Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts

Endpoint summary

Description of key information

According to Annex I of Regulation (EC) 1272/2008, the test item Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts has no obligatory labelling requirement for skin and eye irritation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

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:

equivalent or similar to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Phosphoric acid, C14-15 branched and linear alkyl esters, potassium salts
CAS No.: 1893414-79-3
Physical state: white solid at 20 °C
Batch No.: PU61810016
Re-certification date of batch: 09 March 2018
Purity: 100 % (UVCB, lyophilized solid, water content 0.85 % (w/w))
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult donors

Source strain:

not specified

Justification for test system used:

This test method is able to detect chemicals that cause skin irritation, i.e. produce reversible damage to the skin and allows for hazard identification in accordance with UN GHS “Category 2”. Depending on the regulatory framework it can also be used to identify non-classified chemicals.

Vehicle:

unchanged (no vehicle)

Details on test system:

In the present study the skin irritant potential of Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts was analysed. The EPISKIN-Standard Model (EPISKIN-SMTM), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

10 µL (undiluted)
The test was performed on a total of 3 tissues per dose group.

Duration of treatment / exposure:

15 min of exposure

Duration of post-treatment incubation (if applicable):

42 h of post-incubation

Number of replicates:

The test was performed on a total of 3 tissues per dose group.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

92.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

10 µL DPBS

Positive controls validity:

valid

Remarks:

10 µL 5% SDS solution

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

98.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

10 µL DPBS

Positive controls validity:

valid

Remarks:

10 µL 5% SDS solution

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

105.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

10 µL DPBS

Positive controls validity:

valid

Remarks:

10 µL 5% SDS solution

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

98.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Acceptance Criteria
Value Cut off pass/fail
Mean OD570 nm Blank 0.042 < 0.1 pass
Mean Absolute OD570 nm NC 0.857 0.6 ≤ NK ≤1.5 pass
Mean Relative Viability [%] PC 13.0 ≤ 40% pass
Max. SD of % Viability [%] 3.9 – 11.4 ≤ 18% pass

Acceptance criteria of the OECD test guideline were fulfilled.

Validity controls as provided by the supplier (SkinEthic):

Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.

Morphology:
Histology scoring (HES stained vertical paraffin sections, n = 6):
Specification ≥ 19.5
Result: 21.8 ± 0.7, CV = 3.1%

Barrier function:
IC50 determination (SDS concentration, MTT test, n = 14):
Specification ≥ 1.5 mg/mL
Result: 2.4 mg/mL

Pre-experiment

The mixture of 10 µL test item per 2 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equaled 0%.

The mixtures of 10 µL of the test item per 90 µL aqua dest. and per 90 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSCliving equaled 0%

Experiment

The potential of the test item to induce skin irritation was analysed by using the three-dimensional human skin model EPISKIN-SM (SkinEthic) comprising a reconstructed epidermis with a functional stratum corneum. In the present study Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts was applied topically to the EPISKIN-SM tissue for 15 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay.

Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (98.8%) after 15 min treatment and 42 h post-incubation.

The controls confirmed the validity of the study. The mean OD570 of the six blank values was < 0.1. The mean absolute OD570 of the three negative control tissues was between 0.6 and 1.5. The mean relative tissue viability (% negative control) of the positive control was less than 40% (13.0%). The maximum standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (3.9% - 11.4%).

Table 1. Result of the Test Item Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts

Name	Negative Control	Positive control	Test item
Mean Relative Tissue Viability [%]	100.0	13.0**	98.9
SD Tissue Viability [%]*	3.9	11.4	6.3
CV [% Viability]	3.9	87.4	6.4

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

** Mean relative tissue viability of the three positive control tissues is ≤ 40 %.

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Executive summary:

The potential of the test item to induce skin irritation was analysed by using the three-dimensional human skin model EPISKIN-SM (SkinEthic) comprising a reconstructed epidermis with a functional stratum corneum. In the present study Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts was applied topically to the EPISKIN-SM tissue for 15 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (98.8%) after 15 min treatment and 42 h post-incubation. The controls confirmed the validity of the study.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Phosphoric acid, C14-15 branched and linear alkyl esters, potassium salts
CAS No.: 1893414-79-3
Physical state: white solid at 20 °C
Batch No.: PU61810016
Re-certification date of batch: 09 March 2018
Purity: 100 % (UVCB, lyophilized solid, water content 0.85 % (w/w))
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult donors

Source strain:

not specified

Justification for test system used:

This test uses the EPISKIN-SM reconstructed human epidermis model (SkinEthic) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Test Item 50 ± 3 µL (undiluted)

Duration of treatment / exposure:

3 min, 60 min and 4 h exposure time

Duration of post-treatment incubation (if applicable):

4 h experiment: the tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. Then the 12-well plate(s) were incubated for 4 h ± 10 min at room temperature.

60 min experiment: the tissues were treated with the test item and the negative control in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. Then the 12-well plate(s) were incubated for 60 min ± 5 min at room temperature.

3 min experiment: the tissues were treated with the test item and the negative control in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. A constant time interval of e.g. 30 sec was kept between dosing.

Number of replicates:

The test was performed on a total of 6 tissues for each test item and for the negative control, 2 replicates for each treatment period (3 min, 60 min and 4 h exposure time). For the positive control the test was performed with 2 replicates for the 4 h exposure time.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 - 3 min Experiment

Value:

100

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

0.9% NaCl solution

Positive controls validity:

not examined

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2 - 3 min Experiment

Value:

111.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

0.9% NaCl solution

Positive controls validity:

not examined

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean - 3 min Experiment

Value:

106

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

0.9% NaCl solution

Positive controls validity:

not examined

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 - 60 min Experiment

Value:

98.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

0.9% NaCl solution

Positive controls validity:

not examined

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2 - 60 min Experiment

Value:

95.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

0.9% NaCl solution

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean - 60 min Experiment

Value:

97

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

0.9% NaCl solution

Positive controls validity:

not examined

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 - 4 h Experiment

Value:

102.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.9% NaCl solution

Positive controls validity:

valid

Remarks:

50 µL glacial acetic acid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2 - 4 h Experiment

Value:

97.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.9% NaCl solution

Positive controls validity:

valid

Remarks:

50 µL glacial acetic acid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean - 4 h Experiment

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.9% NaCl solution

Positive controls validity:

valid

Remarks:

50 µL glacial acetic acid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Test Acceptance Criteria:

Value Cut off pass/fail
Mean OD570 nm Blank 0.043 < 0.1 pass
Mean Absolute OD570 nm NK (4 h experiment) 0.988 0.6 – 1.5 pass
Mean Absolute OD570 nm NK (60 min Experiment) 0.968 0.6 – 1.5 pass
Mean Absolute OD570 nm NK (3 min Experiment) 0.911 0.6 – 1.5 pass
Mean Relative Viability PC [%](4 h Experiment) 3 ≤ 20% pass
Max. Difference of Tissue Viability 12.0 ≤ 30% pass

Validity controls as provided by the supplier (SkinEthic):

Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.

Morphology:
Histology scoring (HES stained vertical paraffin sections, n = 6):
specification ≥ 19.5
result: 22.7 ± 0.3, CV = 1.1%

Barrier function:
IC50 determination (SDS concentration, MTT test, n = 14):
specification ≥ 1.5 mg/mL
result: 2.7 mg/mL
 

Pre-Experiments

The mixture of 50 µL test item per 2 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equaled 0%.

The mixture of 10 µL test item per 300 µL Aqua. dest. and per 90 µL isopropanol showed no colouring as compared to the solvent. Therefore NSC equaled 0%.

Experiment

The potential of the test item to induce skin corrosion was analysed by using the three-dimensional human skin model EPISKIN-SM, comprising a reconstructed epidermis with a functional stratum corneum.

In the present study Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts was applied topically to the EPISKIN-SM tissue for 3 min, 60 min and 4 h followed by immediate determination of cytotoxic effects via MTT reduction assay. Corrosivity potential of the test item was predicted from the relative mean tissue viabilities compared to the corresponding negative control tissues concurrently treated with 0.9% NaCl. The test item showed no non-specific MTT-reducing or water-colouring potential. The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was more than 35% (100%) after 4 h treatment.

Relative mean tissue viability was reduced to 97% after 60 min treatment and was 106% after 3 min treatment.

The controls confirmed the validity of the study. The mean OD570 of the two negative control tissues was between 0.6 and 1.5 for each exposure period. The mean relative tissue viability (% negative control) of the positive control was less than 20% (3%) after 4 h treatment. The maximum inter tissue viability difference of replicate tissues of all dose groups was less than 30% (1.0% - 12.0%).

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no corrosive effects. The relative mean tissue viability after 4 h treatment was not decreased to less than 35% of the corresponding negative control tissues. The test item is therefore classified as “non-corrosive“.

Executive summary:

In the present study Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts was applied topically to the EPISKIN-SM tissue for 3 min, 60 min and 4 h followed by immediate determination of cytotoxic effects via MTT reduction assay. Corrosivity potential of the test item was predicted from the relative mean tissue viabilities compared to the corresponding negative control tissues concurrently treated with 0.9% NaCl. The test item showed no non-specific MTT-reducing or water-colouring potential. The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was more than 35% (100%) after 4 h treatment. Relative mean tissue viability was reduced to 97% after 60 min treatment and was 106% after 3 min treatment. The controls confirmed the validity of the study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The Bovine Corneal Opacity and Permeability (BCOP) test method is an in vitro test method that can be used to classify substances as “ocular corrosives / severe irritants” and “non-irritants”. The BCOP is recommended for use as part of a tiered-testing strategy for regulatory classification and labelling within a specific applicability domain. Test substances can be classified as ocular corrosives / severe irritants or non-irritants without further testing in rabbits

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Phosphoric acid, C14-15 branched and linear alkyl esters, potassium salts
CAS No.: 1893414-79-3
Physical state: white solid at 20 °C
Batch No.: PU61810016
Re-certification date of batch: 09 March 2018
Purity: 100 % (UVCB, lyophilized solid, water content 0.85 % (w/w))
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany.
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing penicillin ( 100 IU/mL) / streptomycin (100 µg/mL) 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 (Duratec GmbH) 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.

Vehicle:

physiological saline

Remarks:

physiological saline 0.9% NaCl

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item was suspended with physiological saline 0.9% NaCl to give a 20% (w/w) concentration.

Duration of treatment / exposure:

750 µL 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).

Duration of post- treatment incubation (in vitro):

After the illuminance measurement was performed, 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.

Number of animals or in vitro replicates:

3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive controls treated with imidazole 20% in physiological saline 0.9% NaCl

Irritation parameter:

in vitro irritation score

Run / experiment:

mean - IVIS

Value:

38.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

The in vitro irritation 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.

The eye irritancy potential of Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts was investigated in the bovine corneal opacity and permeability assay. The test item was suspended with physiological saline 0.9% NaCl (see 10.3) to give a 20% concentration. All 3 corneas treated with Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts showed slightly opacity and fissures in the tissue.

The following mean in vitro irritation score was calculated: 38.10

No prediction can be made regarding the classification of the test substance Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts according to the evaluation criteria. Further testing in another suitable method is required. The in vitro irritation 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. The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Interpretation of results:

study cannot be used for classification

Conclusions:

No prediction can be made regarding the classification of the test substance Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts according to the evaluation criteria. Further testing in another suitable method is required.

Executive summary:

The eye irritancy potential of Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts was investigated in the bovine corneal opacity and permeability assay. The test item was suspended with physiological saline 0.9% NaCl (see 10.3) to give a 20% concentration. All 3 corneas treated with Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts showed slightly opacity and fissures in the tissue.

The following mean in vitro irritation score was calculated: 38.10

No prediction can be made regarding the classification of the test substance Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts according to the evaluation criteria.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

A Bovine Corneal Opacity and Permeability Assay (OECD Guideline number 437) with Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts was performed (Eurofins Munich / BSL Munich Project No. 164234). Based on the results of this test further in-vivo testing for assessing acute eye irritation/corrosion is necessary.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Phosphoric acid, C14-15 branched and linear alkyl esters, potassium salts
CAS No.: 1893414-79-3
Physical state: white solid at 20 °C
Batch No.: PU61810016
Re-certification date of batch: 09 March 2018
Purity: 100 % (UVCB, lyophilized solid, water content 0.85 % (w/w))
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Test animal:

Species/strain: healthy New Zealand White Rabbits, Crl: KBL (NZW)
Source: Charles River Deutschland, 97633 Sulzfeld, Germany
Sex: male
Body weight at the beginning of the study: > 2 kg

Age at the beginning of the study: animal no. 1 approximately 36 weeks old
animal no. 2 approximately 37 weeks old
animal no. 3 approximately 38 weeks old
Number of animals: 3

The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes.

Housing and Feeding Conditions:

- Semi barrier in an air-conditioned room
- Temperature: 18 +- 3 °C (recommendations of TVT, GV-SOLAS)
- Relative humidity: 55 +- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Free access autoclaved hay and to Altromin 2123 maintenance diet for rabbits, rich in crude fibre
- Free access to tap water (drinking water, municipal residue control, microbiological controls at regular intervals)
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Housed in ABS-plastic or Noryl rabbit cages, floor 4200 cm2
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Preparation of the Animals:

Approximately 24 hours before the test and immediately prior to the application both eyes of each animal were examined. A health inspection was performed to ensure the good state of health of the animals. Approximately 20.5 hours (animal no. 1), 17 hours (animal no. 2) and 21.5 hours (animal no. 3) before the application the eyes were also examined with the aid of a fluorescein solution. The eyes were rinsed with physiological saline 0.9% NaCl after the examination. None of the animals showed eye irritation, ocular defects, or pre-existing corneal injury.

Amount / concentration applied:

A dose of 0.1 g of the test item was applied to the test site.

Duration of treatment / exposure:

In animal no. 2 and animal no. 3, the treated eye was rinsed with physiological saline 0.9% NaCl 1 hour after the application. The eyes of animal no. 1 were not rinsed after application of the test substance.

Observation period (in vivo):

Observation Period:
The animals were observed for 72 hours after dosing. To determine the reversibility of the observed effects, the observation period was extended up to 8 days after dosing.

Clinical Observation:
The eyes were examined for signs of irritation throughout the observation period. The eye irritation was scored and recorded according to the grades in Table 5.
For the calculation only the 24, 48 and 72-hour readings were used. 72 hours post-application as well as once daily during the prolonged observation period the treated eyes were examined with the aid of a fluorescein solution. The eyes were rinsed with physiological saline 0.9% NaCl after the examination.

Number of animals or in vitro replicates:

Number of animals: 3

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24 h

Score:

2

Max. score:

4

Reversibility:

not reversible

Remarks on result:

probability of moderate irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24 h

Score:

2

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of moderate irritation

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of mild irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

48 h

Score:

2

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of moderate irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

48 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of mild irritation

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

48 h

Score:

2

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of moderate irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of moderate irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of mild irritation

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of moderate irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of moderate irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of mild irritation

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of mild irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of mild irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of mild irritation

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

48 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of mild irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

48 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of mild irritation

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

48 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of mild irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of mild irritation

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of mild irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of mild irritation

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of mild irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of mild irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

48 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

48 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

48 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of mild irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of weak irritation

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of mild irritation

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

48 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

48 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

48 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of mild irritation

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of mild irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

probability of weak irritation

Other effects:

Clinical Observation

Animal 1:
test item 1 h
Local Findings / Comments: hypersecretion with moistening of the lids and surrounding hair
Systemic Findings: no specific findings

test item 24 h – 7 d
Local Findings / Comments: no specific findings
Systemic Findings: no specific findings

Animal 2:
test item 1 h
Local Findings / Comments: hypersecretion with moistening of the lids and surrounding hair
Systemic Findings: no specific findings

test item 24 h
Local Findings / Comments: slight hypersecretion
Systemic Findings: no specific findings

test item 48 h – 8 d
Local Findings / Comments: no specific findings
Systemic Findings: no specific findings

Animal 3:
test item 1 h
Local Findings / Comments: hypersecretion with moistening of the lids and surrounding hair
Systemic Findings: no specific findings

test item 24 h – 7 d
Local Findings / Comments: no specific findings
Systemic Findings: no specific findings

After the application into the eyes of three male NZW rabbits the test item produced irritant but not corrosive ocular effects in all animals. Neither mortalities nor significant clinical signs of toxicity but local effects were observed. At the end of the prolonged observation period no corneal lesions were found. Conjunctival redness, chemosis and hypersecretion were observed in all animals, corneal effects and iris lesion were observed in animal no. 1 and no. 3.

Under the conditions of the present study, a single ocular application of the test item Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts to rabbits at a dose of 0.1 g produced irritant effects, which were fully reversible within 7 days in animal no. 1 and animal no. 3 and within 8 days in animal no. 2.

Interpretation of results:

GHS criteria not met

Conclusions:

According to Annex I of Regulation (EC) 1272/2008, the test item Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts has no obligatory labelling requirement for eye irritation.

Executive summary:

Under the conditions of the present OECD 405 study, a single ocular application of the test item Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts to rabbits at a dose of 0.1 g produced irritant effects, which were fully reversible within 7 days in animal no. 1 and animal

no. 3 and within 8 days in animal no. 2. Neither mortalities nor significant clinical signs of toxicity were observed.

According to Annex I of Regulation (EC) 1272/2008, the test item Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts has no obligatory labelling requirement for eye irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available