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Administrative data

Description of key information

Skin irritation: not irritating (OECD 439, GLP, K, Rel.1)
Eye irritation: irritating (OECD 437/GLP/Rel.1, WoE, worst case)
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:
From June 22 to 26, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to OECD test Guideline No. 439 without any deviation.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected on October 13 and 14, 2014 / signed on April 08, 2015
Species:
other: human skin model EPIDerm™ tissues
Details on test animals or test system and environmental conditions:
Commercially available EPIDerm™-Kit (EPI-200-SIT): The EPIDerm™ tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EPIDerm™ tissues are cultured on specially prepared cell cultures inserts.
Origin: EPIDerm™ tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.
Day of delivery: 23 June 2015
Batch no.: 21677
Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
other: not applicable
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL of the test item was applied to the epidermis surface
- Concentration: The test item was used as supplied.
Number of animals:
Triplicate tissues for test item, negative and positive controls
Details on study design:
TEST VESSELS
All vessels used are made of single use sterile plastic.
The following vessels were used: 6-well-plates; 24-well plates; 96-well-plate

PERFORMANCE OF THE STUDY
Pre-Incubation of Tissues
Eight 6-well-plates were prepared with 0.9 mL assay medium in 3 of the 6 wells (upper row). The tissues were inspected for viability. Viable tissues were transferred (3 per plate) in the wells with the medium using sterile forceps under the clean bench and placed into the incubator at 37 ± 1°C and 5 ± 0.5% CO2 for 1 h. After the pre-incubation (1 h), the other 3 wells of each plate (lower row) were filled with fresh assay medium (0.9 mL). Every tissue was transferred into a well of the lower row. All 6-well-plates were set into the incubator at 37 ± 1 °C and 5 ± 0.5% CO2 for 18 h.

Treatment
The pre-incubated tissues were placed into fresh 6-well-plates containing 0.9 mL assay medium per well, using the upper row only.
One plate was used for treatment with the test item: 30 μL test item were applied, and a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
Tissues were dosed in 1 min. intervals. After dosing the last tissue, all plates are transferred into the incubator for 35 min. at 37 ± 1 °C and 5 ± 0.5% CO2. 60 min. after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-min. intervals. After rinsing, each tissue was dried with a sterile cotton tip and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL).
Then, the tissues were set in the incubator at 37 ± 1 °C and 5 ± 0.5% CO2 for 24 h.

Medium Renewal
For 3 incubated tissues, a new 6-well-plate with 0.9 mL assay medium in the upper row was prepared. The tissues were removed from the incubator and shaken for 10 min (500 rpm). Then the inserts were transferred into the new 6-well-plate and set into the incubator for 18 h and 10 min. for post-incubation at 37 ± 1 °C and 5 ± 0.5% CO2.

MTT Assay
After a total incubation time of 42 h, a 24-well-plate was prepared with 300 μL freshly prepared MTT-reagent in each well. The tissues were blotted on the bottom and then transferred into the 24-well-plate. Then the 24-well-plate was set into the incubator at 37 ± 1 °C and 5 ± 0.5% CO2 for 3 h. After this time, the MTT reagent was aspirated and replaced by DPBS buffer. This was then aspirated, too, and replaced several times.
At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then shaken for 2 h at room temperature.
After 2 h, the inserts in which formazan had been produced were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 μL solution (each) were pipetted into a 96-wellplate which was read in a plate spectral photometer at 570 nm.

CONTROLS (reference substances)
- Negative: One plate (3 tissues) was used as negative control; each tissue was treated with 30 μL DPBS buffer, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
- Positive: One plate was used as positive control; each tissue was treated with 30 μL SDS-solution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
Irritation / corrosion parameter:
% tissue viability
Value:
102.9
Other effects / acceptance of results:
Skin Irritation Potential of the Test Item: The mean tissue viability as percentage of mean OD of the negative control was 102.9% (± 3.2% SD) after the treatment with the test item. This value is above the threshold for skin irritation (50%). Therefore, the test item is considered as not irritant to skin.
Other effects:
None

Table 7.3.1/1: Absorbance Values negative control, test item and positive control (OD at 570 nm)

Designation

Measurement

Negative Control

Test item

Positive Control

Tissue 1

1

2.058

2.029

0.122

2

2.031

2.045

0.121

Tissue 2

1

2.021

2.061

0.107

2

2.001

2.095

0.107

Tissue 3

1

1.844

1.952

0.119

2

1.851

1.964

0.124

 

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol (0.036). Mean and relative standard deviation (comparison of the three tissues) was also calculated.

 

Table 7.3.1/2: Mean Absorbance Values

Designation

Negative Control

Test item

Positive Control

Mean corrected (tissue 1)

2.009

2.001

0.086

Mean corrected (tissue 2)

1.975

2.042

0.071

Mean corrected (tissue 3)

1.812

1.922

0.086

Corrected mean of the three tissues

1.932

1.988

0.081

 

Assessment of viability

For the test item and the positive control, the following percentage values of formazan production were calculated in comparison to the negative control:

 

Table 7.3.1/3: % Formazan Production

Designation

Negative Control

Test item

Positive Control

% Formazan production (tissue 1)

104.0%

103.6%

4.5%

% Formazan production (tissue 2)

102.2%

105.7%

3.7%

% Formazan production (tissue 3)

93.8%

99.5%

4.5%

% Formazan production (mean)

100%

102.9%

4.2%

± SD of mean Formazan

production (%)

5.5%

3.2%

0.5%

 

The mean percentage values of formazan production give the mean viability of the tissues.

Validity and Acceptability

 

Table 7.3.1/4: Validity

Criterion

Demanded

Found

OD of negative control

≥ 0.8 and ≤ 2.8

1.9

% Formazan production

of positive control SDS

20% of negative control

4.2%

SD of mean viability of the tissues

replicates (%)

< 18%

5.5 % (negative control)

0.5 % (positive control)

3.2 % (test item)

 

All validity criteria were met.

Values for negative control and for positive control were within the range of historical data of the test facility. Therefore, the experiment is considered valid.
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the test conditions, test item was not classified as irritant according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP).
Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the human skin model EPIDerm™

 

Three tissues of the human skin model EPIDerm™ were treated for 60 min with 30 μL of the undiluted liquid test item (using a nylon mesh) applied to each tissue and spread to match the tissue size (0.63 cm2). DPBS-buffer was used as negative control and 5 % SDS solution was used as positive control. After rinsing of the test item the tissues were incubated for 42 hours. The cell viability was determined by dehydrogenase conversion of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) to a blue formazan product. The percentage viability values, compared to negative control, were used to identify irritant and non-irritant substances.

 

After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.9. The positive control showed clear irritating effects. Relative absorbance was reduced to 4.2 %. Variation within tissues was acceptable (< 18%). Therefore this assay was valid since negative and positive controls showed results within the acceptable range.

 

After the treatment with test item, the mean tissue viability was 102.9 % (± 3.2 % standard deviation). This value is well above the threshold for irritation potential (50%). Therefore, test item is considered as not skin irritant in the Human Skin Model Test.

 

Under the test conditions, test item was classified as irritant according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP).

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.

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:
October 23, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to OECD test Guideline No. 437 without any deviation.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected on July 13-16, 2015 / signed on September 14, 2015
Species:
other: Bovine eye
Details on test animals or tissues and environmental conditions:
Test System: Freshly isolated bovine cornea (at least 9 to 60 month old donor cattle)
Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany.

Collection of Bovine Eyes: Freshly isolated bovine eyes of at least 9 month old donor cattle (at least 9 month to 60 month) were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in HBSS at cooled temperature. The corneae were isolated on the same day after delivery of the eyes. The corneae were directly used in the BCOP test on the same day.
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea.

Incubation Medium: The incubation medium consisted of MEM, supplemented with 1.1 g sodium bicarbonate, 5 mL L-glutamine, 5 mL penicillin/streptomycin (500 units penicillin, and 500 µg streptomycin per 5mL) per 500 mL medium. Immediately before starting the test, MEM was supplemented with 1% fetal calf serum (FCS).
The OECD recommends the use of EMEM which is in composition and osmolarity equivalent to the MEM, thus MEM can be used without restriction.
Vehicle:
unchanged (no vehicle)
Controls:
other: Negative control: 0.9 % w/v Sodium chloride. Positive control: 2-Ethoxyethanol (purity: 99%)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL of test item was applied on each cornea.
- Concentration (if solution): The test item was tested undiluted.
Duration of treatment / exposure:
Test item was applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1 °C, then followed by an incubation period of 120 minutes at 32 ± 1 °C in a vertical position.
Observation period (in vivo):
- Corneal opacity was measured pre-treatment, post-treatment and post-incubation (after 120 minutes of incubation).
- After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.
Number of animals or in vitro replicates:
3 corneas per treatment group (undiluted test item, negative control and positive control)
Details on study design:
EXPERIMENTAL PERFORMANCE:
Preparation of cornea:
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0).
The basal opacity of all corneae was recorded. Each corneae with a value of the basal opacity > 7 was discarded. Each cornea was examined for defects, such as lacerations, scratches or wrinkling. Corneas with any observed defects were discarded. Sets of three corneae were used for treatment with the test item and the negative and positive controls.

Treatment and incubation:
The anterior compartment received the test item or negative or positive control at a volume of 0.75 mL on the surface of the corneae. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath. The incubation time lasted ten minutes.
After the test item or control items, respectively, were rinsed off from the application side with saline, the corneae were incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a second opacity reading (t130). The opacity measurement is described in chapter 3.7.
In the second step of the assay, permeability of the corneae was determined.

Opacity Measurement:
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea. After exposure of the corneae to the test groups, after rinsing and further incubation of the corneae for two hours, the opacity value was determined again (t130).

Epithelium Condition:
Also, after the 2-hour post-exposure incubation period each cornea was observed visually and pertinent observations recorded (e.g., tissue peeling, residual test item, non-uniform opacity patterns). These observations are important as they may be reflected by variations in the opacitometer readings.

Permeability Determination:
Following to the opacity readings, the permeability was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium was removed from the anterior and from the posterior compartment and replaced by 1 mL of a 0.4% (w/v) sodium fluorescein solution (Sigma-Aldrich) in HBSS (anterior compartment) and by fresh cMEM (posterior compartment). Corneae were incubated again in a horizontal position for 90 ± 10 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer (Versamax® Molecular Devices). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

CONTROLS:
Negative Control: Saline (0.9% NaCl in deionised water, Envigo CRS GmbH) was included as negative control in each experiment in order to detect non-specific changes in the test system, as well as to provide a baseline for the assay endpoints.
Positive Control: A known ocular irritant was included in each experiment to verify that an appropriate response is induced. 2-Ethoxyethanol (purity: 99%, Sigma-Aldrich) served as positive control.
Irritation parameter:
other: In Vitro Irritancy Score (IVIS)
Basis:
mean
Remarks:
3 corneas
Time point:
other: 120 minutes
Score:
45.04
Irritant / corrosive response data:
Relative to the negative control, the test item caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 45.04.
Negative control:The opacity mean change value was 0.33 and the permeability mean of the negative control was 0.067. The negative control acceptance criterion were therefore satisfied.
Positive control: IVIS = 77.72. The positive control IVIS was within the range of 56.25 to 97.96. The positive control acceptance criterion was therefore satisfied.
Other effects:
Visual observation: Shortly after application of the test item the corneae became murky.

Table 7.3.2/1: Summary of results


Test Group

Opacity value = Difference (t130-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

Proposed in vitro Irritancy Score

Individual values

 

Mean

Individual values

Mean

Negative Control

0

0.33

(SD: 0.58)

0.064

0.067

(SD: 0.010)

0.96

1.34

Not categorized

1

0.059

1.89

0

0.078

1.17

Positive Control

58.67*

1.586*

82.46

77.72

Category 1

54.67*

1.680*

79.87

53.67*

1.145*

70.84

Test Item

31.67*

0.678*

41.84

45.04

No hazard prediction can be made

30.67*

1.062*

46.60

22.67*

1.601*

46.68

 

*corrected values

 

Visual observation: Shortly after application of the test item the corneae became murky.

Negative control: With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase ofopacity nor permeability of the corneae could be observed (mean IVIS=1.34).

Positive control:The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS =77.72) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).
Interpretation of results:
other: No prediction can be made based on the decision criteria
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the experimental conditions reported, the calculated mean in vitro irritancy score was 45.04 for the substance Tartrate diethyle and therefore no prediction for ocular irritation can be made as the result is outside the decision criteria (i.e. ≤3 or >55) according to the EU CLP.
Executive summary:

An in vitro eye irritation study was performed according to the OECD Guideline 437 and in compliance with GLP to assess the corneal damage potential of Tartrate diethyle by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contain incubation medium as well. Afterwards, opacity was measured a second time (t130). After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

 

With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS=1.34). The opacity mean change value was 0.33, which was equal to the maximum acceptance value of 0.33. The permeability mean of the negative control was 0.067, which was below the the maximum acceptance value of 0.091.

The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)). The mean IVIS was of 77.72, within the historical range (56.25-97.96) for the 30 assays performed from February 2015 to September 2015.

Relative to the negative control, the test item Tartrate diethyle caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 45.04.

 

Under the experimental conditions reported, the calculated mean in vitro irritancy score was 45.04 for the substance Tartrate diethyle and therefore no prediction for ocular irritation can be made as the result is outside the decision criteria (i.e. ≤3 or >55) according to the EU CLP.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation / corrosion:

An in vitro study was conducted on the substance (Laus, 2015, rel. 1). The skin irritation potential of the test material was evaluated using the EPIDerm TM human skin model after a treatment period of 60 minutes followed by a post-exposure incubation period of 42 hours. This test was designed to be compatible with the OECD Test Guideline No. 439 and was performed in compliance with GLP. The quality criteria required for acceptance of results in the test were satisfied. The relative mean viability of the test item treated tissues was 102.9 %, after the 60‑minute exposure period. With a tissue viability well above 50%, the test material was considered to be not irritant to skin.

Eye irritation:

An in vitro study on the registered substance was available and reliable (Envigo, 2015, rel.1).

This study was performed to assess the ocular irritancy potential of the undiluted test item to the isolated bovine cornea. The study was conducted according to the OECD guideline No. 437 and in compliance with GLP. The quality criteria required for acceptance of results in the test were satisfied. The In Vitro Irritancy Score of the test item was 45.04, after the 10 -minute exposure period followed by 120 -minute incubation period.

With an 3 < IVIS ≤ 55, no prediction can be made.

With an In IVIS < 55.1, the registered substance was considered not to be an ocular corrosive or severe irritant.

Considering the rather high result of this study, as a worst case scenario and to avoid animal testing, the substance was self classified in a worst case as serious eye irritant.


Justification for selection of skin irritation / corrosion endpoint:
A key study is available. The study is GLP-compliant and of high quality (Klimisch score = 1).

Justification for selection of eye irritation endpoint:
A study on the registered substance was available and reliable (Klimisch = 1) but the results were out of criteria for classification as corrosive or no classification. Based on the results of this study, as a worst case scenario and to avoid animal testing, the substance is directly classified as irritating to the eye.

Effects on eye irritation: irritating

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008 including ATP7.

 

Self-classification:

No additional self-classification is proposed regarding skin irritation according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).

Based on the available data, the substance should be classified as inducing serious eye irritation Category 2 (H319: Causes serious eye irritation) according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).

No data was available regarding respiratory irritation.