity of each cornea was read against an air-filled chamber and recorded.
Corneas that have an initial opacity reading above 7 units were not dosed. The medium was removed from the anterior
chamber and replaced with the test item or control.
750 microL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method).
After 4 hours ± 5 minutes incubation at 32 +/- 1 °C either the test substance or the control substance was removed
and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance,
the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI
and an opacity measurement was performed.
After the opacity measurement the medium was removed from both chambers of the holder. The posterior chamber was
refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and
the corneas were incubated for 90 minutes at 32 +/- 1 °C. Then the medium from the posterior chamber was removed and
its optical density at 490 nm (OD490) was determined, using a spectrophotometer.

Test Groups:
3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive control treated with imidazole 20% in physiological saline 0.9% NaCl
The BCOP assay is considered to be valid if the in vitro score obtained with the positive control falls within the two
standard deviations of the current historical mean.

Evaluation of Results:
The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading.
These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas.
The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.
The mean OD490 for the blank wells were calculated. The mean blank OD490 was subtracted from the OD490 of
each well (corrected OD490). Any dilutions that were made to bring the OD490 values into the linear range of the s
pectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of
the dilution by the dilution factor. The final-corrected OD490 of the test article and the positive control were calculated
by subtracting the average corrected OD490 of the negative control corneas from the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490
The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the
treated corneas for that treatment condition.
The following formula was used to determine the in vitro score:
In vitro score = mean opacity value + (15 x mean OD490 value)

Irritation parameter:

other: in vitro irritation score (IVIS)

Basis:

mean

Score:

130.92

Irritant / corrosive response data:

The eye irritancy potential of the test item was investigated in the bovine corneal opacity and permeability assay.
The test item was diluted with physiological saline 0.9% NaCl to gain a 20% concentration.

The following mean in vitro score was calculated: 130.92
Therefore the test item was classified as very severe irritant.

The in vitro score obtained with the positive control fell within the two standard deviations of the current historical mean and
therefore this assay is considered to be valid.

Opazität
Cornea-No.	Test Item	Opacity	Opacity	Change of	Corrected
		blank value	post dose	opacity values	opacity values
1	Negative	3	3	0
2	Control	3	2	-1
3		3	2	-1
MV		3.00	2.33	-0.67
4	Positive	2	222	220	220.67
5	Control	2	194	192	192.67
6		3	210	207	207.67
MV		2.33	208.67	206.33	207.00
7	Test item	2	139	137	137.67
8		2	123	121	121.67
9		2	136	134	134.67
MV		2.00	132.67	130.67	131.33

Permaebilität
Cornea-No.	Test Item	OD490	Corrected
			OD490 values
1	Negative	0.098
2	Control	0.005
3		0.015
MV		0.039
4	Positive	2.090	2.051
5	Control	2.069	2.030
6		2.062	2.023
MV		2.074	2.034
7	Test item	0.014	-0.025
8		0.008	-0.031
9		0.013	-0.026
MV		0.012	-0.028

in vitroScore
Cornea-No.	Test Item	Corrected	Corrected	in vitro
		opacity value	OD490 value	Score
1	Negative	0	0.098
2	Control	-1	0.005
3		-1	0.015
MV		-0.67	0.039	-0.08
4	Positive	220.67	2.051
5	Control	192.67	2.030
6		207.67	2.023
MV		207.00	2.034	237.52
7	Test item	137.67	-0.025
8		121.67	-0.031
9		134.67	-0.026
MV		131.33	-0.028	130.92

Interpretation of results:

highly irritating

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

According to the evaluation criteria the test item is classified as very severe eye irritant.

Executive summary:

The eye irritancy potential of the test item was investigated in the bovine corneal opacity and permeability assay.

Preparation of the test item: diluted with physiological saline 0.9% NaCl to gain a 20% concentration.

Meanin vitroscore: 130.92

Classification:                         

X	very severe irritant

 

Thein vitroscore obtained with the positive control fell within the two standard deviations of the current historical mean

and therefore this assay is considered to be valid.

Conclusion

According to the evaluation criteria the test item is classified as very severe eye irritant.

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation: 1 key study available: not irritating, 1 supporting study available: not irritating

Eye irritation: 1 key study available: severe eye irritant

 

There is one fully reliable study available on the skin irritancy potential. Reaction mass of dihexadecyl hydrogen phosphate and hexadecyl dihydrogen phosphate was tested for its skin irritant properties using the three-dimensional human skin model Episkin-SM. The study was performed according to OECD Guideline 439. 10 mg of the test item and 5 µL distilled water were applied topically for 15 minutes. After 42 h post-incubation cytotoxic effects were determined via MTT reduction assay. The mean relative tissue viability (% negative control) was > 50%. The test item is therefore classified as "non-irritant" (EU CLP and UN GHS: No Category). Furthermore, Dihexadecyl hydrogen phosphate was tested for its skin irritant properties in 6 New Zealand White rabbits. The study was performed equivalent to OECD Guideline 404. Deviation was a prolonged exposure period (24 hours). These conditions make the study design more rigid compared to the regarding OECD Guideline. No effects on skin (erythema grades 0 and edema scores 0) were observed in all animals 24 and 72 hours after application.

There is one fully reliable in vitro study available on the eye irritancy potential. Reaction mass of dihexadecyl hydrogen phosphate and hexadecyl dihydrogen phosphate was tested for its eye irritant properties in a bovine corneal opacity and permeability assay. The study was performed according to OECD Guideline 437.

According to the mean in vitro score of 130.9 the test item is classified as very severe irritant.

Justification for selection of skin irritation / corrosion endpoint:
Well performed guideline study in accordance with GLP.

Justification for selection of eye irritation endpoint:
Well performed guideline study in accordance with GLP.

Effect level: empty Endpoint conclusion: Adverse effect observed

Justification for classification or non-classification

With reference to the reported results of an in vitro and in vivo skin irritation study Reaction mass of dihexadecyl hydrogen phosphate has not to be classified as irritant to the skin according to the criteria laid down in the EU Classification Labelling and Packaging Regulation (1272/2008/EC).

With reference the reported scores Reaction mass of dihexadecyl hydrogen phosphate and hexadecyl dihydrogen phosphate has to be classified as irritant to the eyes (H318 - Causes serious eye damage) according to the criteria laid down in the EU Classification Labelling and Packaging Regulation (1272/2008/EC).