Dialkyl C18 and C18-unsaturated phosphonates

Endpoint summary

Description of key information

1- Skin irritation: not irritating to skin (OECD 439, Kr.1, GLP)

2- Eye irritation: not irritating to eyes (Kr.2, Not GLP)

3- Respiratory irritation: no data available.

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:

25/09/2017 - 08/10/2017

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Alkenyl phosphonate
- Expiration date of the lot/batch: 30 June 2018
- Purity test date: 08/06/2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature
- Stability under test conditions: yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France.

Justification for test system used:

This model is a three-dimensional human epidermis model, which consists of adult humanderived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes are cultured for 13-days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used: On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for approximately 22.5 hours at 37°C.
- Quality control for skin discs: Electrical resistance obtained with two of the isolated skin discs was [complete, e.g. 10 kΩ]
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed.
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
1- Test for the Interference of the Test Item with the MTT Endpoint:
A test item may interfere with the MTT endpoint if it is colored and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.
2- Test for Color Interference by the Test Item:
Alkenyl phosphonate was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the skin tissues during the exposure. To assess the color interference, 10 µl of Alkenyl phosphonate was added to 90 µl Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 µl Milli-Q water was tested concurrently. At the end of the shaking period a color check was performed.
3- Test for Reduction of MTT by the Test Item
Alkenyl phosphonate was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 25 µl of the test item was added to 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, 25 µl sterile Milli-Q water was tested concurrently. At the end of the incubation period a color check was performed.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement):
* The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be =<18.
b) The mean relative tissue viability of the positive control should be =< 50% relative to the negative control and the Standard Deviation value (SD) of the % viability should be =< 18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be =< 18.

* A test item is considered irritant in the skin irritation test if:
- The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test item is considered non-irritant in the in vitro skin irritation test if:
- The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL

VEHICLE: none

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL PBS.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL 5% SDS.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 +/- 0.5 minutes treatment with Alkenyl phosphonate

Value:

103

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Colour interference with MTT: Alkenyl phosphonate was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no color changes were observed it was concluded that Alkenyl phosphonate did not interact with the MTT endpoint.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: yes

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions of this study, the relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Alkenyl phosphonate compared to the negative control tissues was 103%. Since the mean relative tissue viability for Alkenyl phosphonate was above 50% Alkenyl phosphonate is considered to be non-irritant. Therefore, no classification is required according to EU criteria.

Executive summary:

In a skin irritation study (OECD 439, Kr.1, GLP), 25 µL of undiluted Alkenyl phosphonate was applied directly on top of the skin tissue for 15 ± 0.5 minutes (EPISKIN Small model (EPISKIN-SM^TM)).

After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test item. 

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Alkenyl phosphonate compared to the negative control tissues was 103%. Since the mean relative tissue viability for Alkenyl phosphonate was above 50% after 15 ± 0.5 minutes treatment Alkenyl phosphonate is considered to be non-irritant.

The positive control had a mean cell viability of 11% after 15 ± 0.5 minutes exposure. The absolute mean OD₅₇₀(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 6%, indicating that the test system functioned properly.

In conclusion, Alkenyl phosphonate is non-irritant in the in vitro skin irritation test under the test conditions of this study. Therefore, no classification is required according to EU criteria.

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:

1967

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

In accordance to Section 191.12 (a) (1) and (2) of the Final Orders, Enforcement Regulations, Federal Register, Vol. 29, No. 182, P 13009, 17 September 1964.

GLP compliance:

no

Specific details on test material used for the study:

No data available.

Species:

rabbit

Strain:

not specified

Details on test animals or tissues and environmental conditions:

No data available.

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

Amount applied: 0.1 ml

Observation period (in vivo):

3 days

Number of animals or in vitro replicates:

6

Details on study design:

REMOVAL OF TEST SUBSTANCE
no data

SCORING SYSTEM: Draize system

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: see table of results

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

0.05

Max. score:

3

Reversibility:

fully reversible

Remarks on result:

other: see table of results

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

other: see table of results

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: see table of results

Table 1: Irritant/corrosive response data for each animal at each observation time

Score at time point / Reversibility	Cornea	Iris	Conjunctivae	Chemosis
	Max. score: 4	Max. score: 2	Max. score: 3	Max. score: 4
60 min	0/0/0/0/0/0	0/0/0/0/0/0	0/0/0/0/0/0	0/0/0/0/0/0
24 h	0/0/0/0/0/0	0/0/0/0/0/0	1/0/0/0/0/0	0/0/0/0/0/0
48 h	0/0/0/0/0/0	0/0/0/0/0/0	0/0/0/0/0/0	0/0/0/0/0/0
72 h	0/0/0/0/0/0	0/0/0/0/0/0	0/0/0/0/0/0	0/0/0/0/0/0
Average 24h, 48h, 72h	0	0	0.05	0
Reversibility*)	-	-	c	-

 

*) Reversibility: c. = completely reversible

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions of this study, the mean scores (24, 48 and 72 hours) for six rabbits were 0.0, 0.0, 0.0 and 0.05 for chemosis, iris, corneal opacity and conjunctiva respectively. Therefore, Alkenyl phosphonate is not considered as irritating to eyes according to EU criteria.

Executive summary:

In an eye irritation study (Kr.2, Not GLP), 0.1 mL of Alkenyl phosphonate was instilled into the conjunctival sac of the eye of six Albino New-Zealand White rabbits (6). Animals were then observed for 3 days. Ocular irritation was scored by the method of Draize.

The mean scores (24, 48 and 72 hours) for all tested rabbits were 0.0, 0.0, 0.0 and 0.05 for chemosis, iris, corneal opacity and conjunctiva respectively. Therefore, Alkenyl phosphonate is not considered as irritating to eyes according to EU criteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

1- Skin irritation:

One study was available and considered as the key study. In a skin irritation study (OECD 439, Kr.1, GLP), 25 µL of undiluted Alkenyl phosphonate was applied directly on top of the skin tissue for 15 ± 0.5 minutes (EPISKIN Small model (EPISKIN-SM^TM)). The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Alkenyl phosphonate compared to the negative control tissues was 103%. Since the mean relative tissue viability for Alkenyl phosphonate was above 50% after 15 ± 0.5 minutes treatment Alkenyl phosphonate is considered to be non-irritant.

The positive control had a mean cell viability of 11% after 15 ± 0.5 minutes exposure. The absolute mean OD₅₇₀(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. Based on these results, Alkenyl phosphonate is non-irritant to skin.

2- Eye irritation:

One study was available and considered as the key study (Kr.1, Not GLP). In this study, 0.1 mL of Alkenyl phosphonate was instilled into the conjunctival sac of the eye of six Albino New-Zealand White rabbits. Animals were then observed for 3 days. Ocular irritation was scored by the method of Draize. The mean scores (24, 48 and 72 hours) for all tested rabbits were 0.0, 0.0, 0.0 and 0.05 for chemosis, iris, corneal opacity and conjunctiva respectively. Therefore, Alkenyl phosphonate is not considered as irritating to eyes.

3- Respiratory irritation: no data available.

Justification for selection of skin irritation / corrosion endpoint:
The study was conducted according to a recognised guideline and under GLP conditions.

Justification for selection of eye irritation endpoint:
The study was conducted comparable to a recognised guideline.

Effects on skin irritation/corrosion: not irritating to skin.

Effects on eye irritation: not irritating to eyes.

Justification for classification or non-classification

Harmonized classification:

No harmonized classification is available according to the Regulation (EC) No 1272/2008.

Self classification:

1- Skin irritation:

Based on the results of the key study (OECD 439, Kr.1, GLP), Alkenyl phosphonate is not considered as irritating to skin and therefore no classification is required according to EU criteria.

2- Eye irritation:

Based on the results of the key study (Kr.2, Not GLP), Alkenyl phosphonate is not considered as irritating to eyes and therefore no classification is required according to EU criteria.

3- Respiratory irritation:

No classification is proposed due to lack of data.