Decanoic acid, mixed esters with heptanoic acid, octanoic acid and pentaerythritol

Endpoint summary

Description of key information

Skin Corrosion - In vitro

Hatcol 3178 is considered as non-corrosive to skin in the Reconstructed Human Epidermis (RHE) Test Method.

Skin Irritation - In vitro

Hatcol 3178 is considered as non-irritant to skin in the Reconstructed Human Epidermis (RhE) Test Method.

Skin Corrosion/Irritation - In vivo

Not irritating to rabbit skin based on the results of the read across substances.

Eye Irritation - In vitro

Hatcol 3178 showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is -0.35.

Eye Irritation - In vivo

Not irritating to rabbit eyes based on the results of the read across substances.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Nov. 2016 to 01 Dec. 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)

Version / remarks:

OECD Guidelines for the Testing of Chemicals Part 431, adopted 29. Jul. 2016 “In vitro Skin Corrosion: reconstructed human epidermis (RHE) test method”

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU-Method B.40 BIS.

Version / remarks:

Commission Regulation (EU) No.440/2008, EU-Method B.40 BIS. “IN VITRO SKIN CORROSION: HUMAN SKIN MODEL TEST“ dated 30. May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Test system:

human skin model

Remarks:

EpiDerm TM

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

other: Not applicable

Details on animal used as source of test system:

The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
The EpiDermTM 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 EpiDermTM tissues are cultured on specially prepared cell cultures inserts.

Origin
EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.
Designation of the kit: EPI-212-SCT
Day of delivery: 29. Nov.

Justification for test system used:

The skin corrosion test refers to the production of irreversible tissue damage following the application of a test material on a reconstructed human skin model. It allows the identification of corrosive chemical substances and mixtures.
The test item is applied topically to a three-dimensional human skin model, comprised of non-transformed, 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, as well as a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo.
The principle of the human skin model assay is based on the hypothesis that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion and are cytotoxic to the underlying cell layers.
Corrosive chemicals are identified by their ability to decrease cell viability. The viability is measured by enzymatic conversion of the vital dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) into a blue formazan salt, that is quantitatively measured after extraction from tissues.

Vehicle:

unchanged (no vehicle)

Details on test system:

Additional Tests
Nylon mesh compatibility
First, Hatcol 3178 was tested for possible reaction with the nylon mesh which is used to ensure sufficient contact with the tissue surface. 50 μL of the liquid test item was pipetted onto a sample of nylon mesh, and then placed on a pre-cleaned glass microscope slide for analysis. No reaction with the mesh was visible after 1 hour incubation at room temperature.

Assessment of Coloured or Staining Test Items
A preliminary examination was performed to determine whether the test item independently reacts, and develops a colour without the addition of MTT. 50 μL of the test item was aliquoted into a test tube along with 0.3 mL demineralised water, and incubated at 37 ± 1°C and 5.0 ± 0.5% CO2 for 1 hour.
The results showed that the resulting solution was colourless, therefore binding capacity did not have to be tested.

Assessment of Direct Reduction of MTT by the Test Item
The test item Hatcol 3178 was also tested for the ability to reduce formazan directly. To test for this ability, 50 μL of the test item was added to 1 mL of MTT reagent and incubated under dark room conditions at 37 ± 1°C and 5.0 ± 0.5% CO2 for 1 hour. An additional sample of untreated MTT medium was used as control.
The MTT solution did not change its colour within 1 hour. It was therefore concluded that direct MTT reduction had not taken place, and no data correction was necessary in the final analysis.

Preparations
On the day of the start of the experiment, the MTT concentrate was removed from the freezer and allowed to thaw at room temperature. The concentrate was diluted using the assay medium supplied, and the solution was stored at 2-8°C under dark room conditions.
The tissue plate was removed from the refrigerator 1 hour before the treatment.
The assay medium was warmed in the water bath to 37 ± 1°C.

Description of the Method
Four 6-well-plates were prepared with 0.9 mL assay medium in each well. The inserts containing the tissues were transferred to the wells using sterile forceps and the 6-well-plates were set into the incubator at 37 ± 1°C and 5.0 ± 0.5% CO2 for 1 hour (pre-incubation).
For each experiment (“3 minutes” and “1 hour”), one 24-well-plate was prepared as holding plate. 12 wells of each plate were filled with 300 μL assay medium, the other 12 with 300 μL MTT reagent. One additional plate was left empty. The plates were stored in the incubator at 37 ± 1°C and 5.0 ± 0.5% CO2.
For each experiment (“3 minutes” and “1 hour”), two 6-well-plates for the assay were used.
After pre-incubation, the assay medium was replaced by fresh assay medium and the test was started, using two wells as negative control with 50 μL demineralised water, two wells as positive controls with 50 μL potassium hydroxide solution and two other wells for the test item.
The liquid test item was applied without preparation (50 μL).
After the respective incubation time (“3 minutes” and “1 hour”) at 37 ± 1°C and 5.0 ± 0.5% CO2, the inserts were removed from the plates using sterile forceps. The inserts were thoroughly rinsed with DPBS, blotted with sterile cellulose tissue and set into the respective holding plate, using the wells containing assay medium. After transfer of all inserts, they were immediately moved to the wells containing MTT medium, blotting the bottom with cellulose tissue again before setting the insert into the MTT well. The tissues were incubated with MTT medium for 3 hours at 37 ± 1°C and 5.0 ± 0.5% CO2.
Next, the MTT medium was aspirated and replaced by DPBS. This washing step was repeated several times. Finally, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. 2 mL isopropanol was pipetted into each well, taking care to ensure the solution reached the upper rim of the insert. The plate was then covered with Parafilm® and shaken for 2 hours on an orbital shaker at room temperature.
The inserts were then pierced with an injection needle to extract all traces of coloured solution.
The inserts were then discarded and the content of each well was thoroughly mixed.
From each well, three replicates of 200 μL solution (each) was pipetted into a 96-well-plate which was read in a plate spectrophotometer at a wavelength of 570 nm.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The liquid test item was applied without preparation (50 μL).

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

For each experiment (“3 minutes” and “1 hour”), two 6-well-plates for the assay were used.
After pre-incubation, the assay medium was replaced by fresh assay medium and the test was started, using two wells as negative control with 50 μL demineralised water, two wells as positive controls with 50 μL potassium hydroxide solution and two other wells for the test item.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

96.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h

Value:

105.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Corrosivity of the Test Item
The relative absorbance values were reduced to 96.7% after 3 minutes of treatment. This value is above the threshold for corrosivity (50%). After 1 hour of treatment, the relative absorbance values were increased to 105.8%, a value that is above the threshold for corrosivity (15%). Therefore, the test item is considered to be non-corrosive to skin.

Validity
The criterion for optical density of the negative control (≥ 0.8 and ≤ 2.8) was fulfilled: optical density was measured and found to be 1.9 (3 minutes) and 1.7 (1 hour).
The positive control showed clear corrosive effects. The criterion for the viability of the 1 hour experiment, expressed as % of the negative control (< 15%), was also fulfilled. The measured viability was observed to be 10.1%.
Values for negative control and positive control were within the range of historical data of the testing facility.
All measures obtained, and validated procedures conducted lead to the conclusion that the results obtained in relation to the corrosivity of Hatcol 3178 are to be considered as valid.

Absorbance Values

Negative Control		Test Item		Positive Control		Incubation
Tissue 1	Tissue 2	Tissue 1	Tissue 2	Tissue 1	Tissue 2
1.987	1,900	1.896	1.847	0.390	0.359	3 min
1.907	1.887	1.842	1.836	0.390	0.385
1.900	1.868	1.852	1.801	0.389	0.390
1.726	1.687	1.791	1.802	0.175	0.168	1 h
1.703	1.698	1.791	.1802	0.175	0.168
1.707	1.662	1.779	1.808	0.174	0.167
Mean		Mean		Mean
1.908		1.846		0.384		3 min
1.697		1.795		0.171		1 h

 

% Tissue Viability

Test Item	Positive Control	Incubation
96.7%	20.1%	3 min
105.8%	10.1%	1 h

 

Interpretation of results:

GHS criteria not met

Conclusions:

The test item Hatcol 3178 is considered to be non-corrosive to skin when tested in accordance with OECD guideline 431.
After 3 minutes of treatment, the relative absorbance values were decreased to 96.7%.
This value is well above the threshold for corrosivity (50%). After 1 hour treatment relative absorbance values were increased to 105.8%. This value is well above the threshold for corrosivity (15%). In OECD guideline 431, values greater or equal to the threshold are to be considered as “non-corrosive to skin”.

Executive summary:

Title of Study: Determination of Skin Corrosion Potential of Hatcol 3178 in the Reconstructed Human Epidermis (RHE) Test Method following OECD Guideline 431 and EU Method B.40-BIS

 

Findings and Results:

One valid experiment was performed.

Two tissues of the human skin model EpiDermTM were treated with Hatcol 3178 for 3 minutes and 1 hour, respectively. The test item was applied to each tissue and spread to match the tissue size.

Demineralised water was used as negative control, 8 M KOH was used as positive control.

After treatment, the respective substance was rinsed from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to a blue formazan. Formazan production was evaluated by measuring the optical density (OD) of the resulting solution.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals thus showing the quality of the tissues. The OD was 1.9 (3 minutes experiment) and 1.7 (1 hour experiment). The positive control showed clear corrosive effects for both treatment intervals. The relative absorbance value was reduced to 10.1 % for the 1 hour treatment.

After 3 minutes treatment with the test item, the relative absorbance values were reduced to 96.7 %. This value is above the threshold for corrosion potential (50%). After 1 hour treatment, relative absorbance values were increased to 105.8%. This value, too, is above the threshold for corrosion potential (15%). In the guideline, values greater or equal to the threshold are considered as “non-corrosive to skin”.

 

Therefore, Hatcol 3178 is considered as non-corrosive to skin in the Reconstructed Human Epidermis (RHE) Test Method.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Jan. 2017 to 20 Jan. 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)

Version / remarks:

OECD Guideline for the Testing of Chemicals, Version 439, adopted 28. July 2015, “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)

Version / remarks:

Commission Regulation (EU) No. 761/2009 amending Regulation (EC) No. 440/2008, Annex III, EU method B.46 : “IN VITRO SKIN IRRITATION: RECONSTRUCTED HUMAN EPIDERMIS MODEL TEST”, adopted 23. July 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Test system:

human skin model

Remarks:

EpiDerm TM

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

other: Not applicable

Details on animal used as source of test system:

Specification
The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
The EpiDermTM 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 representing main lipid classes analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell culture inserts.

Origin
EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.
Designation of the kit: EPI-212-SIT
Day of delivery: 17. Jan. 2017
Batch no.: 23388

Justification for test system used:

Skin irritation refers to the production of reversible damage to the skin following the application of a test chemical.
The test consists of a topical exposure of the neat test item to a human reconstructed epidermis model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid, present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percentage reduction of cell viability in comparison of untreated negative controls is used to predict the skin irritation potential.

Vehicle:

unchanged (no vehicle)

Details on test system:

Additional Tests
Nylon mesh compatibility
The test item was tested for possible reaction with the nylon mesh, which is used to ensure sufficient contact with the tissue surface. 30 μL of the test item was pipetted onto a sample of nylon mesh, and then placed on a pre-cleaned on a microscope slide for analysis.
No reaction with the nylon mesh was visible after an exposure time of 1 hour.

Assessment of Coloured or Staining Test Items
A preliminary examination was performed to determine whether the test item independently reacts, and develops a colour without the addition of MTT. 30 μL of the test item was aliquoted into a test tube along with 0.3 mL demineralised water, and incubated at 37 ± 1 °C and 5.0 ± 0.5% CO2 for 1 hour.
The results showed that the resulting solution was colourless, therefore binding capacity did not have to be tested.

Assessment of Direct Reduction of MTT by the Test Item
The test item was tested for the ability to reduce MTT directly. To test for this ability, 30 μL test item was added to 1 mL of MTT solution and the mixture was incubated under dark room conditions at 37 ± 1°C and 5.0 ± 0.5% CO2 for 1 hour. An additional sample of untreated MTT medium was used as control.
The MTT solution did not change its colour within 1 hour. It was therefore concluded that direct MTT reduction had not taken place, and no data correction was necessary in the final analysis.

Pre-Incubation of Tissues
All working steps were performed under sterile conditions. For each treatment group (negative control, test item and positive control) a 6-well-plate was prepared with 0.9 mL assay medium in 3 of the 6 wells (upper row). The tissues were inspected for viability. Then, the tissues were transferred into the wells, which contain medium by using sterile forceps and placed into the incubator at 37 ± 1°C and 5 ± 0.5% CO2 for 1 hour.
After 1 hour pre-incubation, 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 ± 0.5% CO2 for 19 hours.

Treatment
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.
One plate was used as positive control; each tissue was treated with 30 μL 5% SDS solution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
One plate was used for treatment with the test item:
30 μL test item was applied, and a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
Tissues were dosed in 1-minute-intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2.
1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.
After rinsing, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL).
Then, the tissues were placed in the incubator for 23 hours and 30 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2.

Medium Renewal
Post-incubation, the tissues were removed from the incubator and shaken for 5 minutes (120 rpm). 0.9 mL assay medium was filled in the lower row of the 6-well-plate. Inserts were then transferred to the lower row of the 6-well-plate and set into the incubator for 19 hours and 20 minutes for post-incubation at 37 ± 1°C and 5.0 ± 0.5% CO2.

MTT Assay
After a total incubation time of 42 hours and 50 minutes, a 24-well-plate was prepared with 300 μL freshly prepared MTT-medium 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 for 3 hours at 37 ± 1°C and 5.0 ± 0.5% CO2.
At this time, the MTT-medium 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-wellplate.
2 mL isopropanol was pipetted into each well, taking care to reach the upper rim of the insert. The plate was then shaken (120 rpm) for 2 hours at room temperature.
After 2 hours, the inserts 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 of 200 μL solution (each) were pipetted into a 96-well-plate which was read in a plate spectrophotometer at a wavelength of 570 nm.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

negative control; 30 μL DPBS buffer
positive control; 30 μL 5% SDS solution
30 μL test item

Duration of treatment / exposure:

Tissues were dosed in 1-minute-intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 minutes.

Duration of post-treatment incubation (if applicable):

23 hours and 30 minutes

Number of replicates:

3 per test group

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item

Value:

101.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Skin Irritation Potential of the Test Item
The relative absorbance values were increased to 101.9% after the treatment. This value is above the threshold for skin irritation (50%). Therefore, the test item is considered as non-irritant to skin.
All validity criteria were met.
The value for negative control was within the range of historical data. The value for positive control was below the range of historical data of the test facility. This can be seen as uncritical, because the value was well within the required validity criterion (≤ 20%) and the positive control showed clear skin irritating effects.
Therefore, the experiment is considered valid.

Absorbance values blank isopropanol (OD 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorbance	0.039	0.038	0.039	0.039	0.038	0.038	0.039	0.038	0.039

 

Absorbance Values negative control, test item and positive control (OD 570 nm)

Designation	Measurement	Negative Control	Hatcol 3178	Positive Control
Tissue 1	1	2.075	2.113	0.078
	2	2.048	2.072	0.079
Tissue 2	1	2.112	2.131	0.071
	2	2.053	2.092	0.073
Tissue 3	1	2.114	2.127	0.079
	2	2.057	2.159	0.078

 

Mean Absorbance Values

Designation	Negative Control	Hatcol 3178	Positive Control
Mean – blank (tissue 1)	2.023	2.054	0.040
Mean – blank (tissue 2)	2.044	2.073	0.033
Mean – blank (tissue 3)	2.047	2.104	0.040
Mean of three tissues	2.038	2.077	0.038

 

% Viability

Desigmation	Hatcol 3178	Positive Control
% Viability (tissue 1)	100.8%	2.0%
% Viability (tissue 2)	101.7%	1.6%
% Viability (tissue 3)	103.2%	2.0%
% Viability (mean)	101.9%	1.8%
± SD of mean viability (%)	1.2%	0.2%

 

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is considered as non-irritating to skin.
After the treatment, the relative absorbance values were increased to 101.9%. This value is above the threshold for skin irritation (50%).
The optical density of the negative control was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8.
The positive control has met the acceptance criterion too, for thus ensuring the validity of the test system.
Variation within replicates was within the accepted range for negative control, positive control and test item (required: ≤ 18%).
For these reasons, the result of the test is considered valid.

Executive summary:

Title of Study: Determination of Skin Irritation Potential of Hatcol 3178 in the Reconstructed Human Epidermis (RhE) Test Method following EU-Method B.46 resp. OECD 439

 

Findings and Results:

Three tissues of the human skin model EpiDermTM were treated with Hatcol 3178 for 60 minutes.

The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier).

DPBS-buffer was used as negative control, 5% SDS solution was used as positive control.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 2.0. The positive control showed clear irritating effects. Relative absorbance was reduced to 1.8% (required: ≤ 20%).

Variation within the tissue replicates was acceptable (required: ≤ 18%).

 

After the treatment with the test item, the relative absorbance values were increased to 101.9 %. This value is above the threshold for skin irritation potential (50%).

 

Therefore, Hatcol 3178 is considered as non-irritant to skin in the Reconstructed Human Epidermis (RhE) Test Method.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Nov - 16 Nov 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

adopted in 1981

Deviations:

yes

Remarks:

Lack of details on test substance

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elevage Cunicole du Val de Selle, France
- Weight at study initiation: 2.3 kg
- Diet: ad libitum; Rabbit certified pellet diet (Rabbit sustenance ref. 112 C), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days
-other: the animals received a preventive coccidiosis treatment during acclimation (Mucoxid, 137.5 mg/kg bw/day), via drinking water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 / 12

Type of coverage:

semiocclusive

Preparation of test site:

other: clipped

Vehicle:

unchanged (no vehicle)

Controls:

other: not required, untreated sites of the same animal served as control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL

Duration of treatment / exposure:

4 h

Observation period:

72 h
Reading time points: 1, 24, 48 and 72 h

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: 6 cm², flanks were clipped a day before treatment
- Type of wrap if used: adhesive hypoallergic aerated semi-occlusive dressing (Laboratoires de Pansements et d´Hygiène, France)


REMOVAL OF TEST SUBSTANCE
- Washing (if done): No


SCORING SYSTEM: According to Draize

Irritation parameter:

erythema score

Basis:

mean

Remarks:

of all three animals

Time point:

other: 24 h, 48 h and 72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: no cutaneous effects observed

Irritation parameter:

edema score

Basis:

mean

Remarks:

of all three animals

Time point:

other: 24 h, 48 h and 72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: no cutaneous effects observed

Irritant / corrosive response data:

No cutaneous reactions were observed in all the animals.

Erythema score

Animal Number	1 h	24 h	48 h	72 h
1	0	0	0	0
2	0	0	0	0
3	0	0	0	0

 

Edema Score

Animal Number	1 h	24 h	48 h	72 h
1	0	0	0	0
2	0	0	0	0
3	0	0	0	0

 

Interpretation of results:

GHS criteria not met

Conclusions:

CLP: not classified
DSD: not classified

Reference 4

Endpoint:

skin irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Justification for type of information:

The substance is a member of a group of pentaerythritol and a mixture of alkyl carboxylic acids which share similar characteristics across the group. Please see attached justification for read across.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Remarks:

CAS 11138-60-6, 70693-33-3

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Remarks:

CAS 11138-60-6, 70693-33-3

Interpretation of results:

GHS criteria not met

Conclusions:

The substance, CAS 68441-67-8 is analogous to the substances to be read across to, in terms of basic form, and the degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of in vivo skin irriation. The substance is considered to be not irritating to skin for the defined endpoints on the basis of read across. This will be confirmed by appropriate study data as soon as this is available.

Executive summary:

The substance, CAS 68441-67-8 is analogous to the substances to be read across to, in terms of basic form, and the degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of in vivo skin irriation. The substance is considered to be not irritating to skin for the defined endpoints on the basis of read across. This will be confirmed by appropriate study data as soon as this is available.

Reference 5

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

17 to 20 December 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD, EU & US EPA test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Species: Albino Rabbit, New Zealand White, (SPF-Quality). Recognised by international guidelines as the recommended test system (e.g. EC, OECD). Source: Charles River Deutschland. Kisslegg, Germany
Number of animals: 3 Animals of one sex.
Age and body weight: Animals used within the study were at least 6 weeks old and body weights were at least 1.0 kg.
Identification: Earmark.

Conditions: A controlled environment was maintained in the room with optimal conditions considered as being approximately 15 air changes per hour, a temperature of 21±3°C, a relative humidity of 30-70% and 12 hours artificial fluorescent light and 12 hours dark per day.
Accommodation: Individually in labelled cages with perforated floors (Scan bur. Denmark, dimensions 56x44x37.5 cm). Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.
Diet: Standard laboratory rabbit diet (Charles River Breeding and Maintenance Diet for Rabbits,
Allromin, Lage, Gennany) approx. 100 g. per day. In addition, pressed hay (BMI, Helmond, the Netherlands) was provided twice a week.
Water: Free access to tap-water.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

The test substance was applied undiluted as delivered by the sponsor.
Each animal was treated by dermal application of 0.5 ml of the test substance.

Duration of treatment / exposure:

Four hours

Observation period:

72 hours

Number of animals:

3 male rabbits treated with the test substance

Details on study design:

TREATMENT
Approximately 24 hours before treatment, the dorsal fur was clipped with electric clippers, exposing an area of approximately 150 square centimetres (10x15 cm2). Whenever considered necessary the treated skin areas were re-lipped at least 3 hours before the observations, to facilitate scoring.
A health inspection was performed prior to the commencement of treatment, to ensure that the animals were in a good state of health. Special attention was paid to the skin to be treated, which was intact and free from abnormalities.
Each animal was treated by dermal application of 0.5 ml of the test substance. The test substance was applied to the skin of one flank, using a metalline patch of 2x3 cm. The patch was mounted on Micropore tape, which was wrapped around the abdomen and secured with Coban elastic bandage.
Four hours after the application, the dressing was removed and the skin cleaned of residual test substance using water.

OBSERVATIONS
Mortality/Viability: Twice daily.
Toxicity: At least once daily.
Body Weight: Day of treatment (prior to application) and at termination.
Irritation: The skin reactions were assessed at approximately 1, 24, 48 and 72 hours after the removal of the dressings and test substance. The irritation scores and a description of all other (local) effects were recorded. Adjacent areas of the untreated skin of each animal served as controls.

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 24-72 hours

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 24-72 hours

Score:

0

Max. score:

4

Irritant / corrosive response data:

Irritation: Four hours exposure to 0.5 ml of HATCOL 5236 resulted in very slight erythema in the treated skin-areas of the three rabbits.
The skin irritation had resolved within 1 day after exposure in all animals.

Corrosion: There was no evidence of a corrosive effect on the skin.

Other effects:

Colouration: Sticky and dry remnants of the test substance were present on the skin on days 1 and 2 in one animal and during the whole observation period in the other two animals.
Toxicity / Mortality: No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

INDIVIDUAL SKIN IRRITATION SCORES

Following exposure, sticky remnants of the test substances were found on the treated skin-area after removal of the test substance.

Animal#	74			76			78
Time after exposure	Erythema	Oedema	Comments	Erythema	Oedema	Comments	Erythema	Oedema	Comments
1 hour	1	0	b	1	0	b	1	0	b
24 hours	0	0	b1	0	0	b1	0	0	b1
48 hours	0	0	b1	0	0	-	0	0	b1
72 hours	0	0	b1	0	0	-	0	0	b1

Comments:

b. Sticky remnants of the test substance present.

b1. Dry remnants of the test substance present.

 

MEAN VALUE IRRITATION SCORES

Animal#	Mean 24-72 hrs
	Erythema	Oedema
74	0	0
76	0	0
78	0	0

 

#Animal specifications:

Animal no	Sex	Age at start (weeks)	Body weights (grams)
			Prior to application	At termination
74	Male	8-10	1678	1871
76	Male	8-10	1803	1936
78	Male	8-10	1742	1936

 

Interpretation of results:

GHS criteria not met

Conclusions:

HATCOL 5236 does not have to be classified and has no obligatory labelling requirement for skin irritation.

Executive summary:

Primary skin irritation/corrosion study with HATCOL 5236 in the rabbit (4-hour semi-occlusiveapplication).

The study was carried out based on the guidelines described in: EC Commission Directive

92/69/EEC, B.4 "Acute Toxicity - Skin irritation", OECD No.404, "Acute Dermal Irritation/Corrosion", US EPA, OPPTS 870.2500, Acute Dermal Irritation, EPA 712-C-98-196, August 1998 and JMAFF, Japanese Test Guidelines (draft), July 2000.

Three rabbits were exposed to 0.5 ml of HATCOL 5236, applied onto clipped skin for 4 hours using a semi-occlusive dressing. Observations were made 1, 24, 48 and 72 hours afterexposure.

Four hours exposure to 0.5 ml of HATCOL 5236 resulted in very slight erythema in the treated skin-areas of the three rabbits.

The skin irritation had resolved within 1 day after exposure in all animals.

Based on the results and according to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Council Directive 67/548/EEC), HATCOL 5236 does not have to be classified and has no obligatory labelling requirement for skin irritation.

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:

11 Jan. 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Version / remarks:

OECD Guideline for the Testing of Chemicals, Method No. 437, edition adopted 26. Jul. 2013: “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:

according to guideline

Guideline:

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

Version / remarks:

Regulation (EU) No. 1152/2010 amending Regulation (EC) No. 440/2008, EU Method B. 47: “Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants” adopted 08. Dec. 2010

Deviations:

no

Principles of method if other than guideline:

OECD Guideline for the Testing of Chemicals, Series on Testing and Assessment No. 160: “GUIDANCE DOCUMENT ON “THE BOVINE CORNEAL OPACITY AND PERMEABILITY (BCOP) AND ISOLATED CHICKEN EYE (ICE) TEST METHODS: COLLECTION OF TISSUES FOR HISTOLOGICAL EVALUATION AND COLLECTION OF DATA ON NON-SEVERE IRRITANTS”; 25. Oct. 2011

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Specification
Species: Bos primigenius Taurus (fresh bovine corneas)

Origin
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hank’s balanced salt solution (supplemented with 0.01% streptomycin and 0.01% penicillin). Then, the corneas were dissected and incubated in medium at 32 ± 1 °C in an incubation chamber for 1 h.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The respective substance (negative control solution, test item or positive control) was applied by pipetting 750 μL of the appropriate liquid.

Duration of treatment / exposure:

Exposure time on the corneas was 10 minutes at 32 ± 1 °C.

Duration of post- treatment incubation (in vitro):

The corneas were stored for additional 2 hours at 32 ± 1 °C (post-incubation).

Number of animals or in vitro replicates:

3 replicates per group (test item, negative control & positive control).

Details on study design:

Preparations
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C.
On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C.
The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate.
After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.

Experimental Parameters
Date of treatment 11. Jan. 2017
Incubation time 10 min.
Negative control HBSS solution
Positive control Dimethylformamide (undiluted)

Method Description
After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used. For each treatment group (negative control solution, test item and positive control), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 μL negative control solution, test item and positive control were applied to each replicate.
According to the characteristics of the test item, the following treatment procedure was performed:
Closed Chamber Method
The “closed chamber-method” is used for liquid substances.
The respective substance (negative control solution, test item or positive control) was applied by pipetting 750 μL of the appropriate liquid through the refill hole in the holder on the cornea. The test item was given on the epithelium in such a manner that as much as possible of the cornea was covered with the test item.
Exposure time on the corneas was 10 minutes at 32 ± 1 °C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chamber was filled with cMEM without phenol red, and the corneas were stored for additional 2 hours at 32 ± 1 °C (post-incubation).
After the post-incubation, the cMEM without phenol red was renewed in both chambers.
Then, the final opacity value of each cornea was recorded. The cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution (concentration: 4 mg/mL) was added to the front chamber.
The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, the permeability of the liquid was measured with the spectrophotometer at 492 nm.

Irritation parameter:

in vitro irritation score

Run / experiment:

Test Item

Value:

-0.35

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Under the conditions of this test, the test item Hatcol 3178 showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is -0.35.
The negative control (HBSS-solution) and the positive control (undiluted dimethylformamide) have met the validity criteria.
No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.

Illuminance Values

Parameter	Negative Control			Test item			Positive Control
(I) Measured values before exposure	1018	989	1016	1023	1033	1053	997	985	956
(I) Measured values after exposure	971	971	1001	1013	1013	1031	311	300	271

 

Opacity Values Negative Control

Parameter	Negative Control
Opacity before exposure	2.17	3.39	2.25
Opacity after exposure	4.18	4.18	2.87
Opacity Difference	2.01	0.79	0.62
Mean Opacity Difference	1.14

 

Opacity Values Test Item and Positive Control

Parameter	Test Item			Positive Control
Opacity before exposure	1.96	1.56	0.79	3.04	3.56	4.87
Opacity after exposure	2.37	2.37	1.64	96.71	101.70	116.80
Opacity Difference	0.41	0.81	0.86	93.66	98.14	111.93
Opacity Difference Corrected	-0.74	-0.33	-0.29	92.52	96.99	110.79

 

Optical density at 492 nm of Blank

Parameter	cMEM without phenol red
1. Measurement	0.033
2. Measurement	0.035
3. Measurement	0.043
Mean	0.037

 

Optical density at 492 nm of Negative Control, Test Item and Positive Control

Parameter	Negative Control			Test Item			Positive Control
1. Measurement	0.046	0.037	0.033	0.037	0.039	0.060	0.534	0.484	0.784
2. Measurement	0.043	0.035	0.033	0.035	0.040	0.060	0.546	0.482	0.784
3. Measurement	0.041	0.040	0.034	0.037	0.037	0.060	0.544	0.480	0.797
1. Measurement – blank	0.009 0	0.000 0	- 0.004 0	0.000 0	0.002 0	0.023 0	0.4970	0.4470	0.7470
2. Measurement – blank	0.006 0	- 0.002 0	- 0.004 0	- 0.002 0	0.003 0	0.023 0	0.5090	0.4450	0.7470
3. Measurement – blank	0.004 0	0.003 0	- 0.003 0	0.000 0	0.000 0	0.023 0	0.5070	0.4430	0.7600
Mean of each replicates	0.006 3	0.000 3	- 0.003 7	- 0.000 7	0.001 7	0.023 0	0.5043	0.4450	0.7513
Mean of the three replicates	0.0010			-			-
Corrected	-	-	-	- 0.0017	0.0007	0.0220	2.5207*	2.2240*	3.7557*

* Note: All values for the positive control were obtained by measurement of a 5-fold diluted solution and multiplication of the absorbances with factor 5.

 

IVIS

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control HBSS-solution	2.11	1.16	71.64%
	0.80
	0.57
Test item Hatcol 3178	-0.76	-0.35	116.10%
	-0.32
	0.04
Positive Control DMF undiluted	130.33	142.60	14.89%
	130.35
	167.13

 

Interpretation of results:

GHS criteria not met

Conclusions:

This in vitro study was performed to assess corneal damage potential of Hatcol 3178 by quantitative measurements of changes in opacity and permeability in a bovine cornea.
The test item Hatcol 3178 was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been determined. The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured.
The test item was tested pure.
Under the conditions of this test, the test item Hatcol 3178 showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is -0.35.
According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.
The negative control (HBSS-solution) and the positive control (undiluted dimethylformamide) have met the validity criteria.
No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.

Executive summary:

Title of Study: Evaluation of Hatcol 3178 in the BCOP Test following OECD Guideline 437 resp. EU Method B.47

 

Findings and Results:

One valid experiment was performed.

Bovine corneas were used. They were collected from slaughtered cattle which were between 12 and 60 months old.

The test item Hatcol 3178 was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C.

After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured.

 

HBSS-solution was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (in vitro irritancy score) is 1.16.

Dimethylformamide (DMF) undiluted was used as positive control. The positive control induced serious eye damage on the cornea and falls within two standard deviations of the current historical mean. The calculated IVIS (in vitro irritancy score) is 142.60.

 

Under the conditions of this study, the test item Hatcol 3178 showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is -0.35.

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

The substance is a member of a group of pentaerythritol and a mixture of alkyl carboxylic acids which share similar characteristics across the group. Please see attached justification for read across.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Remarks:

CAS 11138-60-6, 70693-33-3

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Remarks:

CAS 11138-60-6, 70693-33-3

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Remarks:

CAS 11138-60-6, 70693-33-3

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Remarks:

CAS 11138-60-6, 70693-33-3

Interpretation of results:

GHS criteria not met

Conclusions:

The substance, CAS 68441-67-8 is analogous to the substances to be read across to, in terms of basic form, and the degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of in vivo eye irriation. The substance is considered to be not irritating to skin for the defined endpoints on the basis of read across. This will be confirmed by appropriate study data as soon as this is available.

Executive summary:

The substance, CAS 68441-67-8 is analogous to the substances to be read across to, in terms of basic form, and the degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of in vivo eye irriation. The substance is considered to be not irritating to skin for the defined endpoints on the basis of read across. This will be confirmed by appropriate study data as soon as this is available.

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

17 to 20 December 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD, EU & US EPA test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Species: Albino Rabbit, New Zealand White, (SPF-Quality). Recognised by international guidelines as the recommended test system (e.g. EC, OECD). Source: Charles River Deutschland, Kisslegg, Germany
Number of animals: 3 Animals of one sex.
Age and body weight: Animals used within the study were at least 6 weeks old and body weights were at least 1.0 kg.
Identification: Earmark

Conditions: A controlled environment was maintained in the room with optimal conditions considered as being approximately 15 air changes per hour, a temperature of 21 ± 3°C, a relative humidity of 30-70% and 12 hours artificial fluorescent light and 12 hours dark per day.
Accommodation: Individually in labelled cages with perforated floors (Scanbur, Denmark, dimensions 56x44x37.5 cm). Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.
Diet: Standard laboratory rabbit diet (Charles River Breeding and Maintenance Diet for Rabbits,
A1tromin, Lage, Germany) approx. 100 g. per day. In addition, pressed hay (BMI, Helmond, the Netherlands) was provided twice a week.
Water: Free access to tap-water.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

Each animal was treated by instillation of 0.1 ml of the test substance

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3 male rabbits

Details on study design:

TREATMENT
A health inspection was performed prior to commencement of treatment, to ensure that the animals were in a good state of health. Special attention was paid to the eyes, which were free from any abnormality.
Each animal was treated by instillation of 0.1 ml of the test substance in the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test substance. The other eye remained untreated and served as the reference control.
Immediately after the 24-hour observation, a solution of 2% fluorescein in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area.
Mortality/Viability: Twice daily.
Toxicity: At least once daily.
Body Weight: Day of treatment (prior to instillation) and at termination.
Irritation: The eyes of each animal were examined approximately 1, 24, 48 and 72 hours after instillation of the test substance. The irritation scores and a description of all other (local) effects were recorded.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: 24-72 hours

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Time point:

other: 24-72 hours

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

mean

Time point:

other: 24-72 hours

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: 24-72 hours

Score:

0

Max. score:

4

Irritant / corrosive response data:

Irritation: Instillation of 0.1 ml of the test substance into one eye of each of three rabbits resulted in irritation of the conjunctivae, which was seen as redness on day 1.
No iridial irritation or corneal opacity was observed, and treatment of the eyes with 2% fluorescein, 24 hours after test substance instillation revealed no corneal epithelial damage in any of the animals.
Corrosion: There was no evidence of ocular corrosion.

Other effects:

Colouration / Remnants: No staining of (peri) ocular tissues by the test substance was observed.
Toxicity / Mortality: No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

INDIVIDUAL EYE IRRITATION SCORES

Time after dosing	Cornea			Iris	Conjunctivae			Comments
	Opacity	Area	Fluor area (%)		Redness	Chemosis	Discharge
M No 61#
1 hour	0	0		0	1	0	0	-
24 hours	0	0	0	0	0	0	0	-
48 hours	0	0		0	0	0	0	-
72 hours	0	0		0	0	0	0	-
M No 66#
1 hour	0	0		0	1	0	0	-
24 hours	0	0	0	0	0	0	0	-
48 hours	0	0		0	0	0	0	-
72 hours	0	0		0	0	0	0	-
M No 85#
1 hour	0	0		0	1	0	0	-
24 hours	0	0	0	0	0	0	0	-
48 hours	0	0		0	0	0	0	-
72 hours	0	0		0	0	0	0	-

Fluor area (%): green staining (percentage of total corneal area) after fluorescein treatment

 

MEAN VALUE EYE IRRITATION SCORES

Animal#	Mean 24-72 hours
	Corneal opacity	Iris	Conjunctivae
			Redness	Chemosis
61	0	0	0	0
66	0	0	0	0
85	0	0	0	0

 

#Animal specifications:

Animal no	Sex	Age at start (weeks)	Bodyweights (grams)
			Prior to application	At termination
61	Male	12-14	2950	2953
66	Male	12-14	2535	2622
85	Male	9-11	2260	2361

 

Interpretation of results:

GHS criteria not met

Conclusions:

HATCOL 5236 does not have to be classified and has no obligatory labelling requirement for eye irritation.

Executive summary:

Acute eye irritation/corrosion study with HATCOL 5236 in the rabbit.

The study was carried out based on the guidelines described in: EC Commission Directive92/69/EEC, B.5, "Acute Toxicity - Eye irritation", OECD No 405, "Acute Eye Irritation/Corrosion"and US EPA, OPPTS 870.2400, Acute Eye Irritation, EPA 712-C-98-195, August 1998.

Single samples of 0.1 ml of HATCOL 5236 were instilled into one eye of each of three rabbits.

Observations were made 1, 24, 48 and 72 hours after instillation.

Instillation of 0.1 ml of the test substance into one eye of each of three rabbits resulted in irritation of the conjunctivae, which was seen as redness on day 1.

Based on these results and according to the EC criteria for classffcation and labelling requirements for dangerous substances and preparations (Council Directive 67/548/EEC), HATCOL 5236 does not have to be classified and has no obligatory labelling requirement for eye irritation.

Reference 4

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Nov - 23 Nov 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study; only few details on study substance)

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted in 1987

Deviations:

yes

Remarks:

lack of details on test substance

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Elevage Cunicole du Val de Selle, France
- Weight at study initiation: 2.6 kg
- Housing: individually
- Diet: Certified pellet (Rabbits sustenance ref. 112 C; ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days
-other: the animals received a preventive coccidiosis treatment during acclimation (Mucoxid, 137.5 mg/kg bw/day, via drinking water)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:

unchanged (no vehicle)

Controls:

other: the untreated eye served as control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL

Duration of treatment / exposure:

single dose, no rinsing

Observation period (in vivo):

72 h
Reading time points: 1, 24, 48 and 72 h

Number of animals or in vitro replicates:

3 males

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): No

SCORING SYSTEM: according to 84/499/EEC appendix V B5, i.e., according to the Draize scoring system

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

of all 3 animals

Time point:

other: mean over 24, 48 and 72 h

Score:

0

Max. score:

3

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: only slight exsudation at 1h (2 cases) and 24 h (1 case) was seen

Irritation parameter:

iris score

Basis:

mean

Remarks:

of all 3 animals

Time point:

other: 24 h, 48 h and 72 h

Score:

0

Max. score:

2

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: none of the animals showed effects in the iris

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

of all 3 animals

Time point:

other: 24 h, 48 h and 72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility not applicable

Remarks on result:

other: none of the aniamals showed corneal opacity

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

of all 3 animals

Time point:

other: 24 h, 48 h and 72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility not applicable

Remarks on result:

other: none of the animals showed chemosis

Irritant / corrosive response data:

In 2 animals slight exsudation (grade 1) was obsevered at 1 h. At 24 h slight exsudation (grade 1) was observed in 1 animals. These effects were completely reversible at 48 h.

Animal	Hours after application
	1					24					48					72
	A	B	C	D	E	A	B	C	D	E	A	B	C	D	E	A	B	C	D	E
1	0	0	0	0	1	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0
2	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
3	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

A = Cornea Score

B = Iris Score

C = Erythema Score

D = Chemosis Score

E = Exsudation Score

Interpretation of results:

GHS criteria not met

Conclusions:

CLP: not classified
DSD: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Corrosion - In vitro

One valid experiment was performed.

Two tissues of the human skin model EpiDermTM were treated with Hatcol 3178 for 3 minutes and 1 hour, respectively. The test item was applied to each tissue and spread to match the tissue size.

Demineralised water was used as negative control, 8 M KOH was used as positive control.

After treatment, the respective substance was rinsed from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to a blue formazan. Formazan production was evaluated by measuring the optical density (OD) of the resulting solution.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals thus showing the quality of the tissues. The OD was 1.9 (3 minutes experiment) and 1.7 (1 hour experiment). The positive control showed clear corrosive effects for both treatment intervals. The relative absorbance value was reduced to 10.1 % for the 1 hour treatment.

After 3 minutes treatment with the test item, the relative absorbance values were reduced to 96.7 %. This value is above the threshold for corrosion potential (50%). After 1 hour treatment, relative absorbance values were increased to 105.8%. This value, too, is above the threshold for corrosion potential (15%). In the guideline, values greater or equal to the threshold are considered as “non-corrosive to skin”.

Therefore, Hatcol 3178 is considered as non-corrosive to skin in the Reconstructed Human Epidermis (RHE) Test Method.

Skin irritation - In vitro

Three tissues of the human skin model EpiDermTM were treated with Hatcol 3178 for 60 minutes.

The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier).

DPBS-buffer was used as negative control, 5% SDS solution was used as positive control.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 2.0. The positive control showed clear irritating effects. Relative absorbance was reduced to 1.8% (required: ≤ 20%).

Variation within the tissue replicates was acceptable (required: ≤ 18%).

After the treatment with the test item, the relative absorbance values were increased to 101.9 %. This value is above the threshold for skin irritation potential (50%).

Therefore, Hatcol 3178 is considered as non-irritant to skin in the Reconstructed Human Epidermis (RhE) Test Method.

Skin Corrosion/Irritation - In vivo

2,2-bis[(octanoyloxy)methyl]butyl decanoate (CAS No. 11138-60-6) was tested for its skin irritation potential according to OECD Guideline 404 (Key, Oleon, Clouzeau, 1990, skin irr., rabbit, RL1): The shaved back of three New Zealand White rabbits was exposed to 0.5 mL test material for 4 hours under semi-occlusive conditions. The rabbits were observed for 3 d after exposure and skin reactions were

assessed using the Draize scheme periodically (24, 48 and 72 hours) after removal of the test substance.

No cutaneous reactions were observed in any of the tested animals at any observation interval.

Primary skin irritation/corrosion study with HATCOL 5236 in the rabbit (4-hour semi-occlusive application).

Three rabbits were exposed to 0.5 ml of HATCOL 5236, applied onto clipped skin for 4 hours using a semi-occlusive dressing. Observations were made 1, 24, 48 and 72 hours after exposure. Four hours exposure to 0.5 ml of HATCOL 5236 resulted in very slight erythema in the treated skin areas of the three rabbits.

The skin irritation had resolved within 1 day after exposure in all animals.

Based on the results HATCOL 5236 does not have to be classified for skin irritation.

Eye irritation - In Vitro

One valid experiment was performed.

Bovine corneas were used. The test item Hatcol 3178 was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C.

After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured.

 

HBSS-solution was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (in vitro irritancy score) is 1.16.

Dimethylformamide (DMF) undiluted was used as positive control. The positive control induced serious eye damage on the cornea and falls within two standard deviations of the current historical mean. The calculated IVIS (in vitro irritancy score) is 142.60.

Under the conditions of the study, the test item Hatcol 3178 showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is -0.35.

Eye Irritation: In vivo

2,2-bis[(octanoyloxy)methyl]butyl decanoate (CAS No. 11138-60-6) was tested for its eye irritation potential according to OECD Guideline 405 (Key, Oleon, Clouzeau, 1990, eye irr., rabbit, RL1): 0.1 mL of the test material were instilled into the conjunctival sac of one eye of three New Zealand White rabbits. Animals were observed for 72 hours and observed effects were scored 24, 48 and 72 hours

after instillation according to 84/499/EEC appendix V B5 (Draize). No effects, except for one animal with a very slight exudation reaction at the 24 h time point, were observed in any of the tested animals at any relevant observation interval.

Acute eye irritation/corrosion study with HATCOL 5236 in the rabbit.

Single samples of 0.1 ml of HATCOL 5236 were instilled into one eye of each of three rabbits.

Observations were made 1, 24, 48 and 72 hours after instillation.

Instillation of 0.1 ml of the test substance into one eye of each of three rabbits resulted in irritation of the conjunctivae, which was seen as redness on day 1.

Based on these results HATCOL 5236 does not have to be classified for eye irritation.

Justification for classification or non-classification

Skin Irritation / Corrosion

The test substance was found to be non-corrosive and non-irritant in two seperate in vitro studies, therefore, Hatcol 3178 is not classified as it does not fulfill the criteria for classification in accordance with CLP.

Eye Irritation

Under the conditions of the study, the test item Hatcol 3178 showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is -0.35.

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.