Registration Dossier

Administrative data

Description of key information

eye irritation: not irritating (OECD 405; GLP)

skin irritation: not irritating (OECD 439, GLP)

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:
2017-11-21 to 2017-12-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015-07-28
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012-07-06
Deviations:
no
Qualifier:
according to
Guideline:
other: MatTek Corporation Protocol for: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)
Version / remarks:
2014-11-07
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2015-06-05
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry, < 30 °C
Test system:
human skin model
Source species:
human
Cell type:
other: normal, human-derived epidermal keratinocytes
Cell source:
other: humans
Source strain:
other: not applicable
Details on animal used as source of test system:
not applicable
Justification for test system used:
This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human-derived epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely resembles the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.
Vehicle:
other: Dulbecco's phosphate buffered saline
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (EPI-200-SIT; MatTek)
- Tissue lot number: 25867

TEST FOR DIRECT MTT REDUCTION
- to check the non-specific MTT-reducing capability of the test item 25 mg of the test item was mixed per 1 mL MTT medium and incubated for 60 min at 37 ± 1 °C in the incubator (5 % CO2, 95 % RH).
- untreated MTT medium was used as control.
- if the mixture turns blue/purple, the test item is presumed to have reduced MTT and the part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using freeze-killed tissues.

TEST FOR COLOUR INTERFERENCE
- to check the colouring potential of the test item 25 mg of the test item was mixed per 300 µL aqua dest. and per 300 µL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min (5 % CO2, 95 % RH).
- if the test item is classified as non-irritant and colouring is detected by unaided eye-assessment, and the chemical in water and/or isopropanol absorbs light in the range of 570 ± 30 nm, the test item was checked for its tissue-colouring potential using additional living tissues treated with the test item.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for 35 ± 1 minutes followed by incubation at room temperature until the 60 ± 1 minute treatment period was completed
- Temperature of post-treatment incubation: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- after the end of the treatment period the tissues were washed 15 times with DPBS.
- subsequently, the inserts were submerged three times in DPBS and shaken to remove rests of the test item.
- then inserts were rinsed once from the inside and the outside with sterile DPBS.
- inserts were placed in prepared 6-well plates containing pre-warmed fresh assay medium per well.
- plates were post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing fresh assay medium and incubated for additional 18 ± 2 h.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/well)
- Incubation time: 3 hours ± 5 minutes
- Extraction of formazan: after the MTT incubation period, the tissues were rinsed three times with DPBS and allowed to dry. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol, sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature for at least 2 hours with shaking on a plate shaker.
Before using the extracts, the plate had been shaken for at least 15 minutes on a plate shaker and the inserts were pierced with an injection needle. The extract was pipetted up and down 3 times before 2 x 200 µL aliquots per each tissue were transferred into a 96-well plate. Optical density (OD) was measured with a filter band without reference wavelength in a plate spectrophotometer using isopropanol as a blank.
- Wavelength: 570 nm
- Filter bandwidth: maximum ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1-virus, Hepatitis B virus and Hepatitis C virus
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density (OD) of the three negative control tissues was calculated after blank correction. This value corresponds to 100 % tissue viability in the current test. For each individual tissue treated with the test item or the positive control, the individual relative tissue viability was calculated according to the following formula:
Relative viability (%) = [mean ODtest item or positive control / ODmean of negative control] * 100
For the test item and the positive control the mean relative viability ± relative standard deviation of the three individual tissues will be calculated and used for classification according to the following prediction model:
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues. The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 (UN GHS “Category 2”), if the tissue viability after exposure and post-incubation is less or equal to 50%. The test substance may be considered as non-irritant to skin in accordance with UN GHS “No Category” if the tissue viability after exposure and post-treatment incubation is more than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg (39 mg/cm²) of the test item

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL of the vehicle

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of 5 % SDS solution
Duration of treatment / exposure:
60 ± 1 minutes
Duration of post-treatment incubation (if applicable):
approx. 42 hours
Number of replicates:
triplicates
Irritation / corrosion parameter:
% tissue viability
Remarks:
(mean)
Value:
105.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, no additional controls were necessary.
- Colour interference with MTT: mixture of 25 mg of the test item per 300 µL aqua dest. and/or per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, no additional controls were necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (value: 1.865).
- Acceptance criteria met for positive control: mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.1 %)
- Acceptance criteria met for variability between replicate measurements: standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.2 % - 9.5 %).
Please also refer to the field "An other information on results incl. tables" below.

Table 1: Results of the test item barium 4 -dodecylphenolate

Name

Negative Control (NK)

Positive Control (PC)

Test material (TM)

Tissue

1

2

3

1

2

3

1

2

3

absolute OD570

1956

1.874

1.856

0.096

0.102

0.099

2.205

1.852

1.976

1.969

1.899

1.889

0.097

0.103

0.102

2.215

1.876

1.957

OD570

(blank-corrected)

1.913

1.832

1.814

0.053

0.059

0.057

2.162

1.809

1.934

1.926

1.856

1.846

0.055

0.061

0.059

2.172

1.833

1.914

mean OD570of the duplicates (blank-corrected)

1.920

1.844

1.830

0.054

0.060

0.058

2.167

1.821

1.924

total mean OD570of 3 replicate tissues (blank-corrected)

1.865*

0.057

1.971

SD OD570

0.048

0.003

0.178

relative tissue viability [%]

103.0

98.9

98.1

2.9

3.2

3.1

116.2

97.7

103.2

mean relative tissue viability [%]

100.0

3.1**

105.7

SD tissue viability [%]***

2.6

0.2

9.5

CV [% viabilities]

2.6

5.4

9.0

* Blank-corrected mean OD570 nmof the negative control corresponds to 100% absolute tissue viability.

** Mean relative tissue viability of the three positive control tissues is  20 %.

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

Table 2: Historical data

 

Mean

OD570±30nm

Negative control (NK)

Mean

Relative Viability [%] Positive control (PC)

SD Viability [%]

Mean

1.843

4.3

4.2

SD

0.286

2.2

4.7

n

22

22

84

Historical data were generated from 2015 to 2017.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is not irritating to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is not irritating to the skin.
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:
2018-04-01 to 2018-04-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
2017-10-09
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2015-06-05
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry, < 30 °C

Due to the intrinsic properties of the test material direct application according to the OECD Guideline number 405 (neat or grounded to fine dust) was technically not feasible. The test material is hygroscopic. After preparation of a solution with ground test material it immediately clumped. For this reason it is assumed, that the ground test material immediately clumps after direct application into the eye.
So the test material was suspended with aqua ad injectionem at a concentration of 40 g/L in order to obtain a saturated aqueous solution (the water solubility of the test material is approximately 33 g/L). The suspension was incubated under agitation for 48 hours at room temperature.
After 48 hours the suspension was centrifuged and the saturated aqueous solution of Barium 4-dodecylphenolate was administered.
Species:
rabbit
Strain:
New Zealand White
Remarks:
Crl:KBL
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, 97633 Sulzfeld, Germany
- Age at study initiation: approx. 47 or 48 weeks old
- Weight at study initiation: 4.0 – 4.4 kg
- Housing: housed in ABS-plastic or Noryl rabbit cages, floor 4200 cm2
- Diet (ad libitum): autoclaved hay and to Altromin 2123 maintenance diet for rabbits, rich in crude fibre
- Water (ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 ± 3 °C
- Relative humidity: 55 ± 10%
- Air changes: at least 10 x / hour
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
other: aqua ad injectionem
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL of the saturated aqueous solution of Barium 4-dodecylphenolate
Duration of treatment / exposure:
not applicable
Observation period (in vivo):
1 hour as well as 24, 48 and 72 hours after test item application
Number of animals or in vitro replicates:
2 female rabbits
Details on study design:
INITIAL AND CONFIRMATORY TEST
The in vivo test was performed initially using one animal. Since the results of the initial test did not indicate the saturated aqueous solution of Barium 4-dodecylphenolate of the test item to be corrosive or a severe irritant to the eye using the procedure described. In order to confirm the response, one additional animal was treated in the same manner.

USE OF TOPICAL ANESTHETICS AND SYSTEMIC ANALGESICS
One hour before the application of the saturated aqueous solution of the test item, 0.01 mg/kg of buprenorphine (Temgesic® 0.3 mg/mL) was administered subcutaneously in order to achieve a therapeutic level of systemic analgesia.
Approx. 5 minutes prior to the application of the saturated aqueous solution of the test item, 1-2 drops of an ocular anaesthetic (Proparakaine-POS® hydrochloride ophthalmic 0.5% solution) were administered in both the treated and the control eye of each animal.
Post-application the animals did not show pain and distress and therefore did not receive analgesia.

REMOVAL OF TEST SUBSTANCE
- Washing: eyes were not rinsed to remove residues of the test item supernatant, foreign bodies or incrustation 1 hour after application.

SCORING SYSTEM: according to the Draize scale

TOOL USED TO ASSESS SCORE: slit lamp biomicroscope / fluorescein solution
To detect cornea lesions the treated eyes were examined with the aid of a fluorescein solution and a slit lamp biomicroscope 24 hours post-application and from then on daily until end of the observation period. The eyes were rinsed with physiological saline 0.9% NaCl after the examination.

OBSERVATIONS
- body weight: prior to the administration and at the end of the observation period
- nature, severity and duration of clinical observations were described (systemic effects and local effects)
Irritation parameter:
cornea opacity score
Basis:
animal #1
Remarks:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #1
Remarks:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal #1
Remarks:
mean
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
animal #1
Remarks:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #2
Remarks:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #2
Remarks:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal #2
Remarks:
mean
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
animal #2
Remarks:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritant / corrosive response data:
Initial animal:
The animal showed slight conjunctival redness (score 1) from 1 hour until 24 hours post-application. The effect was fully reversible within 48 hours after application of the test material. The mean score of the animal, following grading at 24, 48 and 72 h after installation of the test material’s supernatant, was calculated to be 0.33.
Iris, conjunctiva and cornea were not affected by administration of the saturated aqueous solution of the test item.

Confirmatory animal:
The animal showed slight conjunctival redness (score 1) from one hour until 24 hours post-application. The effect was fully reversible within 48 hours after application of the test material. The mean score of the animal, following grading at 24, 48 and 72 h after installation of the test material’s supernatant, was calculated to be 0.33.
Iris, conjunctiva and cornea were not affected by administration of the saturated aqueous solution of Barium 4-dodecylphenolate.

Other effects:
- clinical observations: neither mortalities nor significant clinical signs of toxicity were observed in any animal. Local effects were only found in the confirmatory animal, which showed 1 hour post-application redness on the nictitating membrane. The observed effect fully disappeared within 24 hours.
- body weight: the body weight development of all animals was within the expected range.
Interpretation of results:
GHS criteria not met
Conclusions:
The saturated aqueous solution of barium 4-dodecylphenolate is not irritating to the eyes.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the saturated aqueous solution of barium 4-dodecylphenolate does not require classification as eye irritant.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

The substance was not observed to be irritanting to the skin in a reliable in vitro skin irritation study according to OECD 439.

Eye irritation:

The in vitro eye irritation test according to OECD 437 was technically not feasible. Therefore, an in vivo eye irritation study according to OECD 405 (2017) was conducted. The results showed that the substance was not observed to be irritanting to the eyes.

Justification for classification or non-classification

Skin irritation:

The substance does not possess a skin irritating potential based on an in vitro OECD 439 (2015) test and does not require classification as skin irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Eye irritation:

The substance does not possess an eye irritating potential based on an in vivo OECD 405 (2017) test and does not require classification as eye irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations.